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Chemokines are produced in the brain early during the course of experimental African trypanosomiasis
Abstract
African trypanosomiasis is characterized by progressive central nervous system (CNS) involvement. Using single and double immunohistochemistry, we evaluated the induction of alpha- and beta-chemokines in brains of Sprague-Dawley rats infected with Trypanosoma brucei brucei (T. b. brucei) and identified their cellular source. The results showed high production of MIP-2, RANTES and MIP-1alpha and to a lower extend MCP-1 in infected animals compared to controls. MIP-2, RANTES and MIP-1alpha were produced early by astrocytes and microglia and later by macrophages and T-cells. These findings suggest that chemokines may contribute to the immunopathogenesis that occurs in the CNS early during infections.
... As previously stated, the precise mechanisms used by the parasite to gain entry to the brain and induce the associated inflammatory reaction remain unclear. However, mouse model studies indicate that the expression of inflammatory mediators including cytokines, chemokines and adhesion molecules change during the course of the infection and that the balance between pro-and anti-inflammatory mediators is central to the outcome of the disease (Kennedy, 2004(Kennedy, , 2008aSharafeldin et al., 2000;Mulenga et al., 2001). Following parasite CNS invasion in a murine model of HAT, expression of IL-1, IL-6, TNF-α, IFN-γ and MIP-1α was found to be upregulated within the CNS by comparative RT-PCR analysis (Hunter et al., 1991(Hunter et al., , 1992b. ...
... There is also evidence for early CNS production of the chemokines MIP-2, RANTES, MIP-1α and to a lesser extent MCP-1 following T.b. brucei infection in rats (Sharafeldin et al., 2000). Using double Fig. 2. Schematic representation of the possible pathways and cell types involved in the generation of the neuroinflammatory response associated with trypanosome infection and drug treatment. ...
... In addition recent evidence suggest a central role for IFN-γ, T-lymphocyte transmigration pathways and the presence of particular laminins in the BBB basement membranes, in the entry of the parasites to the brain parenchyma. labelling immunocytochemistry the initial source of the MIP-2, RANTES and MIP-1α was shown to be astrocytes and microglia with production switching to macrophages and T-cells later in the course of the disease (Sharafeldin et al., 2000). These results suggest that astrocytes and microglia become activated very quickly following infection and initiate the inflammatory reaction within the brain which is later enhanced by the infiltrating lymphocytes. ...
Trypanosomes have been recognised as human pathogens for over a century. Human African trypanosomiasis is endemic in an area sustaining 60 million people and is fatal without chemotherapeutic intervention. Available trypanocidal drugs require parenteral administration and are associated with adverse reactions including the development of a severe post-treatment reactive encephalopathy (PTRE). Following infection the parasites proliferate in the systemic compartment before invading the CNS where a cascade of events results in neuroinflammation. This review summarises the clinical manifestations of the infection and chemotherapeutic regimens as well as the current research findings and hypotheses regarding the neuropathogenesis of the disease.
... In addition, parasites such as Leishmania promastigotes can release a granulocyte chemotactic factor and inhibit IP-10/CXCL10 production by neutrophil granulocytes (40,41). In another study the induction of RANTES/CCL5, MCP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2 and MCP-1/CCL2 has been observed in footpads of resistant mice infected with L. major and L. amazonensis at early and late phases of infection which enhance parasite killing (42)(43)(44)(45). Furthermore, in vitro infection of murine macrophages with L. major promastigotes rapidly induces expression of several CC chemokines and the parasitic infection in mice triggers the production of inflammatory cytokines including TNFalpha by epidermal keratinocytes and IL-1beta by Langerhans cells in murine epidermis. ...
... Nitric oxide release by addition of L-N(G) monometry L-arginine (L-N), which is a competitive inhibitor of the L-arginine nitric oxide pathway, indicates that these chemokines mediate macrophage activation for nitric oxide release and subsequent parasite clearance, thus playing a role in the containment of Leishmania infection. Treatment of HeLa cells with T. gondii stimulated nuclear factor-kappa B activation and increased the expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) mRNA (43)(44)(45)(46). Also treatment of human peripheral blood neutrophils with T. gondii Ag stimulated the production of IL-12 and TNF-alpha. ...
... In addition to their chemotactic activity, the beta chemokines RANTES, MIP-1alpha and MIP-1beta enhance parasite uptake and cause intracellular destruction of Trypanosoma cruzi trypomastigotes by human macrophages and may play a role in resistance to T. cruzi infection. In another experiment, Sharafeldin et al. evaluated the induction of alpha and beta chemokines in the brains of Sprague-Dawley rats infected with Trypanosoma brucei brucei (44). The results showed high production of MIP-2, RANTES and MIP-1alpha, chemokines that were produced early by astrocytes and microglia and later by macrophages and T cells, and somewhat lower production of MCP-1 in infected animals compared to controls. ...
Chemokines are a group of small secreted proteins (8-10 kDa) produced and released by a wide variety of cell types. They were originally described as mediators of leukocyte recruitment, which is essential in acute and chronic inflammation. They also play a critical role in many pathophysiological processes such as allergic responses, infections and autoimmune diseases, tumor growth and hematopoietic development. This review introduces the three supergene families of chemokines (CXC, CC and C) with emphasis on their important role in different states in humans and in animal models with parasitic diseases. The concentration of transcription and translation of the cytokines and chemokines in the parasitic diseases may be an important marker for evaluation of the inflammatory state.
... However, excessive production is also deleterious to the host [12]. It has been observed that C-C chemokines, such as MIP-1-alpha and RANTES, are significantly upregulated in brains of Trypanosoma brucei brucei infected rats [34]. This increase in expression of these chemokines occurs before brain lesions developed in infected rats, implying that the induction of these chemokines could be directly responsible for the observed rat brain lesions [34]. ...
... It has been observed that C-C chemokines, such as MIP-1-alpha and RANTES, are significantly upregulated in brains of Trypanosoma brucei brucei infected rats [34]. This increase in expression of these chemokines occurs before brain lesions developed in infected rats, implying that the induction of these chemokines could be directly responsible for the observed rat brain lesions [34]. Ultrastructural analysis of mouse brain by electron microscopy at peak parasitaemia in this study, revealed disintegrating microvascular endothelial layer at the blood brain barrier in the cerebellar region of infected mouse brain. ...
Abstract
Background
Malaria afflicts 300–500 million people causing over 1 million deaths globally per year. The immunopathogenesis of malaria is mediated partly by co mplex cellular and immunomodulator interactions involving co-regulators such as cytokines and adhesion molecules. However, the role of chemokines and their receptors in malaria immunopathology remains unclear. RANTES (Regulated on Activation Normal T-Cell Expressed and Secreted) is a chemokine involved in the generation of inflammatory infiltrates. Recent studies indicate that the degradation of cell-cell junctions, blood-brain barrier dysfunction, recruitment of leukocytes and Plasmodium -infected erythrocytes into and occlusion of microvessels relevant to malaria pathogenesis are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, thus suggesting a unique role for RANTES in the generation and maintenance of the malaria-induced inflammatory response. The hypothesis of this study is that RANTES and its corresponding receptors (CCR1, CCR3 and CCR5) modulate malaria immunopathogenesis. A murine malaria model was utilized to evaluate the role of this chemokine and its receptors in malaria.
Methods
The alterations in immunomodulator gene expression in brains of Plasmodium yoelii 17XL-infected mice was analysed using cDNA microarray screening, followed by a temporal comparison of mRNA and protein expression of RANTES and its corresponding receptors by qRT-PCR and Western blot analysis, respectively. Plasma RANTES levels was determined by ELISA and ultrastructural studies of brain sections from infected and uninfected mice was conducted.
Results
RANTES (p < 0.002), CCR1 (p < 0.036), CCR3 (p < 0.033), and CCR5 (p < 0.026) mRNA were significantly upregulated at peak parasitaemia and remained high thereafter in the experimental mouse model. RANTES protein in the brain of infected mice was upregulated (p < 0.034) compared with controls. RANTES plasma levels were significantly upregulated; two to three fold in infected mice compared with controls (p < 0.026). Some d istal microvascular endothelium in infected cerebellum appeared degraded, but remained intact in controls.
Conclusion
The upregulation of RANTES, CCR1, CCR3, and CCR5 mRNA, and RANTES protein mediate inflammation and cellular degradation in the cerebellum during P. yoelii 17XL malaria.
... However, excessive production is also deleterious to the host [12]. It has been observed that C-C chemokines, such as MIP-1-alpha and RANTES, are significantly upregulated in brains of Trypanosoma brucei brucei infected rats [34]. This increase in expression of these chemokines occurs before brain lesions developed in infected rats, implying that the induction of these chemokines could be directly responsible for the observed rat brain lesions [34]. ...
... It has been observed that C-C chemokines, such as MIP-1-alpha and RANTES, are significantly upregulated in brains of Trypanosoma brucei brucei infected rats [34]. This increase in expression of these chemokines occurs before brain lesions developed in infected rats, implying that the induction of these chemokines could be directly responsible for the observed rat brain lesions [34]. Ultrastructural analysis of mouse brain by electron microscopy at peak parasitaemia in this study, revealed disintegrating microvascular endothelial layer at the blood brain barrier in the cerebellar region of infected mouse brain. ...
Malaria afflicts 300-500 million people causing over 1 million deaths globally per year. The immunopathogenesis of malaria is mediated partly by co mplex cellular and immunomodulator interactions involving co-regulators such as cytokines and adhesion molecules. However, the role of chemokines and their receptors in malaria immunopathology remains unclear. RANTES (Regulated on Activation Normal T-Cell Expressed and Secreted) is a chemokine involved in the generation of inflammatory infiltrates. Recent studies indicate that the degradation of cell-cell junctions, blood-brain barrier dysfunction, recruitment of leukocytes and Plasmodium-infected erythrocytes into and occlusion of microvessels relevant to malaria pathogenesis are associated with RANTES expression. Additionally, activated lymphocytes, platelets and endothelial cells release large quantities of RANTES, thus suggesting a unique role for RANTES in the generation and maintenance of the malaria-induced inflammatory response. The hypothesis of this study is that RANTES and its corresponding receptors (CCR1, CCR3 and CCR5) modulate malaria immunopathogenesis. A murine malaria model was utilized to evaluate the role of this chemokine and its receptors in malaria.
The alterations in immunomodulator gene expression in brains of Plasmodium yoelii 17XL-infected mice was analysed using cDNA microarray screening, followed by a temporal comparison of mRNA and protein expression of RANTES and its corresponding receptors by qRT-PCR and Western blot analysis, respectively. Plasma RANTES levels was determined by ELISA and ultrastructural studies of brain sections from infected and uninfected mice was conducted.
RANTES (p < 0.002), CCR1 (p < 0.036), CCR3 (p < 0.033), and CCR5 (p < 0.026) mRNA were significantly upregulated at peak parasitaemia and remained high thereafter in the experimental mouse model. RANTES protein in the brain of infected mice was upregulated (p < 0.034) compared with controls. RANTES plasma levels were significantly upregulated; two to three fold in infected mice compared with controls (p < 0.026). Some distal microvascular endothelium in infected cerebellum appeared degraded, but remained intact in controls.
The upregulation of RANTES, CCR1, CCR3, and CCR5 mRNA, and RANTES protein mediate inflammation and cellular degradation in the cerebellum during P. yoelii 17XL malaria.
... b. brucei-infected rat) of African trypanosomiasis, in which an early increase in CCL-5, CCL-3 and CCL-2 production occurred in the brain. Cell types expressing these chemokines were identified as astrocytes and microglial cells, which may be involved in attracting macrophages and T-lymphocytes to the brain, leading to lesions (Sharafeldin et al., 2000). In contrast with this report, CCL-5 was not increased in patients' CSF, maybe due to the progressive immune suppression associated with parasite development and linked to the switch to a type II cytokine environment (De Baetselier et al., 2001). ...
... CCL-3 in CSF was only expressed in S-2 patients with high values (>100-fold of stage 1 patients) correlated with cell count but not with the presence of trypanosomes. Its early production in the CNS during inflammation processes was described in an experimental model of African trypanosomiasis (Sharafeldin et al., 2000). Increased concentrations of CCL-3 were also detected in CSF of patients with meningitis of purulent, tuberculous or viral aetiology and CCL-3 is believed to contribute to monocyte migration to the CSF Inaba et al., 1997;Lahrtz et al., 1997). ...
Trypanosoma brucei gambiense infection is an important public health challenge in sub-Saharan Africa. This parasitic disease is difficult to diagnose due to insidious clinical signs and transient parasitaemias. The clinical course is marked by two stages of increasing disease severity. An early systemic parasitic invasion is followed by the development of a progressive meningo-encephalitis. During this latter stage, a broad spectrum of neurological signs appears, which finally lead to a demyelinating and fatal stage if untreated. Treatment is toxic and difficult to administer when the CNS is invaded. Therefore, accurate diagnostic methods for stage determination are needed. The classically used criteria are not sufficiently specific and mechanisms of parasite invasion through the blood-brain barrier remain poorly understood. As cytokines/chemokines are involved in the early recruitment of leukocytes into the CNS, this study has focused on their potential value to define the onset of CNS involvement. Levels of monocyte chemoattractant protein-1/CCL-2, macrophage inflammatory protein-1alpha/CCL-3, IL-8/CXCL-8, regulated upon activation T cell expressed and secreted (RANTES)/CCL-5 and IL-1beta were measured in paired sera and CSF from 57 patients and four controls. Patients were classified into three groups (stage 1, intermediate and stage 2) according to current field criteria for stage determination (CSF cell count, presence of trypanosomes in CSF and neurological signs). In sera, cytokine/chemokine levels were poorly related to disease stage. Only CXCL-8 was higher in stage 1 patients when compared with stage 2 and CCL-5 was higher in controls when compared with patients. In contrast, in CSF the expression of the selected cytokines, except CCL-5, was associated with the presence of neurological signs, demonstrating their diagnostic value. We observed a relationship between the presence of trypanosomes or trypanosome-related compounds in CSF and levels of IL-1beta, CXCL-8, CCL-2 and CCL-3. These cytokines and chemokines may be triggered by the parasite and hence are potential markers of CNS invasion.
... Further pathology occurs with secretion of CCL3 and CCL4, which augment lymphocyte migration to the brain and facilitate the breakdown of the blood-brain barrier [118]. CXCR3 dependent migration is required during T. cruzi infection [119][120][121][122], while T. brucei invasion of the central nervous system (CNS) occurs with elevated production of CCL2, CCL3, CCL5, CXCL2, CXCL8, and CXCL10 [123][124][125], facilitating widespread mononuclear cell infiltration within the brain parenchyma surrounding the blood vessels [123]. ...
Parasites are diverse eukaryotic pathogens that can have complex life cycles. Their clearance, or control within a mammalian host requires the coordinated effort of the immune system. The cell types recruited to areas of infection can combat the disease, promote parasite replication and survival, or contribute to disease pathology. Location and timing of cell recruitment can be crucial. In this review, we explore the role chemokines play in orchestrating and balancing the immune response to achieve optimal control of parasite replication without promoting pathology.
... Findings have pointed to an association between cytokine expression, particularly IFN-gamma and TNF-alpha, and the onset and development of the neuroinflammatory reaction. The CSF levels of the chemokines CXCL-2, CCL-5, CCL-3, and CCL-2 have been reported to increase in the brain early after infection; the early source of these inflammatory mediators appeared to be the brain resident cells astrocytes and microglia, with T cells and macrophages taking the pro-duction over later during the disease course [10,63]. This finding suggests that the initial steps in the development of the neuroinflammatory disease are controlled from within the CNS. ...
Human African trypanosomiasis (HAT) is a fatal if untreated fly-borne neuroinflammatory disease caused by protozoa of the species Trypanosoma brucei (T.b.). The increasing trend of HAT cases has been reversed, but according to WHO experts, new epidemics of this disease could appear. In addition, HAT is still a considerable burden for life quality and economy in 36 sub-Saharan Africa countries with 15–20 million persons at risk. Following joined initiatives of WHO and private partners, the fight against HAT was re-engaged, resulting in considerable breakthrough. We present here what is known at this day about HAT etiology and pathogenesis and the new insights in the development of accurate tools and tests for disease staging and severity monitoring in the field. Also, we elaborate herein the promising progresses made in the development of less toxic and more efficient trypanocidal drugs including the potential of medicinal plants and related alternative drug therapies.
... This was paralleled by a significant rise in the level of IFN-c and TGF-b found in the CSF. In a further study Sharafeldin et al. used immunocytochemistry to demonstrate increased staining of the chemokines ; MIP-2, RANTES, MIP-1a and, to a lesser extent, MCP-1 in the brain early after infection (Sharafeldin et al. 2000). Initially astrocytes and microglia were the main source of these chemokines with T cells and macrophages taking over production later in the infection. ...
Neurological involvement following trypanosome infection has been recognised for over a century. However, there are still many unanswered questions concerning the mechanisms used by the parasite to gain entry to the CNS and the pathogenesis of the resulting neuroinflammatory reaction. There is a paucity of material from human cases of the disease therefore the majority of current research relies on the use of animal models of trypanosome infection. This review reports contemporary knowledge, from both animal models and human samples, regarding parasite invasion of the CNS and the neuropathological changes that accompany trypanosome infection and disease progression. The effects of trypanosomes on the blood-brain barrier are discussed and possible key molecules in parasite penetration of the barrier highlighted. Changes in the balance of CNS cytokines and chemokines are also described. The article closes by summarising the effects of trypanosome infection on the circadian sleep-wake cycle, and sleep structure, in relation to neuroinflammation and parasite location within the CNS. Although a great deal of progress has been made in recent years, the advent and application of sophisticated analysis techniques, to decipher the complexities of HAT pathogenesis, herald an exciting and rewarding period for advances in trypanosome research.
... However, the mechanism or mechanisms by which this chemokine effects extravasation into the CNS remain unclear. Confounding this issue is that astrocytes, which project their foot processes onto the microvessels comprising the blood-brain barrier (BBB; Lassmann et al., 1991), are the predominant source of MCP-1 in several neuroinflammatory conditions (Ransohoff et al., 1993;Berman et al., 1996;Glabinski et al., 1996;Van Der Voorn et al., 1999;Sauder et al., 2000;Sharafeldin et al., 2000). That MCP-1 primarily originates from a source located behind the BBB begs the following question: How does MCP-1 reach leukocytes residing in the microvessel lumen? ...
Previous results from this laboratory revealed the presence of high-affinity saturable binding sites for monocyte chemoattractant protein-1 (MCP-1) along human brain microvessels (Andjelkovic et al., 1999; Andjelkovic and Pachter, 2000), which suggested that CC chemokine receptor 2 (CCR2), the recognized receptor for this chemokine, was expressed by the brain microvascular endothelium. To test the role of CCR2 directly in mediating MCP-1 interactions with the brain microvasculature, we assessed MCP-1 binding activity in murine brain microvessels isolated from wild-type mice and from CCR2 (-/-) mice engineered to lack this receptor. Results demonstrate that MCP-1 binding is greatly attenuated in microvessels prepared from CCR2 (-/-) mice compared with wild-type controls. Moreover, microvessels from wild-type mice exhibited MCP-1-induced downmodulation in MCP-1 binding and a recovery of binding activity that was not dependent on de novo protein synthesis. Furthermore, MCP-1 was shown to be internalized within wild-type microvessels, but not within microvessels obtained from CCR2 (-/-) mice, additionally demonstrating that CCR2 is obligatory for MCP-1 endocytosis. Last, internalization of MCP-1, but not transferrin, was observed to be inhibited by disruption of caveolae. Internalized MCP-1 also colocalized at some sites with caveolin-1, a major protein of caveolae, implying that this chemokine is endocytosed, in part, via nonclathrin-coated vesicles. These results prompt consideration that MCP-1 signals may be relayed across the blood-brain barrier by highly specialized interactions of this chemokine with its cognate receptor, CCR2, along brain microvascular endothelial cells.
... This induction was likely of functional signi¢cance because it was associated with subsequent increases in MCP-1 as assessed by quantitative immunoblot analysis and by sandwich ELISA. Enhanced expression of MCP-1 previously has been associated with a variety of neural injury and disease models such as ischemia (Kim et al., 1995;Gourmala et al., 1997), CNS infection Sharafeldin et al., 2000), multiple sclerosis/ EAE (McManus et al., 1998;Glabinski et al., 1997;, experimental autoimmune myasthenia gravis , trauma Ghirnikar et al., 1998;McTigue et al., 1998), axotomy (Schwaiger et al., 1999;Muessel et al., 1999;, and excitotoxic brain injury (Galasso et al., 2000). In most of these previous models, induction of MCP-1 was presumed to play some role in the in£ammatory responses that accompanied neural damage. ...
Enhanced expression of proinflammatory cytokines and chemokines has long been linked to neuronal and glial responses to brain injury. Indeed, inflammation in the brain has been associated with damage that stems from conditions as diverse as infection, multiple sclerosis, trauma, and excitotoxicity. In many of these brain injuries, disruption of the blood-brain barrier (BBB) may allow entry of blood-borne factors that contribute to, or serve as the basis of, brain inflammatory responses. Administration of trimethyltin (TMT) to the rat results in loss of hippocampal neurons and an ensuing gliosis without BBB compromise. We used the TMT damage model to discover the proinflammatory cytokines and chemokines that are expressed in response to neuronal injury. TMT caused pyramidal cell damage within 3 days and a substantial loss of these neurons by 21 days post dosing. Marked microglial activation and astrogliosis were evident over the same time period. The BBB remained intact despite the presence of multiple indicators of TMT-induced neuropathology. TMT caused large increases in whole hippocampal-derived monocyte chemoattractant protein (MCP)-1 mRNA (1,000%) by day 3 and in MCP-1 (300%) by day 7. The mRNA levels for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, cytokines normally expressed during the earliest stage of inflammation, were not increased up to 21 days post dosing. Lipopolysaccharide, used as a positive control, caused large inductions of cytokine mRNA in liver, as well as an increase in IL-1beta in hippocampus, but it did not result in the induction of astrogliosis. The data suggest that enhanced expression of the proinflammatory cytokines, TNF-alpha, IL-1beta and IL-6, is not required for neuronal and glial responses to injury and that MCP-1 may serve a signaling function in the damaged CNS that is distinct from its role in proinflammatory events.
... The results of studies in murine models indicate that T. gondii-infected CNS cells secrete chemokines, which recruit cells during murine Toxoplasma encephalitis [17,20,21]. The involvement of chemokines is also observed during another protozoan infection -in the model of experimental African trypanosomiasis -in which the accumulation of inflammatory cells in brain lesions was observed [19]. ...
In immunocompromised hosts, disruption of toxoplasmic cysts and conversion from bradyzoites to tachyzoites occur in brain. In these areas, infiltrates of mononuclear cells are observed. In the murine toxoplasmosis model, recent data suggest that chemokines may play a role in leukocyte recruitment in the central nervous system (CNS). This study analyzed the monocyte chemotactic protein-1 (MCP-1) secretion and chemokine expression after Toxoplasma gondii infection of human astrocytes, glioblastoma cells (U373) and fibroblasts (MRC5) in vitro. T. gondii infection of these CNS cells, astrocytes and glioblastoma cells significantly increased MCP-1 secretion, particularly for astrocytes. In our cellular models, the pattern of chemokine gene expression is dominated by MCP-1 expression. MCP-1 mRNAs were also quantified by real-time-PCR (LightCycler). The behavior of cells studied after T. gondii infection was different (invasion and growth) and the cell mechanisms of chemokine regulation could be dependent on the type of cells infected, while MCP-1 may contribute to the cell recruitment during human cerebral reactivation of T. gondii.
... While the cellular response to SARS-CoV in the brains of K18-hACE2 mice was minimal, proinflammatory cytokines and chemokines are upregulated in the brains of infected mice at day 4 p.i. (32,49). In JHMV-infected mice and in other CNS infections, cytokines are generally expressed by glia and infiltrating cells, especially astrocytes and macrophages (3,42,47). In general, these cytokine-producing cells are uninfected, and cytokines are expressed as part of the proinflammatory response. ...
Infection of humans with the severe acute respiratory syndrome coronavirus (SARS-CoV) results in substantial morbidity and mortality, with death resulting primarily from respiratory failure. While the lungs are the major site of infection, the brain is also infected in some patients. Brain infection may result in long-term neurological sequelae, but little is known about the pathogenesis of SARS-CoV in this organ. We previously showed that the brain was a major target organ for infection in mice that are transgenic for the SARS-CoV receptor (human angiotensin-converting enzyme 2). Herein, we use these mice to show that virus enters the brain primarily via the olfactory bulb, and infection results in rapid, transneuronal spread to connected areas of the brain. This extensive neuronal infection is the main cause of death because intracranial inoculation with low doses of virus results in a uniformly lethal disease even though little infection is detected in the lungs. Death of the animal likely results from dysfunction and/or death of infected neurons, especially those located in cardiorespiratory centers in the medulla. Remarkably, the virus induces minimal cellular infiltration in the brain. Our results show that neurons are a highly susceptible target for SARS-CoV and that only the absence of the host cell receptor prevents severe murine brain disease.
Many parasitic diseases (including cerebral malaria, human African trypanosomiasis, cerebral toxoplasmosis, neurocysticercosis and neuroschistosomiasis) feature acute or chronic brain inflammation processes, which are often associated with deregulation of glial cell activity and disruption of the brain blood barrier’s intactness. The inflammatory responses of astrocytes and microglia during parasite infection are strongly influenced by a variety of environmental factors. Although it has recently been shown that the gut microbiota influences the physiology and immunomodulation of the central nervous system in neurodegenerative diseases like Alzheimer’s disease and Parkinson’s, the putative link in parasite-induced neuroinflammatory diseases has not been well characterized. Likewise, the central nervous system can influence the gut microbiota. In parasite infections, the gut microbiota is strongly perturbed and might influence the severity of the central nervous system inflammation response through changes in the production of bacterial metabolites. Here, we review the roles of astrocytes and microglial cells in the neuropathophysiological processes induced by parasite infections and their possible regulation by the gut microbiota.
Human African trypanosomiasis (HAT), which is also known as sleeping sickness, has for several decades been one of the most important parasitic infections affecting man in the African continent. The disease is caused by protozoan parasites of the Trypanosoma species, with the East African form of the disease caused by Trypanosoma brucei rhodesiense and the West African form caused by Trypanosoma brucei gambiense. The disease is fatal if left untreated. The parasites in both cases are transmitted by the tsetse fly of the genus Glossina so that the control of this insect vector is a major challenge in the fight against HAT. A landmark World Health Organization (WHO) report published in 1986 (1) estimated that 50 million people worldwide are at risk of developing HAT, and it is likely that the current figure is closer to 60 million. The disease is a major health problem in Africa and occurs in no less than 36 countries in subSaharan Africa between latitudes 14(N and 29(S reflecting the distribution of the tsetse fly (2) (Fig. 1). The area of land which is effectively “held captive” by the tsetse fly is massive and in the region of 10 million square km (3). Precise figures for the incidence of HAT are very difficult to define in large part because only a small percentage (about 5-15%) of the susceptible population is usually under active surveillance and the disease is often inadequately reported (2). In an insightful analysis of the problem, Kuzoe (2) reported in 1993 that about 25,000 new cases are reported annually. However, it is likely that the true incidence of the disease is in the region of 300,000 cases per year (4).
Human infection with Trypanosoma brucei may result in meningo-encephalitis, neuronal demyelination, blood-brain-barrier dysfunction, peri-vascular infiltration, astrocytosis and neuronal apoptosis. Prevention of the short- or long-term, parasite-induced, neuronal assault requires a better understanding of the host's responses to the infection at the molecular level. Northern analysis, cDNA micro-arrays, reverse-transcrip-tase-PCR (RT-PCR), SDS-PAGE and immunohistology were therefore used to investigate global gene and protein expression in the brains of mice infected with T. brucei. Temporal and spatial expression of neuroleukin (NLK), a predominant neurotrophin which is associated with neuronal protection and regeneration during neuronal assault in the brain, was then assessed. Expression of 20 of the 588 genes investigated (representing pro- and anti-inflammatory immuno-modulators, growth factors, neurotransmitters, and pro- and anti-apoptosis factors) was significantly altered (P < 0.05). TUNEL analysis revealed extensive apoptosis at peak parasitaemia, mainly in the cerebellum. RT-PCR analysis of two regulators of apoptosis, Bcl-x(L) (anti-apoptotic) and Bax (pro-apoptotic), revealed equivalent increases in levels of expression. NLK expression was up-regulated in punctated fashion in brain and was mainly localized to abnormal (stellate) catecholamine neurons (CN) in the locus coeruleus (LC) of infected [and, to a lesser degree, the normal (polygonal) cells of uninfected] brainstem. Expression of NLK receptor (NLK-R) was inversely correlated with that of NLK. At peak parasitaemia, trypanosome infection apparently induces cerebellar apoptosis and a corresponding increase in NLK expression. NLK may be modulating inflammation and is probably involved in protecting CN and the cerebellum against apoptosis.
Unicellular microorganisms developed on Earth approximately 3–4 billion years ago, and since the evolution of modern man (Homo sapiens) about 200,000 years ago, there been a close interaction between them which has not always been beneficial for the host. Diseases resulting from microbial infections have for long been a bane of human society and with the discovery of viruses and prions, the array of infectious agents has further widened. An infectious agent may target either specific or multiple cell types, organs, or organ systems. As a response to the infections, the body ‘fights back’ with its own set of defenders, i.e., the immune system. In this chapter, we focus on the various types of infections that can affect our central nervous system (CNS), arguably the most complicated organization of matter that we have the knowledge about, and the immune responses against them. The CNS had been long considered to be ‘immune-privileged’ due to its apparent separation from the rest of the body by specialized barriers. However, these barriers have been found to be dynamic in nature, regulating the flow of material across them. Also, the cells in the brain are themselves equipped with various mechanisms to detect the presence of the infectious agents and respond accordingly to contain or neutralize the threat posed by them. The response mechanism often results in a condition termed as inflammation, which in itself is a complex process involving multiple mediators. Inflammation is often referred as ‘double-edged’ sword as, if un-controlled, it results in severe damage to the host itself. In a non-regenerating organ system such as the CNS this has detrimental ramifications that are commonly termed as neurodegeneration. Thus, in this chapter we have strived to provide the reader not only on the different types of pathogens infecting human CNS but also the immune response associated with them. More specifically, we have tried to provide information about how these pathogens are detected/recognized by cells of the CNS, how the cells respond following the detection, and how is the response regulated (if there is any regulation at all). Even though we have separated the response against bacteria, viruses, fungi, parasites, and prions in different sections, the readers will no doubt notice a certain degree of overlap in the mechanism of response of these different types of pathogens indicating the plasticity of the immune system. However, there is some uniqueness associated with each pathogen infection which makes the immune systems task even more difficult. In this chapter, we have strived to incorporate multitude of information in a concise manner; however, we do stress that this is by no means all-encompassing. Hence, the readers are encouraged to follow-up any particular point of interest from the cited publications.
This chapter discusses the role of microglial chemokines and chemokine receptors in central nervous system (CNS) inflammation. Microglia are invariably activated during CNS pathology and expresses macrophage-specific surface markers. Microglia contribution to the synthesis of chemokines in the CNS during pathological conditions has also been presented in this chapter. Microglia, therefore, are a substantial part of the chemokinergic network within the CNS. Since most of the endogenous cells of the CNS express receptors for chemokines, a broad spectrum of chemokines in CNS physiology and pathophysiology has been proposed. Chemokines are most likely involved in development and patterning of the brain. Furthermore, chemokines may contribute to CNS synaptic transmission and plasticity. Chemokine signaling in the CNS is involved in physiological as well as pathological processes. Since the scope of functions of chemokines and their receptors has broadened considerably to extend beyond leukocyte-chemoattraction, microglial chemokines, and chemokine receptors might be important factors in controlling the activity of microglia. The identification of the precise relationship of chemokines and microglial activity and the determination, whether microglial activation induced by chemokines will have protective or pathogenic consequences, will be the future challenge.
CUEDC2, a CUE domain-containing protein, is highly expressed in many tumors, which also may be associated with inflammation.
In this study, we studied whether CUEDC2 plays a role in the progress of inflammatory bowel disease using CUEDC2 knockout (KO) mice and discussed the effects of CUEDC2 on cell proliferation in colonic mucosa.
CUEDC2 KO mice were administered with drinking dextran sodium sulfate (DSS) to establish colitis mice model. At different time points after DSS administration, body weight and stool consistency of mice were graded. Cytokines in colon tissue such as IL-6 were measured by RT-PCR. NF-κB and STAT3 signaling pathways in colon tissue were assessed by western blotting. Besides, cell proliferation of intestinal mucosa was analyzed by immunohistochemical staining.
CUEDC2 alleviated the colonic inflammation, showing elevated body weight loss, worse diarrhea, and more severe colonic mucosal injury in CUEDC2 KO mice than WT mice. Moreover, pro-inflammatory cytokines such as IL-6, TNFα, COX2, and MIP2 were significantly elevated. In CUEDC2 KO mice, the NF-κB and STAT3 signaling pathways were increasingly activated in different stages of progression of the colonic inflammation, and the percentage of proliferating cells as indicated by Ki67, CyclinD1, and BrdU in the inflammatory tissues was significantly increased.
Our findings demonstrate that CUEDC2 plays an important role in protection from colonic inflammation, primarily by inhibiting the NF-κB and STAT3 signaling pathways and preventing excessive proliferation of the inflammatory epithelial cell.
This chapter focuses on the expression of chemokine receptors along the brain microvasculature, and their potential role in neuroinflammatory and neuroinfectious processes. The unique properties of the cerebral microvasculature might significantly dictate the course and extent of chemokine action within the central nervous system (CNS), and critically modulate both physiologic and pathophysiologic processes. In numerous inflammatory, traumatic, and infectious conditions of the CNS, astrocytes and infiltrating leukocytes have been determined to be major sources of chemokine production. Chemokine receptors have been reported to be expressed by endothelial cells from a wide variety of tissues and implicated in mediating several aspects of angiogenesis and angiostasis. Such expression by endothelial cells prompts consideration that specialized interactions of chemokines with the brain microvascular endothelium might constitute the incipient steps in directing leukocyte traffic into the CNS. Bilateral expression of chemokine binding sites along both abluminal and luminal surfaces of brain in vivo, may carry important implications for both chemokine and pathogen transport across the blood–brain barrier (BBB). Additional studies have attested to the functional competence of these chemokine binding sites. Confirmation of a role for chemokine receptors at the BBB will expand the endothelium as a target for therapeutics in the treatment of inflammatory, infectious and vascular diseases of the brain.
The aim of this study was to evaluate new methods to improve detection and investigation of the effects of chronic or subclinical infection with Trypanosoma evansi in various mammalian species. Some of the more resistant host species, including pigs and buffaloes, are present in large feral populations in the northern parts of Australia, the area where T. evansi is most likely to gain entry to the country. Existing tests are not sufficiently reliable to detect all cases of disease and they cannot distinguish acute from chronic infections. Furthermore, the tests have different sensitivities in different host species.
Surveillance for trypanosomiasis in Australia is problematic because of the need to work in remote parts of northern Australia where provision of a cold-chain for traditional blood and serum storage is difficult. An existing dried blood storage system was modified by treating cotton lint filter paper (Whatman #903) with a commercial post coating buffer (TropBio, Queensland). This treatment increased the longevity of antibodies to T. evansi in serum and blood stored on the paper (detected using an antibody-detection ELISA) compared to samples stored on plain paper, especially when the papers were stored under humid conditions and at high ambient temperatures.
Attempts were made to improve the diagnostic utility and repeatability of antibody-ELISAs through the use of 2 recombinant T. brucei antigens (PFRA and GM6) and to optimize a competitive ELISA using RoTat 1.2 variable surface antigen and its monoclonal antibody. Antibody-detection using the two recombinant proteins was not sufficiently specific to enable their use for the detection of T. evansi. The RoTat 1.2 cELISA had good sensitivity and specificity (75% and 98% respectively) when used to test serum from cattle and buffaloes experimentally infected with T. evansi and uninfected animals. However, the test was not able to detect anti-T. evansi antibodies in serum from wallabies, pigs, a dog or a horse that were experimentally infected with T. evansi. The inability of the cELISA to detect anti-T. evansi antibodies may be due to the small number of samples tested or the lack of RoTat 1.2 specific antibodies in the animals tested.
The feasibility of using an enzymatic test to detect trypanosome aminotransferase or antibodies to this enzyme was evaluated. Prior publications suggested that the detection of TAT was an appropriate diagnostic tool for the detection of T. evansi infection in camels. However, the results from this study did not support the use of this test for the detection of T. evansi infection in cattle or buffaloes with low to moderate parasitaemia.
Trypanosomiasis is an immunological disease that affects most of the body’s organs, with more severe disease developing over time. Attempts were made to determine key cytokine and biochemical patterns that would distinguish infected from uninfected animals and acute from chronic infections. The results from this study showed that there was no specific pattern in serum cytokines or serum biochemistry that could be used to distinguish infected from uninfected animals, or different stages of disease.
Immunohistochemistry was used on tissues from buffaloes and mice experimentally infected with T. evansi and T. brucei gambiense respectively to characterise the cellular immune response that was present. The immune response was predominantly cell mediated, with CD3+ T lymphocyte and macrophage infiltration occurring in most tissues. In end stage disease there was often suppression of the immune system with disruption of the architecture of the spleen and a decrease in B lymphocytes in the circulation.
Trypanosomes were rarely visible in the tissues and were only seen in those animals with high parasitaemia. Lesions generally became more severe over time, but there was a large variation between animals, which suggests that immunohistochemistry is unsuitable as a diagnostic tool.
Trypanosoma brucei is exposed to the adaptive immune system and complement in the blood of its mammalian hosts. The aim of this review is to analyse the role and regulation of the proteins present on the external face of the plasma membrane in the long-term persistence of an infection and transmission. In particular, the following are addressed: (1) antigenic variation of the variant surface glycoprotein (VSG), (2) the formation of an effective VSG barrier shielding invariant surface proteins, and (3) the rapid uptake of VSG antibody complexes combined with degradation of the immunoglobulin and recycling of the VSG.
Chemokines are a family of small, structurally related proteins that may participate in immune and inflammatory responses through the chemoattraction and activation of leukocytes. These chemotactic cytokines mediate their effects by binding to the seven transmembrane domain superfamily of receptor proteins. Besides their role in leukocyte trafficking, chemokines and their receptors have been found to be implicated in a number of inflammatory and infectious diseases. Chemokines also appear to have beneficial effects involving tissue repair or tumor progression, angiogenesis, hematopoiesis, and antimicrobial mechanisms. These have recently been found to be involved in coordinating the cellular communication in the central nervous system as well. Based on these findings, chemokines agonists and antagonists targeted against chemokines and their receptors have the potential to become therapeutically important in the treatment of various diseases. This review explores the role of the chemokine system in the pathophysiology of diseases and their potential as targets for therapy.
Here, we review the interactions between parasites and chemokines and chemokine receptors in toxoplasmosis, trypanosomiasis, leishmaniasis, malaria and other diseases caused by protozoan parasites. The potential roles of chemokines after infection by these intracellular pathogens include host defence functions such as leukocyte recruitment, participation in cell-mediated immunity and antiprotozoal activity. However, these interactions can also help the parasite in, for example, the penetration of host cells.
Human infection with Trypanosoma brucei may result in meningo-encephalitis, neuronal demyelination, blood-brain-barrier dysfunction, peri-vascular infiltration, astrocytosis and neuronal apoptosis. Prevention of the short- or long-term, parasite-induced, neuronal assault requires a better understanding of the host's responses to the infection at the molecular level. Northern analysis, cDNA micro-arrays, reverse-transcriptase-PCR (RT-PCR), SDS-PAGE and immunohistology were therefore used to investigate global gene and protein expression in the brains of mice infected with T. brucei. Temporal and spatial expression of neuroleukin (NLK), a predominant neurotrophin which is associated with neuronal protection and regeneration during neuronal assault in the brain, was then assessed. Expression of 20 of the 588 genes investigated (representing pro- and anti-inflammatory immuno-modulators, growth factors, neurotransmitters, and pro- and anti-apoptosis factors) was significantly altered (P < 0.05). TUNEL analysis revealed extensive apoptosis at peak parasitaemia, mainly in the cerebellum. RT-PCR analysis of two regulators of apoptosis, Bcl-x(L) (anti-apoptotic) and Bax (pro-apoptotic), revealed equivalent increases in levels of expression. NLK expression was up-regulated in punctated fashion in brain and was mainly localized to abnormal (stellate) catecholamine neurons (CN) in the locus coeruleus (LC) of infected [and, to a lesser degree, the normal (polygonal) cells of uninfected] brainstem. Expression of NLK receptor (NLK-R) was inversely correlated with that of NLK. At peak parasitaemia, trypanosome infection apparently induces cerebellar apoptosis and a corresponding increase in NLK expression. NLK may be modulating inflammation and is probably involved in protecting CN and the cerebellum against apoptosis.
Astrocytes from different sources bind the chemokine monocyte chemoattractant factor (MCP-1), yet functional expression in these cells of CCR2, the major receptor for this ligand, has been a matter of controversy. Here we show that cultured human fetal astrocytes express CCR2 at the mRNA and protein levels, and display chemotaxis and calcium flux in response to MCP-1. Surface CCR2 protein expression and MCP-1 binding activity were observed to undergo near parallel downmodulation and recovery following MCP-1 exposure, supporting the argument that CCR2, and not another receptor, mediates MCP-1 ligation in these cells. Downmodulation was further determined to occur via receptor internalization, and to apparently proceed via both clathrin-coated vesicles and caveolae, the latter being a novel mode for the endocytosis of chemokine receptors. Insofar as MCP-1 is thought to mediate inflammatory and developmental processes within the central nervous system (CNS), such astrocyte responses to this chemokine are likely to significantly impact physiological and pathophysiological events at the blood-brain barrier and within the CNS parenchyma.
The contribution of cytokines and chemokines to resistance and susceptibility to African trypanosomiasis remains controversial. In the present study, the levels of type I and type II cytokines and of the MCP-1 chemokine were compared during the early and late stages of Trypanosoma congolense infection in susceptible BALB/c and resistant C57BL/6 mice. Moreover, the status of macrophage activation was compared in these animals by analyzing the inducible nitric oxide synthase-arginase balance, tumor necrosis factor secretion, and expression of the FIZZ1 and YM genes. Data show that changing from a predominant type I cytokine environment in the early stage of infection to a predominant type II cytokine environment and an enhanced MCP-1 secretion in the late stage of infection correlates with resistance to T. congolense. Concomitantly, macrophage activation evolves from a classical to a predominant alternative phenotype. We further confirmed that the simultaneous occurrence of type I/type II cytokines in the early stage of infection in susceptible BALB/c mice, reflected by the presence of macrophages exhibiting a mixed classical/alternative activation phenotype, is associated with uncontrolled parasite growth and early death. Interleukin-4 (IL-4) and IL-13 signaling did not influence the susceptibility of BALB/c mice to T. congolense infection and interestingly were not the main trigger to alternative macrophage activation. In T. congolense-resistant C57BL/6 mice, our results corroborated the induction of FIZZ1 and YM gene expressions with the alternative pathway of macrophage activation. In susceptible BALB/c mice, however, YM but not FIZZ1 induction reflected the emergence of alternatively activated macrophages. Hence, the FIZZ1 and YM genes may be useful markers to discriminate between distinct populations of alternatively activated macrophages.
Mimosine is a plant amino-acid which has been reported to block DNA replication in mammalian cells and to arrest cell reversibly towards the end of the G1 phase or at the beginning of the S phase. In this study, 42 mice were infected with T. spiralis a nematode parasite, and treated with the anti-inflammatory compound L-mimosine, to determine if any alteration in the chronic inflammatory state occurred, by investigating the hosts immunological response. MCP-1, a C-C chemokine and MIP-2, a C-X-C chemokine were tested and calculated in the sera of infected animals, after 1, 10, 20, 30, 40, 50 and 60 days post infection, by ELISA method. The diaphragm and the masseters of the infected mice, were tested for inflammatory response. Here we found, that MCP-1 was partially inhibited by L-mimosine, while MIP-2 was totally inhibited. Moreover in sections of the diaphragm and masseters, the infiltration of inflammatory cells, such as macrophages, lymphocytes and eosinophils were more intense in untreated animals compared to those treated with L-mimosine. These findings show, that L-mimosine may have an inhibitory effect on MCP-1 and MIP-2 serum levels in Trichinellosis and may influence the recruitment of inflammatory cells and the intensity of the inflammatory reaction in this parasitic disease.
While the expression of chemokine receptors by endothelial cells is now well established, little is known of the function of these receptors at this cellular locale. However, given that chemokines are instrumental in directing leukocytes to specific parenchymal sites, one possibility is that endothelial chemokine receptors play a role in the process of leukocyte extravasation. To test this hypothesis, we investigated the contribution of CCR2, the major cognate receptor for the chemokine CCL2 (formerly known as MCP-1), to CCL2-stimulated transendothelial migration of macrophages (mØ) across cultured brain microvascular endothelial cells (BMEC). Specifically, we prepared both BMEC and mØ from wild-type (WT) mice and mice deficient in CCR2; i.e., CCR2 (-/-), and compared the ability of WT and CCR2 (-/-) BMEC to support CCL2-stimulated transendothelial migration of WT and CCR2 (-/-) mØ. In response to CCL2, WT mØ, but not CCR2 (-/-) mØ, were stimulated to migrate across WT BMEC, consistent with the recognized obligatory role for CCR2 in mediating CCL2-stimulated responses. Remarkably, however, neither WT nor CCR2 (-/-) mØ were stimulated by CCL2 to migrate across CCR2 (-/-) BMEC. In contrast, both types of mØ were able to migrate similarly across both types of BMEC in response to another chemokine--CCL3 (formerly known as MIP-1alpha)--which utilizes receptors other than CCR2. Lastly, CCL2-induced mØ transendothelial migration was blocked by treatment of WT BMEC with pertussis toxin, suggesting that CCR2 is functionally coupled to the inhibitory G protein Galphai, much as it is in other cell types. These results highlight a heretofore-unrecognized role for endothelial CCR2 in mediating transendothelial migration.
Neurocysticercosis, caused by infection with larval Taenia solium, is a major cause of epilepsy worldwide. Larval degeneration, which is symptomatic, results in inflammatory cell influx. Astrocytes, the most abundant cell type and major cytokine-producing cell within the CNS, may be important in orchestrating inflammatory responses after larval degeneration. We investigated the effects of direct stimulation and of conditioned medium from T. solium larval Ag (TsAg)-stimulated monocytes (CoMTsAg) on neutrophil and astrocyte chemokine release. CoMTsAg, but not control conditioned medium, stimulated astrocyte CCL2/MCP-1 (161.5 +/- 16 ng/ml), CXCL8/IL-8 (416 +/- 6.2 ng/ml), and CXCL10/IFN-gamma-inducible protein (9.07 +/- 0.6 ng/ml) secretion after 24 h, whereas direct astrocyte or neutrophil stimulation with TsAg had no effect. There was rapid accumulation of CCL2 and CXCL8 mRNA within 1 h, with somewhat delayed expression of CXCL10 mRNA initially detected 8 h poststimulation. Neutralizing anti-TNF-alpha inhibited CoMTsAg-induced CCL2 mRNA accumulation by up to 99%, causing total abolition of CXCL10 and up to 77% reduction in CXCL8 mRNA. CoMTsAg induced maximal nuclear binding of NF-kappaB p65 and p50 by 1 h, with IkappaBalpha and IkappaBbeta decay within 15 min. In addition, CoMTsAg induced transient nuclear binding of AP-1, which peaked 4 h poststimulation. In NF-kappaB blocking experiments using pyrrolidine dithiocarbamate, CoMTsAg-induced CCL2 secretion was reduced by up to 80% (p = 0.0006), whereas CXCL8 was inhibited by up to 75% (p = 0.0003). In summary, the data show that astrocytes are an important source of chemokines following larval Ag stimulation. Such chemokine secretion is NF-kappaB dependent, likely to involve AP-1, and is regulated in a paracrine loop by monocyte-derived TNF-alpha.
Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major cause of mortality and morbidity in sub-Saharan Africa. Current therapy with melarsoprol for CNS HAT has unacceptable side-effects with an overall mortality of 5%. This review discusses the issues of diagnosis and staging of CNS disease, its neuropathogenesis, and the possibility of new therapies for treating late-stage disease.
Astrocytes regulate the integrity of the blood-brain barrier and influence inflammatory processes in the central nervous system. The pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1), which is both released by and stimulates astrocytes, is thought to play a crucial role in both these activities. Because astrocytes have been shown to possess caveolae, vesicular structures that participate in intracellular transport and signal transduction events, we reasoned that expression of the major structural protein of these organelles, caveolin-1, might feature critically in the cellular responses to MCP-1. To test this hypothesis, caveolin-1 level was "knocked down" in human astrocyte cultures by using a small interfering RNA approach. This method resulted in efficient (>90% loss) and specific knockdown of caveolin-1 expression while sparring glial fibrillary acidic protein as well as several other proteins involved in endocytosis. Astrocytes suffering caveolin-1 loss showed diminished ability to down-modulate and internalize the MCP-1 receptor (CCR2) in response to exposure to this chemokine and also demonstrated significantly reduced capacity to undergo chemotaxis and calcium flux when MCP-1-stimulated. The results highlight a potentially prominent role for caveolae and/or caveolin-1 in mediating astrocyte responses to MCP-1, a feature that might significantly dictate the progression of inflammatory events at the blood-brain barrier.
Amoebic liver abscess (ALA) is an acute inflammatory disease caused by Entamoeba histolytica.
The key player in the pathogenesis and immunoprotection is the Gal/GalNAc inhibitable lectin, possessed by both pathogenic (Entamoeba histolytica) and nonpathogenic (Entamoeba dispar) strains, but only E. histolytica infection is associated with an acute inflammation and subsequently the disease.
The stimulation with the lectin induces the secretion of various proinflammatory cytokines/chemokines from intestinal epithelial cells. The differential induction of cytokine/chemokine network by the two strains can further regulate the immunoregulatory functions of the immune cells (monocytes and neutrophils) of the host. The soluble levels of IL-1beta, IL-6, IL-8, IL-10, MIP-1alpha, MCP-1, RANTES, GRO-alpha, GMCSF were quantitated to be significantly higher in pathogenic lectin-induced conditioned media (LCM) compared to nonpathogenic LCM (NP-LCM). The monocytes from ALA patients responded to the presence of pathogenic-LCM (P-LCM) by lowering of intracellular Ca(2+) (which was still higher than control). The proinflammatory MCP-1, GRO-alpha, and GMCSF levels in the monocytes were recorded both (quantitatively and mRNA quantitation) to be tremendously higher along with their respective receptors, with P-LCM compared to NP-LCM. A significant increase in the reactive oxygen intermediates and chemotactic index (CI) was observed in the monocytes when treated with P-LCM. Similarly, in neutrophils of ALA patients, an increase in intracellular Ca(2+), ROS and chemotaxis was observed with P-LCM.
The study is a step towards understanding the mechanism of immunopathogenesis of amoebiasis, on one hand, and points to the central role of cytokine/chemokine network in the process, on the other hand.
The immunopathology of Trypanosoma brucei brucei in the central nervous system was studied by using an experimental model of chronic meningoencephalitis in outbred CD-1 mice. Mice infected with T. b. brucei were treated with a subcurative dose of the trypanocidal compound diaminazine aceturate. These mice relapsed and were again drug treated. The brains were examined histologically and by immunocytochemistry to identify activated astrocytes. The polymerase chain reaction was used to detect cytokine RNA transcripts. The infected and treated animals developed severe chronic meningoencephalitis characterized by large numbers of inflammatory cells and widespread astrocyte proliferation. In uninfected controls, only interleukin 1 and beta-actin RNA transcripts were detected while transcripts for beta-actin, tumor necrosis factor alpha, macrophage inflammatory protein 1 and interleukins 1 and 4 were demonstrated in the brains of infected animals. Gamma interferon and interleukin 6 were also detected in a few of the infected animals, but interleukin 2 was not found in any of these animals.
A common feature of human melanoma is infiltration by monocytes at early stages of tumorigenesis. This infiltration may be highly significant since macrophages have the capacity to alter the behavior of tumor cells. The authors previously demonstrated that the predominant monocyte chemoattractant produced by tumor cells in vitro was monocyte chemotactic protein-1 (MCP-1). The authors identify the expression of MCP-1 in pathologic specimens of both primary and metastatic human melanoma but not in normal skin. The finding that MCP-1 is produced by malignant melanoma suggests that specific genes are expressed in tumor cells that can induce the recruitment of monocytes in vivo.
RANTES (regulated on activation, normal T expressed and secreted) is a member of the chemotactic cytokine (chemokine) beta subfamily. High affinity receptors for RANTES have been identified on a human monocytic leukemia cell line THP-1, which responded to RANTES in chemotaxis and calcium mobilization assays. Steady-state binding data analyses revealed approximately 700 binding sites/cell on THP-1 cells with a Kd value of 400 pM, comparable to that expressed on human peripheral blood monocytes. The RANTES binding to monocytic cells was competed for by monocyte chemotactic and activating factor (MCAF) and macrophage inflammatory protein 1 (MIP-1) alpha, two other chemokine beta cytokines. Although MCAF and MIP-1 alpha competed for RANTES binding to monocytes with apparent lower affinity (with estimated Kd of 6 and 1.6, nM respectively) both of these cytokines effectively desensitized the calcium mobilization induced by RANTES. The chemotactic response of THP-1 cells to RANTES was also markedly inhibited by preincubation with MCAF or MIP-1 alpha. In contrast, RANTES did not desensitize the THP-1 calcium mobilization and chemotaxis in response to MCAF or MIP-1 alpha. These results, together with our previous observations that RANTES did not compete for MCAF or MIP-1 alpha binding on monocytic cells, indicate the expression of promiscuous receptors on monocytes that recognize one or more cytokines within the chemokine beta family.
We have utilized a transendothelial lymphocyte chemotaxis assay to identify and purify a lymphocyte chemoattractant in supernatants of mitogen-stimulated peripheral blood mononuclear cells. Amino acid sequence analysis revealed identity with monocyte chemoattractant protein 1 (MCP-1), a chemoattractant previously thought to be specific for monocytes. Recombinant MCP-1 is chemoattractive for purified T lymphocytes and for CD3+ lymphocytes in peripheral blood lymphocyte preparations. The T-cell response to MCP-1 is dose-dependent and chemotactic, rather than chemokinetic. Phenotyping of chemoattracted T lymphocytes shows they are an activated memory subset. The response to MCP-1 by T lymphocytes can be duplicated in the absence of an endothelial monolayer and the majority of T-lymphocyte chemotactic activity in mitogen-stimulated peripheral blood mononuclear cell supernatants can be neutralized by antibody to MCP-1. Thus, MCP-1 is the major lymphocyte chemoattractant secreted by mitogen-stimulated peripheral blood mononuclear cells and is capable of acting as a potent T-lymphocyte, as well as monocyte, chemoattractant. This may help explain why monocytes and T lymphocytes of the memory subset are always found together at sites of antigen-induced inflammation.
Mononuclear leukocytes preferentially accumulate in the central nervous system (CNS) during the course of experimental autoimmune encephalomyelitis (EAE). To address factors that govern leukocyte trafficking in EAE, we monitored expression of mRNAs encoding IP-10 and JE/MCP-1, which are members of a family of chemoattractant cytokines. A transient burst of IP-10 and JE/MCP-1 mRNA accumulation in the CNS occurred, in close relation to the onset of histologic and clinical disease. In situ hybridizations showed, unexpectedly, that astrocytes were the major source of mRNAs encoding IP-10 and JE/MCP-1. These observations implicate astrocyte-derived cytokines as potential chemoattractants for inflammatory cells during EAE.
Mouse monocyte chemoattractant protein-1 (MCP-1), previously termed JE, is a member of the beta chemokine gene family and a homologue of the human monocyte chemoattractant protein, MCP-1. Mouse rMCP-1 was used to immunize hamsters for the production of mAb. Seven mouse MCP-1-specific mAbs were characterized: two of these mAbs cross-reacted with the human MCP-1, as determined by ELISA. A sensitive and specific capture ELISA for MCP-1 quantitation, which allowed measurement of mouse MCP-1 levels in supernatants from cells stimulated with inflammatory agents, was developed. LPS-stimulated astrocytes produce the highest levels of MCP-1 (80 ng/ml); macrophages and mesangial cells produce lower levels of MCP-1 (2 to 14 ng/ml) after LPS stimulation. IL-1 and TNF-alpha stimulation also can induce low levels of MCP-1 production. Western blot analysis demonstrated that the predominant native form of mouse MCP-1 is a 30-kDa glycoprotein. Two mAbs (2H5 and 6C7) demonstrated dose-dependent neutralization of mouse MCP-1 chemotactic activity. To localize the epitope recognized by one of these neutralizing Abs, the mAb was used to bind a series of genetically engineered truncated variants of human MCP-1. The C-terminal residues 62 to 67 on human MCP-1 molecules seem to be critical to express the epitope recognized by the neutralizing 2H5 anti-MCP-1 mAb. However, multiple sites on the MCP-1 molecule seem to be critical for bioactivity. Thus, these Ab reagents provide a useful tool to explore the biology of the mouse MCP-1 beta chemokine.
Inflammatory cell recruitment to the central nervous system (CNS) is a cardinal feature of physiological and pathological processes, including multiple sclerosis (MS). Despite recent progress, the soluble signals that attract inflammatory cells from the vascular compartment into the CNS parenchyma remain obscure. We favor the hypothesis that chemoattractant cytokines termed ‘chemokines' are uniquely important for mediating leukocyte entry into CNS tissues during immunemediated inflammation. Three lines of evidence supporting this hypothesis will be reviewed. The first regards expression of chemokines in animal models of immune-mediated CNS inflammation and in the human disease, multiple sclerosis. The second line of evidence involves interventional studies of chemokine blockade in such model disorders. The third line of evidence comprises function of chemokines in the CNS, as analysed in transgenic mice. Investigation of CNS chemokine function will enhance our understanding of leukocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.
RANTES (regulated on activation, normal T expressed and secreted) is a member of the chemotactic cytokine (chemokine) beta subfamily. High affinity receptors for RANTES have been identified on a human monocytic leukemia cell line THP-1, which responded to RANTES in chemotaxis and calcium mobilization assays. Steady-state binding data analyses revealed approximately 700 binding sites/cell on THP-1 cells with a Kd value of 400 pM, comparable to that expressed on human peripheral blood monocytes. The RANTES binding to monocytic cells was competed for by monocyte chemotactic and activating factor (MCAF) and macrophage inflammatory protein 1 (MIP-1) alpha, two other chemokine beta cytokines. Although MCAF and MIP-1 alpha competed for RANTES binding to monocytes with apparent lower affinity (with estimated Kd of 6 and 1.6, nM respectively) both of these cytokines effectively desensitized the calcium mobilization induced by RANTES. The chemotactic response of THP-1 cells to RANTES was also markedly inhibited by preincubation with MCAF or MIP-1 alpha. In contrast, RANTES did not desensitize the THP-1 calcium mobilization and chemotaxis in response to MCAF or MIP-1 alpha. These results, together with our previous observations that RANTES did not compete for MCAF or MIP-1 alpha binding on monocytic cells, indicate the expression of promiscuous receptors on monocytes that recognize one or more cytokines within the chemokine beta family.
The recruitment of various leukocyte populations to an area of injured and inflamed tissue is one of the most fundamental host defense responses. Historic evidence supports the concept that the pathology of acute inflammation is characterized by the elicitation of neutrophils, while the leukocyte composition of more chronic inflammatory responses is mononuclear in nature. Interestingly, little is known regarding the mechanism involved in the 'switch' from an acute neutrophil-mediated response to a chronic mononuclear-cell-directed immune reaction. Recent studies demonstrate that two supergene families of chemokines play a key role in dictating the recruitment of specific leukocyte populations necessary for the appropriate inflammatory response. The expression of specific chemokines appears to be under the control of other cytokines, such as interleukins-1, -4 and -10, and tumor necrosis factor, that serve as either positive or negative regulatory mediators in the control of chemokine production, thus, controlling the recruitment of leukocyte subpopulations.Copyright © 1996 S. Karger AG, Basel
LYMPHOCYTE migration from blood into tissue depends on integrin-mediated adhesion to endothelium1-4. Adhesion requires not only integrin ligands on the endothelium, but also activation signals because T-cell integrins cannot bind well until they are activated. The physiological 'triggers' for T-cell adhesion are unknown, but cytokines may be good candidates as they are released during inflammation and trigger adhesion in neutrophils and monocytes2,5,6. We have identified a cytokine, macrophage inflammatory protein-1beta (MIP-1beta), that induces both chemotaxis and adhesion of T cells; MIP-1beta is most effective at augmenting adhesion of CD8+ T cells to the vascular cell adhesion molecule VCAM-1. We reasoned that, as cytokines in vivo will be rapidly washed away, MIP-1beta might be bound to endothelial surfaces and so induce adhesion in its immobilized form. Here we show that: (1) M IP-1beta is present on lymph node endothelium; (2) immobilized MIP-1beta induces binding of T cells to VCAM-1 in vitro. M IP-1beta was immobilized by binding to proteoglycan: a conjugate of heparin with bovine serum albumin and cellular proteoglycan CD44 were both effective. We propose that MIP-1beta and other cytokines with glycosaminoglycan-binding sites will bind to and be presented by endothelial proteoglycans to trigger adhesion selectively not only of lymphocyte subsets, but also of other cell types.
Adult deer mice (Peromyscus maniculatus) were inoculated intraperitoneally with 1103
Trypanosoma brucei organisms. Chronic disease was induced consistently in those mice not killed early in infection. Infected mice were killed between 6 and 222 post-inoculation days (PID) and brains were prepared for light, immunofluorescent, and electron microscopy.Light microscopic examination revealed extreme variability in brain lesion composition in deer mice infected withT. brucei. Considering all cases, several histopathologic features were frequently encountered, including: (1) lymphocytic and histiocytic meningitis, choroiditis, and encephalitis, (2) astrocytic and microglial gliosis, (3) spongiform leukoencephalopathy (lower white matter), (4) presence ofT. brucei in choroids, blood vasculature, and neuropil, and (5) thrombosis with infarction. The importance of these factors, both in quantity and distribution, varied from case to case, especially in mice inoculated longer than PID 30.Frozen sections of brains from deer mice infected withT. brucei were stained with fluorescein-conjugated goat-anti-mouse IgM and IgG. In no case was specific fluorescence found as compared with positive control renal sections from the same infected animals. Thus, we are unable to confirm the improtance of immune complexes in the pathogenesis of brain lesions in this laboratory animal model of African sleeping sickness.Electron microscopy (EM) was used to examine one lesion only, that being the spongiform lesion of lower white matter. Ultrastructurally, the spongiform lesion was defined as splitting of myelin lamellae at the interperiod line with subsequent vacuole formation.
LYMPHOCYTE migration from blood into tissue depends on integrin-mediated
adhesion to endothelium1-4. Adhesion requires not only
integrin ligands on the endothelium, but also activation signals because
T-cell integrins cannot bind well until they are activated. The
physiological 'triggers' for T-cell adhesion are unknown, but cytokines
may be good candidates as they are released during inflammation and
trigger adhesion in neutrophils and monocytes2,5,6. We have
identified a cytokine, macrophage inflammatory protein-1β
(MIP-1β), that induces both chemotaxis and adhesion of T cells;
MIP-1β is most effective at augmenting adhesion of CD8+
T cells to the vascular cell adhesion molecule VCAM-1. We reasoned that,
as cytokines in vivo will be rapidly washed away, MIP-1β might be
bound to endothelial surfaces and so induce adhesion in its immobilized
form. Here we show that: (1) M IP-1β is present on lymph node
endothelium; (2) immobilized MIP-1β induces binding of T cells to
VCAM-1 in vitro. M IP-1β was immobilized by binding to
proteoglycan: a conjugate of heparin with bovine serum albumin and
cellular proteoglycan CD44 were both effective. We propose that
MIP-1β and other cytokines with glycosaminoglycan-binding sites
will bind to and be presented by endothelial proteoglycans to trigger
adhesion selectively not only of lymphocyte subsets, but also of other
cell types.
The present study was designed to investigate the capacity of human mononuclear phagocytes to produce a cytokine chemotactic for monocytes (monocyte chemotactic protein (MCP), alternative acronyms JE, monocyte chemotactic and activating factor, MCP-1, and tumor-derived chemotactic factor). Human PBMC exposed in vitro to bacterial LPS expressed high levels of MCP transcripts. Monocyte-depleted lymphoid cells were not induced to express MCP by LPS. Percoll-gradient purified monocytes were able to express high levels of MCP transcripts. In an effort to exclude a role of contaminating non-monocytic cells, mononuclear phagocytes were separated by flow cytometry and sorting: CD14+ cells exposed to LPS showed high levels of MCP mRNA. LPS-stimulated monocytes released chemotactic activity for monocytes that could be inhibited by absorption with anti-MCP antibodies. IL-1, TNF, IFN-gamma, granulocyte-macrophage-CSF and, to a lesser extent, macrophage-CSF, as well as inactivated streptococci, also induced MCP gene expression. Actinomycin D experiments indicated that induction of MCP in monocytes was gene transcription-dependent. The protein synthesis inhibitor cycloheximide (Cy) blocked IL-1-, TNF-, or LPS-induced MCP gene expression in monocytes. In contrast, expression of the structurally related chemotactic cytokine IL-8 was superinduced by Cy. Moreover, Cy superinduced MCP gene expression in cells other than monocytes, including endothelial cells, smooth muscle cell and fibrosarcoma cells, indicating different mechanisms of regulation in mononuclear phagocytes vs cells of other lineages. The capacity of cells of the monocyte-macrophage lineage to produce a cytokine that recruits and activates circulating monocytes may be of considerable importance in inflammatory and immunologic reactions. Thus, the mononuclear phagocyte system can autonomously regulate the extravasation and activation of immature elements of the same lineage, a key event in inflammation and immunity.
During the late-stage disease associated with human African trypanosomiasis, caused by infection with either Trypanosoma gambiense or T. rhodesiense, parasites invade the central nervous system (CNS), eventually leading to development of CNS pathology. This can be exacerbated by subcurative chemotherapy. The mechanisms through which the inflammatory processes within the CNS are controlled remain unclear.
Mice infected with T. b. brucei were treated with a trypanocidal drug regimen on day 28 postinfection that cleared parasites from all sites except the brain. Brains of mice killed at different times during infection and after chemotherapy were analyzed, using immunocytochemistry for astrocyte activation and polymerase chain reaction assisted amplification of RNA to detect cytokine transcripts.
Drug-treated animals developed a posttreatment meningoencephalitis similar to that which can occur in humans with late-stage African trypanosomiasis. Between days 14 and 21 postinfection, before chemotherapy and the subsequent development of inflammatory lesions in the brain, astrocytes became activated. The production of several cytokines correlated with this astrocyte activation. Low levels of interleukin-1 alpha transcripts were detected in uninfected controls, but levels increased with astrocyte activation in the infected animals. Transcripts for the macrophage inflammatory protein-1 and tumor necrosis factor-alpha were first detected on day 21 postinfection, with higher levels in mice after development of the posttreatment meningoencephalitis, whereas granulocyte macrophage-colony stimulating factor was detected only in animals that developed posttreatment reaction. Interleukin-6 and interferon-gamma were also first detected on day 21 postinfection, correlating with astrocyte activation but subsequently declined with time in both treated and untreated mice.
These results indicate that cytokines are being produced within the CNS before any inflammation and that astrocytes may be the source of some of these cytokines. Thus astrocyte activation may be key in the control and development of the CNS inflammatory processes that occur in African sleeping sickness.
An important process in the immune response is the migration of different populations of lymphocytes at the proper time to sites of antigenic challenge. Although several chemoattractants are known for broad classes of lymphocytes, such as T and B cells, the process by which lymphocytes of specific subsets, such as helper, cytotoxic or memory T cells, migrate to the appropriate sites remains obscure. Interleukin-8 is a chemoattractant for T cells and neutrophils and is a member of a superfamily of soluble molecules related by a conserved motif containing four cysteine residues. IL-8 and related molecules, including platelet factor 4, constitute the C-X-C class of the superfamily and a group of cytokines produced by haematopoietic cells constitute the RANTES/sis or C-C class. The roles of most of these molecules are not well known, although murine MIP-1 alpha of the C-C branch is a specific inhibitor of haematopoietic stem cell proliferation and some members of the C-X-C branch are neutrophil-targeted inflammatory agents. Here we report that the RANTES protein of the C-C class causes the selective migration of human blood monocytes and of T lymphocytes expressing the cell surface antigens CD4 and UCHL1. CD4+/UCHL1+T cells are thought to be prestimulated or primed helper T cells involved in memory T cell function. The preferential attraction of T-cell subsets by specific cytokines could in part explain how lymphocytes are targeted, and may provide insight into the workings of T cell memory.
To help to elucidate the changes induced by Trypanosoma brucei gambiense in the central nervous system (CNS) in advanced sleeping sickness patients, levels of interleukin-1 (IL-1) and prostaglandins D2 (PGD2) and E2 (PGE2) were measured by radioimmunoassay in the cerebrospinal fluid (CSF) from 24 patients diagnosed on the criteria of CSF protein, leucocyte count and parasite presence as having CNS (i.e. late stage) involvement, and from 12 patients without CNS involvement. PGD2 concentrations were selectively and markedly elevated in the late stage patients. The increased PGD2 may in part account for the increased somnolence and the immunosuppression within the CNS. Measurement of PGD2 levels in CSF may be a useful criterion for CNS involvement.
Cells that produce interleukin-6 (IL-6) require the presence of signaling molecules since this cytokine is not normally constitutively expressed. It is now established that astrocytes produce IL-6; however, the precise inducing molecules and the kinetics of their action have not yet been clearly identified. In the current study, we show that either interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) exert a strong inducing signal for IL-6 in primary rat astrocytes. When the two cytokines are added together the response is synergistic, suggesting that each cytokine may induce IL-6 gene expression by different pathways. Interferon-gamma (IFN-gamma) does not affect IL-6 expression although if it is added in conjunction with IL-1 beta, an augmented induction of IL-6 occurs. In addition to the cytokines, bacterial lipopolysaccharide (LPS) and the calcium ionophore, A23187, induce IL-6 expression. IL-6 expression can be blocked by the glucocorticoid analogue, dexamethasone. IL-6 induction by LPS/Ca2+ ionophore is more sensitive to the suppressive effects of dexamethasone than is IL-6 induction by TNF-alpha/IL-1 beta. Cycloheximide (CHX), an inhibitor of protein synthesis, markedly increased levels of IL-6 mRNA in both unstimulated and stimulated astrocytes, indicating that ongoing protein synthesis is not required for astrocyte IL-6 gene expression. We propose that astrocyte-produced IL-6 may have a role in augmenting intracerebral immune responses in neurological diseases such as multiple sclerosis (MS), AIDS dementia complex (ADC), and viral infections. These diseases are characterized by infiltration of lymphoid and mononuclear cells into the central nervous system (CNS), and intrathecal production of immunoglobulins. IL-6 may act to promote terminal differentiation of B cells in the CNS, leading to immunoglobulin synthesis.
A number of studies of inflammation and of cell growth and transformation have recently converged by defining two related families of cytokines. The first, represented by macrophage inflammatory protein 1, is composed of several gene products that have been identified in activated T cells, macrophages, and fibroblasts. The biological activities of this family are still being characterized but so far include effects on neutrophils, monocytes, and hematopoietic cells. The second, represented by macrophage inflammatory protein 2, includes platelet products such as platelet factor 4 and beta-thromboglobulin as well as several other recently described gene products that have effects on a number of cell types including neutrophils, fibroblasts, hematopoietic cells, and melanoma cells. The two families are structurally related and may have evolved from a common ancestral gene that duplicated and then diverged. Their differential control and expression in a wide variety of cell types suggests that they may have multiple functions in regulating inflammation and cell growth.
The principal clinical and pathological findings in 16 fatal cases of human African trypanosomiasis caused by T.b. Gambiense are described. The changes in the brain took the form of a non-specific lymphoplasmacytic meningo-encephalitis of varying intensity. Other features included morular cells, diffuse microglial hyperplasia, and large reactive astrocytes in the white matter. Carditis was identified in 10 cases. Acute reactive arsenical encephalopathy appeared to be the principal cause of death in 10 patients. Convulsions figured prominently in this type of encephalopathy in seven patients and were sufficiently severe to produce hypoxic brain damage. In three cases of acute reactive arsenical encephalopathy the structural changes in the brain were those of acute haemorrhagic leucoencephalopathy.
Mice infected with Trypanosoma brucei can be easily cured using the available trypanocidal drugs, provided treatment is given soon after the infection; however, if infection is allowed to continue for some time then it is extremely difficult to obtain a permanent cure, and after an aparasitaemic period the infection eventually relapses. It has been shown that a relatively small number of infective trypanosomes in the brain have escaped the action of the drug and these are able to replicate and eventually they re-establish a circulating parasitaemia. The number of infective trypanosomes remaining after chemotherapy varied according to the stabilate of T. brucei, but using the stabilate T. brucei GVR 35 there were up to, 5,000 infective trypanosomes 2 days after treatment, and this rapidly increased up to 50,000 trypanosomes at 7 days. Infected mice on histological examination exhibited a mild meningitis and this was exacerbated by Berenil therapy and 7 days posttreatment the mice had an acute meningoencephalitis. It would appear that if the infective trypanosomes are completely eliminated from the brain this exacerbated reaction does not occur. It has been found that combined treatment of chronic T. brucei infections with either diminazene aceturate or suramin followed by 5-substituted nitroimidazoles at appropriate dose levels will completely eliminate the infective trypanosomes from the brain and the mice are permanently cured.
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Mouse monocyte chemoattractant protein-1 (MCP-1), previously termed JE, is a member of the beta chemokine gene family and a homologue of the human monocyte chemoattractant protein, MCP-1. Mouse rMCP-1 was used to immunize hamsters for the production of mAb. Seven mouse MCP-1-specific mAbs were characterized: two of these mAbs cross-reacted with the human MCP-1, as determined by ELISA. A sensitive and specific capture ELISA for MCP-1 quantitation, which allowed measurement of mouse MCP-1 levels in supernatants from cells stimulated with inflammatory agents, was developed. LPS-stimulated astrocytes produce the highest levels of MCP-1 (80 ng/ml); macrophages and mesangial cells produce lower levels of MCP-1 (2 to 14 ng/ml) after LPS stimulation. IL-1 and TNF-alpha stimulation also can induce low levels of MCP-1 production. Western blot analysis demonstrated that the predominant native form of mouse MCP-1 is a 30-kDa glycoprotein. Two mAbs (2H5 and 6C7) demonstrated dose-dependent neutralization of mouse MCP-1 chemotactic activity. To localize the epitope recognized by one of these neutralizing Abs, the mAb was used to bind a series of genetically engineered truncated variants of human MCP-1. The C-terminal residues 62 to 67 on human MCP-1 molecules seem to be critical to express the epitope recognized by the neutralizing 2H5 anti-MCP-1 mAb. However, multiple sites on the MCP-1 molecule seem to be critical for bioactivity. Thus, these Ab reagents provide a useful tool to explore the biology of the mouse MCP-1 beta chemokine.
Chemokines are a family of low molecular mass proteins with chemotactic and cell activating activities. Reverse transcription-polymerase chain reaction and Northern hybridization were used to examine their expression during murine experimental allergic encephalomyelitis (EAE), an autoimmune disease used as a model of multiple sclerosis. The mRNAs encoding RANTES, MIP-1 alpha, MIP-1 beta, TCA3 (I-309), IP-10, JE (MCP-1), KC (MGSA/gro), and MARC (MCP-3) were induced in the spinal cord 1-2 days before clinical signs were apparent. SDF, a cDNA predicted to encode a chemokine-like product, was expressed in normal as well as diseased spinal cords. No expression of C10 or MIP-2 was detected. Activated encephalitogenic T cells expressed message for RANTES, MIP-1 alpha, MIP-1 beta, and TCA3. These results define a subset of chemokines that may play an important role in the inflammatory process during murine EAE.
A novel family of structurally and functionally related polypeptides has recently been detected that are now referred to as chemokines. Within this family, a peptide with the acronym RANTES was shown to be chemotactic for memory T cells, monocytes, and eosinophilic and basophilic granulocytes, thus suggesting it plays an important role in chronic inflammatory and allergic diseases. Murine monoclonal antibodies as well as cDNA probes specific for human RANTES were raised and extensively characterized. With these antibodies, stimulated human dermal fibroblasts were shown to express intracellular RANTES peptide by immunocytochemistry. Furthermore, similar kinetics could be demonstrated in fibroblasts for both RANTES mRNA expression and secretion of RANTES peptide using Northern blot hybridization and sandwich-enzyme-linked immunosorbent assay, respectively. RANTES expression was induced upon stimulation with tumor necrosis factor-alpha as well as with interleukin-1 alpha and -beta in a concentration- and time-dependent manner. These results reinforce the role of both resident and circulating cells in the production and release of RANTES and their participation in inflammatory processes.
The chemokine RANTES induced biphasic mobilization of Ca2+ in T cells. The initial peak, a transient increase in cytosolic
Ca2+ mediated by a heterotrimeric guanine nucleotide-binding protein (G protein)--coupled pathway, was associated predominantly
with chemotaxis. The second peak, Ca2+ release and sustained influx dependent on protein tyrosine kinases, was associated
with a spectrum of cellular responses--Ca2+ channel opening, interleukin-2 receptor expression, cytokine release, and T cell
proliferation--characteristic of T cell receptor activation. Other chemokines did not produce these responses. Thus, in addition
to inducing chemotaxis, RANTES can act as an antigen-independent activator of T cells in vitro.
The level of macrophage inflammatory protein-1 alpha (MIP-1 alpha), a newly discovered cytokine of chemokine family, was determined in cerebrospinal fluid (CSF) from 18 patients with multiple sclerosis (MS) and from control patients with other neurological disorders by an enzyme-linked immunosorbent assay (ELISA). The concentration of MIP-1 alpha in CSF was significantly elevated in MS in relapse (4.4 pg/ml) compared with non-inflammatory neurological disease control samples (0.3 pg/ml) (p < 0.0002). These concentrations in MS patients correlated well with leukocyte cell counts and protein content in CSF (r = 0.845, p < 0.0001; r = 0.853, p < 0.0001, respectively). In other inflammatory neurological disorders such as Behçet's disease and HTLV-1 associated myelopathy, significantly increased CSF levels of MIP-1 alpha were also observed. Chemokines are reported to play an important role in an early event of inflammation such as lymphocyte traffic. This report is the first study which confirmed the involvement of a chemokine in MS and other inflammatory neurological disorders.
Recombinant human macrophage inflammatory protein-1 alpha (rhMIP-1 alpha) and rhMIP-1 beta were potent chemoattractants of
human T lymphocytes. These rhMIP-1 cytokines attracted only T cells activated by monoclonal antibody to CD3 and did not attract
unstimulated lymphocytes. Phenotypic analysis revealed that CD4+ T cells were capable of migrating in response to rhMIP-1
beta, whereas rhMIP-1 alpha induced chemotaxis of predominantly CD8+ T lymphocytes. Activated naive and memory T cells also
migrated in response to rhMIP-1 cytokines. Furthermore, these cytokines enhanced the ability of T cells to bind to an endothelial
cell monolayer. These results suggest that rhMIP-1 cytokines preferentially recruit specific T cell subsets during the evolution
of the immune response.
We have examined the expression of factors associated with the growth, differentiation, and chemotaxis of cells of the monocyte/macrophage series in the central nervous system of Lewis rats sensitized to develop experimental allergic encephalomyelitis. CSF-1 mRNA increased significantly over that found in control animals (sensitized with OVA in CFA or CFA alone). The elevation in the levels of this growth factor commenced immediately before the onset of early clinical signs and peaked immediately before maximal clinical incidence of disease. Expression of CSF-1 message declined to base-line values with resolution of the disease process. CSF-1 protein was also detected in the central nervous system at the height of clinical disease. Expression of the receptor for CSF-1, the proto-oncogene c-fms, also paralleled the early disease process. Elevated levels of c-fms mRNA were detected immediately before the onset and peaked at the height of clinical signs of disease. In contrast to CSF-1 levels, elevated c-fms message expression persisted after resolution of the acute phase of experimental allergic encephalomyelitis. Levels of macrophage chemotactic factor-1 message were also elevated immediately before the onset of clinical signs, peaked with the height of clinical disease, and declined with resolution of the disease. Unlike CSF-1 or c-fms, no endogenous macrophage chemotactic factor-1 message was detected in control animals. Macrophage chemotactic factor-1 protein was demonstrated by Western blot in the central nervous system at the height of clinical disease. The results support the conclusion that expression of factors that specifically target cells of the monocyte/macrophage series are an important component of the disease process in experimental allergic encephalomyelitis.
Potent chemotactic activity for neutrophils was detected in rat inflammatory exudate induced by a subcutaneous injection of lipopolysaccharide in a carboxymethyl-cellulose suspension. We purified and characterized chemoattractants from the exudate by the following procedures: carboxymethyl-Sephadex C-25 ion-exchange chromatography; G3000SW gel-filtration chromatography; preparative reverse-phase high-pressure liquid chromatography; rechromatography on reverse-phase HPLC. Two chemotactic factors were purified and their N-terminal amino acid sequences were determined. One factor was a protein in which the first 20 N-terminal amino acids were identical to those of rat cytokine-induced neutrophil chemoattractant (CINC), a counterpart of human gro/melanoma growth-stimulating activity (MGSA). The other factor was highly similar to mouse macrophage inflammatory protein 2 (MIP-2). Mouse MIP-2, a chemotactic factor for neutrophils, is a member of the interleukin-8 family; however the protein we purified had higher similarity to human gro/MGSA than to human interleukin-8. These results indicate that, in rats, chemotactic factors for neutrophils induced by lipopolysaccharide stimulation are not counterparts of interleukin-8, but are gro/CINC-related peptides.
African trypanosome infections result in lymphocyte unresponsiveness and anemia in the mammalian host. In murine infections, these effects are mediated by suppressor macrophages releasing nitric oxide (NO). We investigated the mechanism of activation of macrophages to produce NO during trypanosomiasis in vitro. A soluble component of trypanosome lysates induced NO synthesis in peritoneal macrophage cultures only when the macrophages were co-stimulated with interferon-gamma (IFN-gamma). The macrophage-activating factor was also released in a soluble form by live bloodstream-form trypanosomes, but not procyclic trypanosomes. When splenocyte cultures were exposed to IFN-gamma and trypanosomes, an NO-dependent suppression of T cell proliferation occurred. This is similar to the suppression observed in the spleens of trypanosome-infected mice, suggesting that a combination of trypanosome-released macrophage-activating factors and IFN-gamma are a trigger of immune dysfunction in trypanosomiasis.
Meningitis is accompanied by a differential immigration of leukocytes into the subarachnoid space. Since the mechanisms regulating leukocyte invasion are still incompletely understood, we studied the release of the neutrophil-attracting alpha-chemokines IL-8 and GRO-alpha and the mononuclear cell-attracting beta-chemokines MCP-1, MIP-1alpha, and RANTES during meningitis. In 48 paired CSF and serum samples from patients hospitalized for meningitic symptoms, high levels of IL-8, GRO-alpha, and MCP-1 were detected in the CSF during bacterial and abacterial meningitis. Elevated chemokine levels were not found in the blood serum samples taken in parallel. The release of MIP-1alpha or RANTES was below detection limits. The IL-8 and GRO-alpha levels significantly correlated with the number of immigrated granulocytes in the CSF of patients with bacterial meningitis. A similar correlation was found when MCP-1 levels and the mononuclear cell count were analyzed in abacterial meningitis. These findings suggest that the local production of the alpha-chemokines IL-8 and GRO-alpha and of the beta-chemokine MCP-1 represents the major chemoattractant stimulus for the differential recruitment of leukocytes into the subarachnoid space during meningitis.
The chemokine superfamily is composed of at least 20 different leukocyte chemoattractants that act by binding to a family of G protein-coupled receptors. Leukocyte subtypes respond preferentially to unique but overlapping subsets of chemokines as determined by the receptor distribution, yet the receptors appear to signal through a common Gi-type G protein. Since chemokines appear to play major roles in inflammatory pathology, their receptors may be good targets for developing leukocyte selective anti-inflammatory drugs. Two chemokine receptors, CC CKRS and ONCC, function pathologically as cell entry factors respectively for human immunodeficiency virus 1, the cause of AIDS, and Plasmodium vivax, the major cause of malaria.
Inflammatory cell recruitment to the central nervous system (CNS) is a cardinal feature of physiological and pathological processes, including multiple sclerosis (MS). Despite recent progress, the soluble signals that attract inflammatory cells from the vascular compartment into the CNS parenchyma remain obscure. We favor the hypothesis that chemoattractant cytokines termed 'chemokines' are uniquely important for mediating leukocyte entry into CNS tissues during immune-mediated inflammation. Three lines of evidence supporting this hypothesis will be reviewed. The first regards expression of chemokines in animal models of immune-mediated CNS inflammation and in the human disease, multiple sclerosis. The second line of evidence involves interventional studies of chemokine blockade in such model disorders. The third line of evidence comprises function of chemokines in the CNS, as analysed in transgenic mice. Investigation of CNS chemokine function will enhance our understanding of leukocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.
The recruitment of various leukocyte populations to an area of injured and inflamed tissue is one of the most fundamental host defense responses. Historic evidence supports the concept that the pathology of acute inflammation is characterized by the elicitation of neutrophils, while the leukocyte composition of more chronic inflammatory responses is mononuclear in nature. Interestingly, little is known regarding the mechanism involved in the "switch' from an acute neutrophil-mediated response to a chronic mononuclear-cell-directed immune reaction. Recent studies demonstrate that two supergene families of chemokines play a key role in dictating the recruitment of specific leukocyte populations necessary for the appropriate inflammatory response. The expression of specific chemokines appears to be under the control of other cytokines, such as interleukins-1, -4 and -10, and tumor necrosis factor, that serve as either positive or negative regulatory mediators in the control of chemokine production, thus, controlling the recruitment of leukocyte subpopulations.
Central nervous system (CNS) expression of two chemokine mRNAs, encoding monocyte chemoattractant protein-1 (MCP-1) and IFN-gamma-inducible protein (IP-10), was previously shown to be closely related to the onset of clinical signs of murine experimental autoimmune encephalomyelitis (EAE). Chemokine mRNAs accumulated in a striking, transient burst within astrocytes, near inflammatory leukocyte infiltrates. It remained unclear if chemokines functioned to initiate leukocyte entry into CNS tissues, or to amplify the intrathecal inflammatory reaction. To address this issue, we determined the expression of chemokine mRNAs at the earliest evidence of CNS immune-mediated inflammation. For these experiments, mice were sacrificed in pairs at varying times after immunization. Only one member of each pair was symptomatic for EAE at the time of sacrifice. Symptom presence correlated well with histological inflammation at the time of sacrifice. RNA was prepared from two CNS sites, brain and spinal cord, and expression of chemokine mRNAs was analyzed by a sensitive and quantitative reverse transcriptase/polymerase chain reaction dot-blot hybridization assay. CNS expressions of MCP-1 and IP-10 gene were correlated tightly with histological inflammation; indeed, chemokine expression was never detected in the absence of leukocyte infiltrates. In situ hybridizations showed that astrocytes expressed chemokine transcripts. These findings provide new information about mechanisms controlling chemokine mRNA expression during immune-mediated inflammation in EAE and are consistent with a role for chemokines as amplifiers of CNS inflammatory reactions.
We examined the induction of 4 chemokines during early experimental African trypanosomiasis using in situ hybridization and immunocytochemistry. mRNA expression and protein production of Rantes, MCP-1, MIP-1a and MIP-2 were studied in splenocytes obtained at 0 h, 4 h and 12 h post-infection. Splenic denervation was performed to study the role of the central nervous system in early infection. The mRNA for Rantes increased at 4 h and declined at 12 h, but the protein level was high at both time-points. MCP-1 and MIP-la had elevated mRNA and protein levels at 12 h post-infection. MIP-2 mRNA was high at both 4 h and 12 h, but the protein level was only increased at 12 h. Splenic denervation, but not sham operation, suppressed these responses. The upregulation of these chemokines during very early infection suggests a chemokine role in the developing immunopathology The sympathetic nervous system may, however, participate in modulation of such early immune responses.
The advanced stages of sleeping sickness are correlated with a spread of trypanosomes into the central nervous system (CNS), producing a disseminated encephalitis. Inflammatory reactions extend along the blood vessels causing perivascular cuffing, which consists of in filtrations and proliferations of lymphocytes and also increased numbers of astrocytes and microglia. Progress in our understanding of the functions of astrocytes suggests that they are efficient antigen-presenting cells, initiating and regulating the intracerebral inflammatory response and limiting parasite spread to the perivascular spaces.
Pathogenesis and pathology of trypanosomiasis in Ž . man The African Trypanosomiasis
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The role of chemokines and their receptor in leukocyte Ž . activation Chemotactic Cytokines; Targets for Therapeutic Development
- P Gray
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Pathogenesis and pathology of trypanosomiasis in man
- W E Ormerod
The role of chemokines and their receptor in leukocyte activation
- P Gray