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Induction of macrophage antiprotozoal activity by monocyte chemotactic and activating factor
Abstract
To determine if monocyte chemotactic and activating factor (MCAF) induces intracellular antimicrobial activity, human monocyte-derived macrophages were treated with MCAF and challenged with Toxoplasma gondii and Leishmania donovani. Pretreatment with MCAF induced macrophages to inhibit protozoal replication by approximately 50%. These findings suggest a potential host defense role for MCAF in the inflammatory response to intracellular pathogens.
... Many chemokines (CCL2, CCL3, CXCL8, CXCL9, CXCL10) are important as chemotactic agents and through their activation of different types of leukocytes in leishmaniasis [1]. There is also evidence that certain chemokines (CCL3, CCL4, CCL5, MCAF) are antiparasitic and increase phagocytosis and NO-mediated killing of different protozoa, but to date such effects have been attributed to indirect activity on the macrophages and not to a possible direct effect on the parasites [2,3,4,5]. Infection with Leishmania parasites alters expression of chemokines and chemokine receptors in the host [6,7]. Leishmania major has been shown to up-regulate genes for IL-8, CXCL2, CXCL3,CCL20 and down-regulate CXCL9, CXCL10 and CCL2 in human macrophages in vitro [7], possibly part of an immune evasion response. ...
... Although the mechanisms for the action of each chemokine needs further investigation, our results demonstrate a direct antimicrobial effect by several chemokines on Leishmania mexicana, something that has not been shown for parasites before. Previous studies have shown increased phagoctyosis and NO-mediated killing of protozoa in the presence of chemokines (CCL2, CCL3 and CCL5) in macrophages [2,3,4,5]. Although our study did not show cytotoxic activity of CCL2 and CCL3, and has not investigated the effects on promastigotes of CCL5, it is possibleFigure 5. Cytotoxicity of CCL3 and CCL28 on human cells. ...
Chemokines and chemokine receptor-mediated effects are important mediators of the immunological response and cure in human leishmaniasis. However, in addition to their signalling properties for leukocytes, many chemokines have also been shown to act directly as antimicrobial peptides on bacteria and fungi. We screened ten human chemokines (CXCL2, CXCL6, CXCL8, CXCL9, CXCL10, CCL2, CCL3, CCL20, CCL27, CCL28) for antimicrobial effects on the promastigote form of the protozoan parasite Leishmania mexicana, and observed direct parasiticidal effects of several, CCL28 being the most potent. Damage to the plasma membrane integrity could be visualised by entrance of propidium iodide, as measured with flow cytometry, and by scanning electron microscopy, which showed morphological changes and aggregation of cells. The findings were in concordance with parasiticidal activity, measured by decreased mitochondrial activity in an MTT-assay. This is the first report of direct antimicrobial activity by chemokines on parasites. This component of immunity against Leishmania parasites identified here warrants further investigation that might lead to new insight in the mechanisms of human infection and/or new therapeutic approaches.
... Chemokines also play an important role in killing parasites from host cells [51,[62][63][64]. Antileishmanial activity of chemokines has been demonstrated in both in vitro and in vivo infections in murine VL by induction of respiratory burst as well as by induction of Th1 cytokines [65,66]. Another CD4+ T-cell lineage called Th17 cells was initially characterized as producing IL-17 family cytokines [67]. ...
... We also determined the role of STAT4 in the induction of the inflammatory chemokines CCL2 and CXCL10. Both CCL2 and CXCL10 are required for the recruitment of Th1 cells to sites of inflammation [19][20][21] and activation of host immune response pathways against Leishmania [22][23][24]. Although there were no significant differences in CXCL10 and CCL2 mRNA expression in WT and stat4 −/− BALB/c mice, there was a significant decrease in both CXCL10 and CCL2 mRNA expression in stat4 −/− C57BL/6 mice compared to WT at day 60 post-infection ( Figures 2I-L). ...
We and others have previously shown that IL-12 is indispensable for immunity and is required for the optimal antiparasitic activity of antimonials in experimental visceral leishmaniasis caused by Leishmania donovani. Here we investigated the role of STAT4 in immunity against L. donovani using STAT4 knockout mice and also determined the effect of STAT4 deficiency in response to antimonial therapy. Upon infection with L. donovani, stat4(-/-) BALB/c and C57BL/6 mice showed enhanced susceptibility to Leishmania during late time points of infection which was associated with a marked reduction in Th1 responses and hepatic immunopathology. Interestingly, these defects in Th1 responses in stat4(-/-) did not impair the antimonial chemotherapy as both stat4(-/-) and WT mice showed comparable levels of parasite clearance from the liver and spleen. These findings highlight the role of STAT4 in immunity to L. donovani infection and also provide evidence that STAT4 is dispensable for antimonial-based chemotherapy.
... In human cutaneous leishmaniasis, the diffuse form of the disease has been associated with lower levels of the macrophage chemotactic factor CCL2 (MCP-1), which is higher in the localized form of the disease . Since CCL2 also induces antiparasitic activity in macrophages (Mannheimer, Hariprashad, Stoeckle, & Murray, 1996), inhibition of this chemokine by Leishmania could potentially facilitate its survival within the host. The Leishmania virulence factor LPG can also inhibit migration of monocytes across the endothelial wall by inhibiting the synthesis of CCL2 and the expression of cell surface adhesion molecules including E-selectin, ICAM-1, and VCAM-1 by endothelial cells (Lo et al., 1998). ...
Diseases caused by Leishmania present a worldwide problem, and current therapeutic approaches are unable to achieve a sterile cure. Leishmania is able to persist in host cells by evading or exploiting host immune mechanisms. A thorough understanding of these mechanisms could lead to better strategies for effective management of Leishmania infections. Current research has focused on parasite modification of host cell signaling pathways, entry into phagocytic cells, and modulation of cytokine and chemokine profiles that alter immune cell activation and trafficking to sites of infection. Immuno-therapeutic approaches that target these mechanisms of immune evasion by Leishmania offer promising areas for preclinical and clinical research.
... IFN-γ from Th1 cells, mediates macrophage activation, nitric oxide production, and parasite killing (reviewed Cunningham, 2002 ). Macrophage leishmanicidal activity induced by IFN-γ may be enhanced by other cytokines such as TNF-α and migration inhibition factor (MIF; Mannheimer et al., 1996; Jüttner et al., 1998; Xu et al., 2003 ) and type-1 interferons (Diefenbach et al., 1998) as well as CD40/CD40L interactions (Campbell et al., 1996; Kamanaka et al., 1996; Soong et al., 1996; Heinzel et al., 1998). The exquisite susceptibility of BALB/c mice has been attributed to intrinsic defects associated with their Th1 cells such as an inability to upregulate CXCR3 and home to lesions (Barbi et al., 2008). ...
Work in large part on Leishmania major in the 1980s identified two distinct apparently counter-regulatory CD4⁺ T cell populations, T helper (h)1 and Th2, that controlled resistance/susceptibility to infection respectively. However, the generation of IL-4−/− mice in the 1990s questioned the paramount role of this Th2 archetypal cytokine in the non-healing response to Leishmania infection. The more recent characterization of CD4⁺ T cell regulatory populations and further effector CD4⁺ T helper populations, Th17, Th9, and T follicular (f)h cells as well as the acknowledged plasticity in T helper cell function has further added to the complexity of host pathogen interactions. These interactions are complicated by the multiplicity of cells that respond to CD4⁺ T cell subset signatory cytokines, as well as the diversity of Leishmania species that are often subject to significantly different immune-regulatory controls. In this article we review current knowledge with regard to the role of CD4⁺ T cells and their products during Leishmania infection. In particular we update on our studies using conditional IL-4Rα gene-deficient mice that have allowed dissection of the cell interplay dictating the disease outcomes of the major Leishmania species infecting humans.
... Chemokines also play an important role in killing parasites from host cells [51, 62–64]. Antileishmanial activity of chemokines has been demonstrated in both in vitro and in vivo infections in murine VL by induction of respiratory burst as well as by induction of Th1 cytokines [65, 66]. Another CD4+ T-cell lineage called Th17 cells was initially characterized as producing IL-17 family cytokines [67]. ...
Leishmaniasis is a protozoan parasitic disease endemic to the tropical and subtropical regions of the world, with three major clinical forms, self-healing cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL). Drug treatments are expensive and often result in the development of drug resistance. No vaccine is available against leishmaniasis. Subunit Leishmania vaccine immunization in animal models has shown some efficacy but little or none in humans. However, individuals who recover from natural infection are protected from reinfection and develop life-long protection, suggesting that infection may be a prerequisite for immunological memory. Thus, genetically altered live-attenuated parasites with controlled infectivity could achieve such memory. In this paper, we discuss development and characteristics of genetically altered, live-attenuated Leishmania donovani parasites and their possible use as vaccine candidates against VL. In addition, we discuss the challenges and other considerations in the use of live-attenuated parasites.
... Antileishmanial activity mediated by chemokines has been demonstrated in both in vitro and in vivo infections with L. donovani. In macrophages primed with the CC chemokines MIP-1α and CCL2, multiplication of L. donovani amastigotes is inhibited through the induction of the respiratory burst and nitric oxide (Mannheimer et al., 1996; Bhattacharyya et al., 2002; Dey et al., 2005). This is also observed in L. infantum infected human macrophages via the same mechanism (Brandonisio et al., 2002). ...
Successful immunity to Leishmania depends on recruitment of appropriate immune effector cells to the site of infection and chemokines play a crucial role in the process. At the same time, Leishmania parasites possess the ability to modify the chemokine profiles of their host thereby facilitating establishment of progressive infection. Therapeutic and prophylactic strategies targeted at chemokines and their receptors provide a promising area for further research. This review highlights our current knowledge concerning the role of chemokines and their receptors in modulating leishmaniasis in both clinical settings and experimental disease models.
... Thus, the increase in RANTES production correlated with the increase in parasitaemia and pathological changes observed in the P. yoelii 17XL-infected mice in this investigation. Chemokines have been shown to have a direct antiprotozoal activity for three protozoans: Toxoplasma gondii, Leishmania donovani and Trypanosoma cruzi [32,33]. Chemokine production is important for defending the host against infection. ...
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.
... Macrophages secrete other cytokines, in addition to IL-12, that not only regulate macrophage function in an autocrine manner but also play an important role in the modulation of acquired immune responses (Fig. 2). For instance, in vitro studies have demonstrated that TNF-α, and monocyte chemotactic and activating factor (MCAF), can enhance the leishmanicidal activity of macrophages ( Mannheimer et al., 1996). Similarly, migration inhibitory factor (MIF) also plays a protective role during L. major infection in vivo and in vitro, possibly by inducing NO production ( Juttner et al., 1998;Xu et al., 1998). ...
Leishmania species are obligate intracellular parasites of cells of the macrophage-dendritic cell lineage. Indeed, the ability to survive and multiply within macrophages is a feature of a surprising number of infectious agents of major importance to public health, including Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, Salmonella typhimurium, Toxoplasma gondii and Trypanosoma cruzi. The relationship between such organisms and their host cells is particularly intriguing because, not only are macrophages capable of potent microbicidal activity, but in their antigen-presenting capacity they can orchestrate the developing immune response. Thus, to initiate a successful infection parasites must gain entry into macrophages, and also withstand or circumvent their killing and degradative functions. However, to sustain a chronic infection, parasites must also subvert macrophage-accessory-cell activities and ablate the development of protective immunity. The leishmanias produce a wide spectrum of disease in mice, and as such they have provided excellent models for studying problems associated with intracellular parasitism. In recent years, largely using these organisms, we have made enormous progress in elucidating the mechanisms by which successful intracellular infection occurs. Furthermore, characterization of immunological pathways that are responsible for resistance or susceptibility to Leishmania has given rise to the Th1/Th2 paradigm of cellular/humoral dominance of the immune response.
... These cells could then become activated by locally produced CCL2/MCP-1 and IFN-γ. This activation could be based on a local synergism between cytokines and chemokines because it has been shown that CCL2/MCP-1 acts synergistically with IFN-γ and induces antiprotozoan activities in differentiated macrophages (31,40). However, these data are controversial. ...
Human leishmaniasis is caused by protozoan Leishmania (L.) parasites and comprises a heterogeneous group of clinical appearances ranging from visceral to cutaneous leishmaniasis. In the New World, L. mexicana mediates American cutaneous leishmaniasis, one of the most common forms of this disease. Two different disease progressions can be observed: (i) self-healing localized cutaneous leishmaniasis (LCL) and (ii) progressive diffuse cutaneous leishmaniasis (DCL). These different forms are associated with a T helper 1 (Th1) or Th2 response, respectively, and are additionally characterized by opposing dermal chemokine profiles. Lesions of LCL show high expression of CCL2/MCP-1, CXCL9/MIG, CXCL10/IP-10 and only low amounts of CCL3/MIP-1alpha. In contrast, lesions of chronic DCL are dominated by the expression of CCL3/MIP-1alpha. This finding implies that CCL2/MCP-1 contributes to the healing process. Indeed, CCL2/MCP-1 induces leishmanicidal activities in human monocytes in contrast to CCL3/MIP-1alpha. This effect is enhanced by interferon-gamma and abrogated by interleukin-4. In the murine model of leishmaniasis, the impact of CCL2/MCP-1 is well documented. Normally resistant mice become susceptible for Leishmania infections if CCR2, the receptor for CCL2/MCP-1, is knocked out. Based on this evidence, we propose that tissue specific expression of these small molecules actively regulates cell traffic and tissue localization of effector cells and, additionally, has direct immunological effects.
... IFN-from Th1 cells, and probably to a lesser extent CD8 + T cells as part of the acquired immune response, but also from IL-12 activated NK cells as part of the innate response, mediates macrophage activation, nitric oxide production and parasite killing [21]. Macrophage leishmanicidal activity induced by IFN-has been shown to be enhanced by other cytokines such as [22], migration inhibition factor (MIF) [23,24] and type-1 interferons [25] as well as CD40/CD40L interactions [19,262728. Interestingly a recent study demonstrated that C57BL/6 mice can control L. mexicana independently of IL- 12 [29]. ...
Studies on Leishmania major have been largely responsible for the characterisation of the Th1/Th2 paradigm of healing/non-healing associated with intracellular infection. IFN-gamma and IL-4 were identified respectively as the counter-regulatory Th1 and Th2 cytokines promoting resistance and susceptibility to infection. While resistance against infection remains largely associated with an IL-12 induced type-1 response studies using in particular gene-deficient mice have questioned the paramount role of IL-4 in the non-healing disease and implicated several alternative candidates. Indeed IL-4 has been shown to have no exacerbatory role in murine visceral leishmaniasis while its contribution to the progression of cutaneous disease has been clearly shown to be influenced not only by the parasite species but also the mouse strain used. Furthermore, it is now well established that not only can Th2 responses be induced independently of IL-4 but IL-4 under certain circumstances can prime for IL-12 production and a type-1 response. Clearly a reappraisal of our current understanding of the role of IL-4 and the Th2 response in the immunobiology of leishmaniasis is required and this review seeks to address this issue.
... Thus, the increase in RANTES production correlated with the increase in parasitaemia and pathological changes observed in the P. yoelii 17XL-infected mice in this investigation. Chemokines have been shown to have a direct antiprotozoal activity for three protozoans: Toxoplasma gondii, Leishmania donovani and Trypanosoma cruzi [32,33]. Chemokine production is important for defending the host against infection. ...
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.
... Nitric oxide has been reported to inhibit T. cruzi replication [37]. CCL2 enhanced the antiprotozoal activity in monocyte-derived macrophages infected with Leishmania donovani or Toxoplasma gondii [38]. It also stimulates the production of superoxide and killing of Leishmania major in human monocytes [39]. ...
Only a subset of individuals infected with Trypanosoma cruzi develop chronic Chagas cardiomyopathy (CCC). Familial aggregation of CCC in areas of endemicity indicates that susceptibility may be genetic, which may be a plausible explanation for why only one-third of T. cruzi-infected individuals develop CCC. The monocyte chemoattractant protein-1 (CCL2/MCP-1) has been shown to enhance the uptake of T. cruzi in murine macrophages and to up-regulate the inducible nitric oxide synthase/nitric oxide system, with a consequent increased production of nitric oxide that controls the replication of the parasite.
We assessed CCL2 variants at position -2518A/G, which are known to influence transcriptional activity, by polymerase chain reaction and restriction fragment-length polymorphism in 245 individuals, all of whom were infected with T. cruzi. One hundred sixty-nine patients had CCC, and 76 were asymptomatic.
Genotype distributions differed between the CCC and asymptomatic groups (chi2 = 9.4; P = .009), with an excess of genotypes with the A allele (AA + AG) in the CCC group. Among patients with CCC, 5% were homozygous for the G allele, compared with 16% of the asymptomatic subjects (odds ratio [OR], 4.1; 95% confidence interval [CI], 1.7-11; P = .001). A similar trend was observed when individuals heterozygous for the G allele were compared with individuals homozygous for the G allele between the CCC and asymptomatic groups (OR, 2.7; 95% CI, 0.97-7.2; P = .026). The A allele seems to confer susceptibility to CCC (OR, 1.9; 95% CI, 1.3-2.9; P = .001).
The CCL2 variant correlated with a low transcriptional level behaves as a genetic modifier of clinical outcome for T. cruzi infection, and subjects with the CCL2 -2518AA genotype have a 4-fold greater risk of developing CCC than do those without this genotype.
... CC chemokine receptor 2 (CCR2) the receptor that binds MCP-1 (28,29), has been shown to be vital for host defense to a number of pathogens (11,12,17,30,36,38,41,42). Likewise, it has been suggested that MCP-1/CCR2 may play a variety of roles in host defense against Leishmania (38). It has been reported that high doses of MCP-1 activate anti-Leishmania macrophage killing mechanisms either directly (23), by inducing reactive oxygen intermediates (33), or via nitric oxide production (5)(6)(7). In addition, MCP-1 has been reported to activate NK cells (47). ...
Transgenic Leishmania parasites that encode the murine chemokine monocyte chemoattractant protein 1 (MCP-1) were generated. These parasites transcribed MCP-1 mRNA and secreted MCP-1 protein. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with these parasites resulted in minimal lesion development with fewer parasites in the infected foot, lymph node, and spleen compared to wild-type-infected mice. In contrast, transgenic parasites caused substantial lesions with relatively high numbers of parasites in CC chemokine receptor 2 (CCR2) KO mice, indicating that the parasites are viable and healthy and that the lack of lesion development is CCR2 dependent. Prior infection of mice with transgenic parasites offered no protection to subsequent wild-type L. major challenge, suggesting that the transgenic parasites are controlled by an early innate immune response. Consistent with innate immunity, flow cytometry of cells from the ears of mice infected with transgenic parasites revealed an increase in the number of CCR2-positive macrophages by day 7 postinfection. The enumeration of transgenic parasites in ear lesions demonstrated a significant reduction in parasite numbers, which coincided with the increased CCR2-positive macrophage migration. CCR2-positive macrophages isolated from ears of mice infected with transgenic parasites contained virtually no parasites. In vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages, followed by stimulation with a combination of both MCP-1 and gamma interferon (IFN-gamma). This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-gamma to efficiently kill Leishmania parasites.
Visceral leishmaniasis (VL; also known as kala-azar), caused by the protozoan parasite Leishmania donovani is characterized by the inability of the host to generate an effective immune response. The manifestations of the disease depends on involvement of various immune components such as activation of macrophages, cell mediated immunity, secretion of cytokines and chemokines, etc. Macrophages are the final host cells for Leishmania parasites to multiply, and they are the key to a controlled or aggravated response that leads to clinical symptoms. The two most common macrophage phenotypes are M1 and M2. The pro-inflammatory microenvironment (mainly by IL-1β, IL-6, IL-12, IL-23, and TNF-α cytokines) and tissue injury driven by classically activated macrophages (M1-like) and wound healing driven by alternatively activated macrophages (M2-like) in an anti-inflammatory environment (mainly by IL-10, TGF-β, chemokine ligand (CCL)1, CCL2, CCL17, CCL18, and CCL22). Moreover, on polarized Th cells, chemokine receptors are expressed differently. Typically, CXCR3 and CCR5 are preferentially expressed on polarized Th1 cells, whereas CCR3, CCR4 and CCR8 have been associated with the Th2 phenotype. Further, the ability of the host to produce a cell-mediated immune response capable of regulating and/or eliminating the parasite is critical in the fight against the disease. Here, we review the interactions between parasites and chemokines and chemokines receptors in the pathogenesis of VL.
In livers of C57BL/6 mice, IFN-γ controls intracellular Leishmania donovani infection and the efficacy of antimony (Sb) chemotherapy. Since both responses usually correlate with granulomatous inflammation, we tested six prominently-expressed, IFN-γ-regulated chemokines – CXCL9, CXCL10, CXCL13, CXCL16 and CCL2 and CCL5 – for roles in: (a) mononuclear cell recruitment and granuloma assembly and maturation, (b) initial control of infection and self-cure, and (c) responsivenesss to Sb treatment. Together, results in L. donovani -infected livers of chemokine-deficient mice (CXCR6 -/- mice were used as CXCL16-deficient surrogates) indicated that individual IFN-γ-induced chemokines have diverse affects and: (a) may be entirely dispensable (CXCL13, CXCL16), (b) promote (CXCL10, CCL2, CCL5) or downregulate (CXCL9) initial granuloma assembly, (c) enhance (CCL2, CCL5) or hinder (CXCL10) early parasite control, (d) promote granuloma maturation (CCL2, CCL5), (e) exert granuloma-independent action that enables self-cure (CCL5), and (f) have no role in responsiveness to chemotherapy. Despite near-absent tissue inflammation in early-stage infection, parasite replication could be controlled (CXCL10 -/- mice) and Sb fully-active (CXCL10 -/- , CCL2 -/- and CCL5 -/- mice). These results characterize chemokine action in the response to L. donovani , and also re-emphasize that: (a) recruited mononuclear cells and granulomas are not required to control infection or respond to Sb chemotherapy, (b) granuloma assembly, control of infection and Sb's efficacy are not invaria bly-linked expressions of the same T cell-dependent, cytokine-mediated antileishmanial mechanism, and (c) that granulomas are not necessarily hallmarks of protective antileishmanial immunity.
Leishmaniasis is a major vector-borne parasitic disease affecting 12 million people worldwide. With a broad range of clinical manifestations, ranging from self-healing skin ulcers to disfiguring mucosal lesions to life-threatening infections of visceral organs (liver and spleen), the disease has become a serious human health issue, particularly in developing countries. Among all of its forms, visceral leishmaniasis (VL, also known as kala-azar), caused by the Leishmania donovani complex (i.e., L. donovani and L. infantum in Old World and L. chagasi in New World), is often fatal in the absence of treatment. Although humans are the principal hosts for L. donovani, canine species are the main reservoirs of L. infantum. Canine VL affects millions of dogs and is associated with outbreaks of human VL and hence has become a major public health issue. The lack of vaccines to prevent and/or treat these infections, as well as the emergence of drug resistant parasites, is serious impediments to control leishmaniasis. Therefore, developing new prophylactic and therapeutic strategies against this disease is urgently required. However, for this to occur, a better understanding of the complex immune mechanisms generated in response to infection and defining those involved in resistance to infection is required. In this special issue on “Immunity to visceral leishmaniasis”, several selected papers will discuss these issues.
Esta Tesis consiste en la valoración de nuevas formulaciones de anfotericina B frente a la leishmaniosis visceral causada por L. infantum. Para ello, primero se ha establecido un modelo animal adecuado, que ha permitido fijar las condiciones óptimas para su aplicación a nuestros objetivos. Posteriormente, se han ensayado distintas pautas de tratamiento, distintos estados de agregación de la anfotericina B, distintos polímeros de encapsulación y distintas dosis de principio activo, valorándose la eficacia parasitológica, así como la respuesta inmune humoral y celular, realizándose un seguimiento de la evolución del tratamiento a nivel inmunológico.De todas las formulaciones ensayadas, la mejor resultó ser la de albúmina sérica humana conteniendo anfotericina B en estado poliagregado, ya que se consigue una alta eficacia, con ausencia de efectos tóxicos y con disminución de los niveles de anticuerpos, de los inmunocomplejos renales y con recuperación de la proliferación celular. Además, se han puesto a punto técnicas de Biología Molecular para la determinación de parámetros inmunológicos como la expresión de citoquinas en criceto y para el recuento de parásitos en órganos.
Secretion of Monocyte Chemotactic Protein-1 (MCP-1) by fibroblasts infected with Toxoplasma gondii was studied in vitro. A significantly higher MCP-1 secretion was observed 24 h after infection by live tachyzoites. Analysis of chemokine mRNA transcripts by RNase protection assay revealed that this MCP-1 secretion seems associated with increased MCP-1 mRNA expression. However, these increased levels of MCP-1 secretion and expression were not obtained after stimulation by heat-killed tachyzoites or parasites pre-treated by a specific inhibitor of phosphatidylcholine-specific phospholipase C (D609). Inhibition of parasite multiplication by pyrimethamine did not modify MCP-1 secretion. Thus, it appeared that the active penetration of T. gondii in cells was of major importance in the induction of MCP-1 secretion. None of the other chemokines studied by RNase protection assay (lymphotactin, RANTES, IP-10, MIP-1alpha, MIP-1beta, IL-8, and I-309) were expressed after infection by live tachyzoites. We also found that MCP-1 secretion induced by live T. gondii is blocked by inhibitors of nuclear factor (NF)-kappaB activation, ALLN and MG132. Such data indicate that NF-kappaB could be involved in T. gondii-induced MCP-1 production. MCP-1 secretion may contribute to the recruitment of monocytes and lymphocytes and thus participate in the control of T. gondii infection and in its pathogenesis.
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.
Toxoplasma gondii infection results in an infiltration of immune cells. The mechanisms responsible for triggering inflammatory cell infiltration in T. gondii infection are not fully understood. We report that T. gondii-infected HeLa cells induced nuclear factor-kappa B (NF-kappaB) activation and increased the expression of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA. An inhibitor of NF-kappaB activation, calpain-1 inhibitor, blocked the chemokine secretion induced by live T. gondii. Activation of the IL-8 and NF-kappaB transcriptional reporters was suppressed in cells co-transfected with IkappaB kinase beta and the IkappaBalpha super-repressor plasmids. Moreover, the addition of IL-1alpha increased NF-kappaB activation and IL-8 mRNA expression in T. gondii-infected HeLa cells. These results suggest that NF-kappaB is a central regulator of the chemokine response in T. gondii-infected human epithelial cells and that chemokine IL-8 and MCP-1 secretion might be involved in the pathogenesis of T. gondii, via the recruitment of neutrophils, monocytes, and lymphocytes.
Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1α significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-γ. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L-N
Gmonomethylarginine (L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1α mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection.
The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In the case of
Leishmania donovani infection, the impairment of PKC-mediated signaling is one of the crucial events for the establishment of parasite into the
macrophages. Earlier reports established that C-C chemokines mediated protection against leishmaniasis via the generation
of nitric oxide after 48 h. In this study, we investigated the role of MIP-1α and MCP-1 in the regulation of impaired PKC
activity in the early hours (6 h) of infection. These chemokines restored Ca2+-dependent PKC activity and inhibited Ca2+-independent atypical PKC activity in L. donovani-infected macrophages under both in vivo and in vitro conditions. Pretreatment of macrophages with chemokines induced superoxide
anion generation by activating NADPH oxidase components in infected cells. Chemokine administration in vitro induced the migration
of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly
restricted the parasitic burden in livers as well as in spleens. Collectively, these results indicate a novel regulatory role
of C-C chemokines in controlling the intracellular growth and multiplication of L. donovani, thereby demonstrating the antileishmanial properties of C-C chemokines in the disease process.
Human blood mononuclear leukocytes stimulated with toxoplasma antigen, concanavalin A, mezerein plus lentil lectin, or staphylococcal enterotoxin A secreted a factor (macrophage-activating factor, or MAF) that enhanced the capacity of human macrophages to release H2O2 and to kill toxoplasmas. The same lymphoid supernatants contained IFN gamma but not IFN alpha or IFN beta. The MAF activity of six of seven unfractionated supernatants was completely eliminated by a monoclonal antibody that neutralizes IFN gamma, and MAF in the remaining supernatant was almost completely neutralized. Native IFN gamma partially purified by two independent protocols to specific activities of 1 X 10(6) and 10(7) U/mg protein was enriched in MAF activity at least as much as in antiviral activity. The capacity of macrophages to secrete H2O2 after incubation in partially purified native IFN gamma (mean peak stimulation, 8.8-fold) was greater than with unpurified lymphokines (3.8-fold) and sometimes equaled or exceeded the capacity of freshly harvested monocytes. The MAF activity of the partially purified native IFN gamma preparations was abolished by monoclonal anti-IFN gamma. Finally, IFN gamma of greater than 99% estimated purity was isolated (at Genentech, Inc.) from bacteria transformed with the cloned human gene for this lymphokine. Recombinant IFN gamma had potent MAF activity, stimulating the peroxide-releasing capacity of macrophages an average of 19.8-fold at peak response and enhancing their ability to kill toxoplasmas from 2.6 +/- 1.3% for untreated cells to 54 +/- 0.4% for treated cells. Attainment of 50% of the maximal elevation in peroxide-releasing capacity required a geometric mean concentration of 0.1 antiviral U/ml of recombinant IFN gamma, which is estimated to be approximately 6 picomolar for this preparation. Peroxide secretory capacity and toxoplasmacidal activity of macrophages peaked 2-4 d after exposure to IFN gamma. Peroxide-secretory capacity remained elevated during at least 6 d of continuous exposure, but the effect of IFN gamma was reversed within about 3 d of its removal. Activation was usually but not invariably accompanied by characteristic changes in cell morphology. Thus, IFN gamma activates human macrophage oxidative metabolism and antimicrobial activity, and appeared to be the only factor consistently capable of doing so in the diverse LK preparations tested.
A monocyte chemotactic and activating factor (MCAF) has been purified from TNF-stimulated 8387 human fibrosarcoma cell line-conditioned media. The purified MCAF showed microheterogeneity yielding two bands on SDS-PAGE analysis. Fibrosarcoma-derived MCAF specifically competed with THP-1 (a human monocytic cell line)-derived 125I-labeled MCAF in binding to human PBMC, whereas a similar basic heparin-binding leukocyte chemoattractant, IL-8, did not. The purified MCAF stimulated superoxide anion and N-acetyl beta-D glucosaminidase-releasing activity in human monocytes, as well as monocyte cytostatic augmenting activity against tumor cells and chemotactic activity for monocytes. When injected subcutaneously into Lewis rat ears, the purified human MCAF also induced considerable in vivo local monocyte infiltration beginning at 3 h and becoming maximal at 18 h. In conclusion, the data presented in this paper indicate that MCAF is a potent activator of monocytes as well as a monocyte recruitment factor that acts through receptors that are specific for this novel molecule. This novel cytokine might have an important role in tumor growth control due to its ability to attract and activate monocytes.
The early growth response gene JE encodes a monocyte chemoattractant, MCP-1. The JE/MCP-1 protein attracts and stimulates
human monocytes and induces monocyte-mediated inhibition of tumor cell growth in vitro. Expression of human or murine JE/MCP-1
in Chinese hamster ovary (CHO) cells completely suppressed their ability to form tumors in nude mice. Coinjection of JE/MCP-1-expressing
cells with nonexpressing CHO cells or with HeLa cells also prevented tumor formation. Since JE/MCP-1 expression had no discernible
effect on the tranformed phenotype of these cells in vitro, the suppressive effect depends on host animal factors. These factors
are likely to be components of the inflammatory response, because JE/MCP-1-expressing cells elicited a predominantly monocytic
infiltrate at the site of injection. Our results suggest that JE/MCP-1 protein may be useful in cancer therapy.
A monocyte chemotactic and activating factor (MCAF) has been purified from TNF-stimulated 8387 human fibrosarcoma cell line-conditioned media. The purified MCAF showed microheterogeneity yielding two bands on SDS-PAGE analysis. Fibrosarcoma-derived MCAF specifically competed with THP-1 (a human monocytic cell line)-derived 125I-labeled MCAF in binding to human PBMC, whereas a similar basic heparin-binding leukocyte chemoattractant, IL-8, did not. The purified MCAF stimulated superoxide anion and N-acetyl beta-D glucosaminidase-releasing activity in human monocytes, as well as monocyte cytostatic augmenting activity against tumor cells and chemotactic activity for monocytes. When injected subcutaneously into Lewis rat ears, the purified human MCAF also induced considerable in vivo local monocyte infiltration beginning at 3 h and becoming maximal at 18 h. In conclusion, the data presented in this paper indicate that MCAF is a potent activator of monocytes as well as a monocyte recruitment factor that acts through receptors that are specific for this novel molecule. This novel cytokine might have an important role in tumor growth control due to its ability to attract and activate monocytes.
Cytokines such as tumor necrosis factor alpha (TNF) profoundly affect endothelial cell function, promoting for example interaction with leukocytes and inducing a procoagulant phenotype. Changes of this nature are likely to be central to the proinflammatory effects of TNF. In order to elucidate molecular mechanisms by which TNF alters endothelial cell function we utilized differential plaque hybridization to identify TNF-responsive genes. Forty TNF-inducible cDNAs were identified which on cross-hybridization were found to arise from six unique genes. DNA sequencing of these cDNAs revealed two encoded known cytokine-induced genes, endothelial leukocyte adhesion molecule 1 and neutrophil chemotactic factor. One of the cDNAs encodes a recently described monocyte-specific chemotactic factor not previously associated with endothelium. The production of a monocyte chemotaxin by cytokine-activated endothelium has important implications for understanding the role of the vessel wall in disease states such as atherosclerosis and may also in part explain the indirect angiogenic activity of TNF. The three other cDNAs are completely novel as judged by data bank searches of partial DNA sequences and remain unidentified. On exposure of endothelial cells to TNF there is a rapid and substantial increase in levels of mRNA encoding the six genes, which are further superinduced by cycloheximide. Thus these represent primary response genes as their induction does not depend on protein synthesis. Interleukin-1 beta and lipopolysaccharide are also potent inducers. Nuclear run-on studies revealed that in most cases induction by TNF is mediated largely at the transcriptional level.
Human malignant cells secrete low molecular size proteins that attract peripheral blood monocytes and may be responsible for the accumulation of tumor-associated macrophages observed in vivo. Similar chemotactic proteins are secreted by cultured vascular smooth muscle cells. The predominant monocyte chemoattractants produced by tumor cells of differing origin were demonstrated to be related to smooth muscle cell-derived chemotactic factor. Thus, a single class of chemotactic proteins is produced by different cell types, which suggests a common mechanism for the recruitment of monocytes and macrophages. These results are significant in view of the potential of macrophages to affect tumor growth.
A novel basic heparin-binding monocyte chemotactic factor (MCF) was purified to homogeneity from the conditioned media of human myelomonocytic cell line THP-1 based on its in vitro monocyte chemotactic activity. The purified MCF was homogenous and estimated to be 15 kD on SDS-PAGE. Purified MCF stimulated normal human monocytes to be growth inhibitory in vitro at 2-3 d for several human tumor cell lines. This represents the first report of the identification and purification of a chemoattractant cytokine that also activates monocytes but is distinct from interferons and other known cytokines.
Human blood mononuclear leukocytes stimulated with toxoplasma antigen, concanavalin A, mezerein plus lentil lectin, or staphylococcal enterotoxin A secreted a factor (macrophage-activating factor, or MAF) that enhanced the capacity of human macrophages to release H2O2 and to kill toxoplasmas. The same lymphoid supernatants contained IFN gamma but not IFN alpha or IFN beta. The MAF activity of six of seven unfractionated supernatants was completely eliminated by a monoclonal antibody that neutralizes IFN gamma, and MAF in the remaining supernatant was almost completely neutralized. Native IFN gamma partially purified by two independent protocols to specific activities of 1 X 10(6) and 10(7) U/mg protein was enriched in MAF activity at least as much as in antiviral activity. The capacity of macrophages to secrete H2O2 after incubation in partially purified native IFN gamma (mean peak stimulation, 8.8-fold) was greater than with unpurified lymphokines (3.8-fold) and sometimes equaled or exceeded the capacity of freshly harvested monocytes. The MAF activity of the partially purified native IFN gamma preparations was abolished by monoclonal anti-IFN gamma. Finally, IFN gamma of greater than 99% estimated purity was isolated (at Genentech, Inc.) from bacteria transformed with the cloned human gene for this lymphokine. Recombinant IFN gamma had potent MAF activity, stimulating the peroxide-releasing capacity of macrophages an average of 19.8-fold at peak response and enhancing their ability to kill toxoplasmas from 2.6 +/- 1.3% for untreated cells to 54 +/- 0.4% for treated cells. Attainment of 50% of the maximal elevation in peroxide-releasing capacity required a geometric mean concentration of 0.1 antiviral U/ml of recombinant IFN gamma, which is estimated to be approximately 6 picomolar for this preparation. Peroxide secretory capacity and toxoplasmacidal activity of macrophages peaked 2-4 d after exposure to IFN gamma. Peroxide-secretory capacity remained elevated during at least 6 d of continuous exposure, but the effect of IFN gamma was reversed within about 3 d of its removal. Activation was usually but not invariably accompanied by characteristic changes in cell morphology. Thus, IFN gamma activates human macrophage oxidative metabolism and antimicrobial activity, and appeared to be the only factor consistently capable of doing so in the diverse LK preparations tested.
Chemotactic factors regulate the recruitment of neutrophils, lymphocytes, or monocytes-macrophages to infectious and inflammatory sites. The purpose of this study was to determine whether monocyte-chemotactic and -activating factor (MCAF [MCP-1], a JE gene product) also influences the host defense mechanism against microbial infection. We evaluated the effect of recombinant human MCAF on the survival rate of mice systemically infected with Pseudomonas aeruginosa or Salmonella typhimurium. The administration of 2.5 micrograms of MCAF 6 h before infection completely protected the mice from lethal infection. Mice with cyclophosphamide-induced leukopenia exhibiting increased susceptibility to P. aeruginosa were also endowed with resistance by the same dose of MCAF. Administration of MCAF at -6 h was critical, since MCAF given either earlier or later than -6 h failed to rescue mice from lethal infection. The in vivo effect on the survival of mice paralleled the reduced recovery of viable P. aeruginosa or S. typhimurium from the peritoneal cavity, i.e., the number of recovered bacteria from the MCAF (2.5 micrograms per mouse)-treated mice was reduced to less than 2% of control mice for P. aeruginosa and 4% of control mice for S. typhimurium at 24 h. Since MCAF exhibited chemotaxis on murine macrophages as well as enhanced phagocytosis and killing of bacteria in vitro, the activation of macrophages, followed by the recruitment into the peritoneal cavity, is responsible for eliminating bacteria and thus enhancing the survival rate.
A novel basic heparin-binding monocyte chemotactic factor (MCF) was purified to homogeneity from the conditioned media of human myelomonocytic cell line THP-1 based on its in vitro monocyte chemotactic activity. The purified MCF was homogenous and estimated to be 15 kD on SDS-PAGE. Purified MCF stimulated normal human monocytes to be growth inhibitory in vitro at 2-3 d for several human tumor cell lines. This represents the first report of the identification and purification of a chemoattractant cytokine that also activates monocytes but is distinct from interferons and other known cytokines.
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.
The L-arginine-dependent generation of reactive nitrogen intermediates (RNI) has been identified as a key intracellular antimicrobial
mechanism of activated mouse macrophages. To determine the role of this mechanism in the activity of human mononuclear phagocytes,
monocytederived macrophages activated in vitro by interferon (IFN)-γ and monocytes from patients receiving IFN-γ as therapy
were treated with NG-monomethyl-l-arginine (NMA) or arginase. Neither competitive inhibition of l-arginine metabolism (NMA) nor depletion of l-arginine (arginase) altered intracellular antimicrobial activity against Toxoplasma gondii, Chlamydia psittaci, or Leishmania donovani. In contrast, NMA and arginase readily reversed the antimicrobial effect of mouse peritoneal macrophages stimulated either
in vitro or in vivo by IFN-γ, and activated mouse but not human cells could be induced to release enhanced levels of nitrite.
These results suggest that the l-arginine-dependent generation of RNI is a species-restricted macrophage mechanism unlikely to participate in the intracellular
antimicrobial activity of IFN-γ stimulated human mononuclear phagocytes.
The JE gene was first described as a platelet-derived growth factor (PDGF)-inducible gene in mouse 3T3 cells. The human homologue of JE encodes a protein whose predicted amino acid sequence is identical to that of the monocyte chemoattractant MCP-1 (also called MCAF and SMC-CF), which belongs to a recently identified family of small secretory proteins with cytokine properties. We purified recombinant human MCP-1/JE (hMCP-1/JE) produced in COS cells and demonstrated that it is chemotactic for human monocytes with a specific activity similar to natural MCP-1. In addition, pure recombinant hMCP-1/JE stimulates monocytes, inducing an increase in cytosolic free calcium and the respiratory burst, but is completely inactive on human neutrophils. These results help to define functionally a well-known growth factor-inducible gene and a member of a new family of cytokines.
Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.
A family consisting of at least ten distinct novel 8-10 kd cytokines has been identified over the past 12 years. These cytokines exhibit from 20 to 45% homology in amino acid sequence, are probably all basic heparin-binding polypeptides, and have proinflammatory and reparative activities. The cDNA for these cytokines are characterized by conserved single open reading frames, typical signal sequences in the 5' region, and AT rich sequences in the 3' untranslated regions. Those human cytokines known as interleukin 8, platelet factor 4, beta thromboglobulin, IP-10 and melanoma growth stimulating factor or GRO can be assigned to a subfamily based on their location on chromosome 4 and unique structural features, whereas the second subset consisting of LD78, ACT-2, I-309, RANTES, and macrophage chemotactic and activating factor (MCAF) are all closely linked on human chromosome 17. In this review we have summarized and discussed the available information concerning the regulation and structure of the genes, the structure and biochemical properties of the polypeptide products, their receptors, signal transduction, cell sources, and in vitro as well as in vivo activities of these cytokines.
We recently purified human monocyte chemoattractant protein-1 (MCP-1) from culture fluids of either human glioma cell lines or mitogen-stimulated human peripheral blood mononuclear leukocytes. It has now been shown that MCP-1 is the product of the gene JE, which was first recognized by its expression in fibroblasts stimulated with platelet-derived growth factor (PDGF). We therefore studied secretion of MCP-1 by three human fibroblast cell lines. Monocyte chemotactic activity was found in culture fluids of all three lines after growth to confluence in DMEM-10% FCS, and the amounts secreted per cell were comparable for the three lines. The MRC-5 line was chosen for further study. Monocyte chemotactic activity secretion by confluent MRC-5 cultures continued after a switch to serum-free medium and was not inhibited by anti-PDGF antibody, indicating that secretion may not have been caused by autocrine release of PDGF. When concentrated serum-free MRC-5 culture fluid was injected into an HPLC gel filtration column, only one chemotactic activity peak was observed, which was in the same location as glioma-derived MCP-1. The activity was completely absorbed out by an anti-MCP-1 affinity column, which indicates that all the chemotactic activity in MRC-5 culture fluid was accounted for by MCP-1. PDGF caused a marked increase in chemotactic activity over that found in serum-free culture fluid of MRC-5 or 501T cells. Immunoprecipitation by anti-human MCP-1 showed two bands, corresponding to the two forms of MCP-1 previously described (MCP-1 alpha and beta); and the amounts increased in response to PDGF stimulation. Thus, the reported increase in human fibroblast JE mRNA in response to PDGF-containing serum stimulation is reflected in increased secretion of the MCP-1 gene product.
Normal human dermal fibroblasts rapidly expressed (less than 30 min.) considerable mRNA for monocyte chemotactic and activating factor (MCAF) and released high levels of biological activity in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF). In contrast, cultured normal human keratinocytes did not express MCAF mRNA when stimulated with IL 1 or TNF. These results suggest the important role of dermal fibroblasts, the predominant cells in dermal connective tissue, in the recruitment of monocytes during inflammation. This is the first report of the induction of MCAF by IL 1 or TNF in any cell type.
The capacity of BALB/c mice to acquire resistance to and eliminate intracellular visceral Leishmania donovani is T cell dependent, associated with a granulomatous tissue reaction, and correlates with the ability to secrete the macrophage-activating lymphokine, IFN-gamma. These responses appear by 4 wk after infection and are fully established by 8 wk. To examine the role of endogenous IFN-gamma, BALB/c mice were injected with anti-IFN-gamma mAb before and for 8 wk after infection. At 4 wk, mAb treatment inhibited the acquisition of resistance to L. donovani and abolished mature granuloma formation. Although liver parasite burdens in mAb-treated mice were fivefold higher than in controls at 8 wk, continually treated mice nevertheless began for form tissue granulomas and decreased their parasite loads by 50% from peak values. The levels of anti-IFN-gamma antibody in the serum of mice injected for 8 wk were appreciably reduced, thus raising the possibilities of either insufficient neutralization of endogenous IFN-gamma at this time point or a pathway independent of IFN-gamma. Although the role of IFN-gamma and the potential effect of an IFN-gamma-independent mechanism in the resolution of visceral infection remain to be defined, these results indicate that IFN-gamma plays a critical role in the early immune response that both optimally controls L. donovani infection and induces the tissue granuloma.
During the first 2 to 4 weeks of progressive visceral infection with the intracellular protozoan, Leishmania donovani, spleen cells from BALB/c mice failed in response to leishmanial antigen to produce either of the activating T cell-derived lymphokines, interleukin 2 (IL 2) or gamma-interferon (IFN-gamma). Four weeks after infection, however, antigen-induced IL 2 and IFN-gamma secretion emerged and coincided with the onset of control over parasite replication and the subsequent killing of greater than 80% of intrahepatic L. donovani. The development of this immunosecretory activity correlated with the hepatic tissue response at the site of parasitized Kupffer cells. This response progressed from Kupffer cell fusion (week 1) to fusion plus a mononuclear cell infiltrate (week 2) to well-organized granuloma formation (weeks 4 to 8). In contrast, T cell-deficient nude BALB/c mice exerted no control over L. donovani, their spleen cells failed to generate antigen-induced IFN-gamma, and at 4 weeks, their livers were devoid of any tissue reaction. Since spleen cells from 2-week infected normal mice did not produce antigen-stimulated IL 2 or IFN-gamma, these mice were treated with recombinant (r) lymphokines. Various protocols using both high and low dose human rIL 2 had no antileishmanial effect. Hepatic parasite replication was completely halted, however, by macrophage-activating doses of murine rIFN-gamma. These results reemphasize that an intact T cell-dependent response is required for successful defense against L. donovani, indicate that this immune response can be measured at both the cellular (secretory) and tissue levels, and confirm that IFN-gamma can exert an antileishmanial effect in vivo.
Recent research on human macrophage activation has reemphasized the critical role of the lymphokine-secreting T cell in converting quiescent macrophages to efficient microbicidal phagocytes. Interferon-gamma, a key lymphokine secreted by antigen-triggered T4+ helper cells, is capable of inducing the macrophage to act against a diverse group of microbial targets, in particular, intracellular pathogens. In animal models, treatment with recombinant interferon-gamma is beneficial in systemic intracellular infections, and inhibition of endogenous interferon-gamma activity impairs host resistance. Trials in patients with cancer, leprosy, and the acquired immunodeficiency syndrome (AIDS) have shown that interferon-gamma can activate the mononuclear phagocyte in humans. This research and the identification of patients whose T cells fail to produce interferon-gamma properly has set the stage for evaluating the role of macrophage-activating immunotherapy using interferon-gamma in various human infectious diseases.
To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.
TNF-alpha has been implicated in cytokine-induced macrophage activation and tissue granuloma formation, two activities linked to control of intracellular visceral infection caused by Leishmania donovani. To determine the role of TNF-alpha in L. donovani-infected BALB/c mice, we measured TNF-alpha levels and treated mice with either anti-TNF-alpha antiserum or TNF-alpha. TNF-alpha activity in infected livers was increased by 2.7-fold 2 wk after challenge and by 5.5-fold at wk 8. In parallel, although control mice acquired resistance by wk 4 and resolved infection by wk 8, liver parasite burdens steadily increased in anti-TNF-alpha-treated animals. Hepatic granuloma formation, however, was not impaired by anti-TNF-alpha. Endogenous TNF-alpha levels provoked by L. donovani appeared sufficient and optimal because exogenous TNF-alpha administration had no beneficial effect on established infection and continuous high-dose treatment impaired antileishmanial activity. Thus, although not required for granuloma formation, endogenous TNF-alpha appears to be critical to both initial acquisition of resistance to L. donovani and resolution of experimental visceral infection.
Cytokines in the treatment of leishmaniasis. Baillere's
- H W Murray
Murray, H.W. (1994) Cytokines in the treatment of leishmaniasis. Baillere's Clin. Infect. Dis. 1, 127-143.
Properties of the novel proinflammatory supergene 'intercrine' cytokine family Secretion by human fibroblasts of monocyte chemoattractant protein-l, the product of gene JE
- J J Oppenheim
- C O C Zachariae
- N Mukaida
- K Matsushima
- E J Leonard
Oppenheim, J.J., Zachariae, C.O.C., Mukaida, N. and Matsushima, K. (1991) Properties of the novel proinflammatory
supergene 'intercrine' cytokine family. Annu. Rev. Immunol.
9, 617-648.
[191
DO1
ml
[7] Yoshimura, T. and Leonard, E.J. (1990) Secretion by human
fibroblasts of monocyte chemoattractant
protein-l, the product of gene JE. J. Immunol. 144, 2377-2382.
Experimental visceral leishmaniasis: production of interleukin 2 and interferon-y
- H W Murray
- J J Stem
- K Welte
- B Y Rubin
- S M Caniero
- C F Nathan
Murray, H.W., Stem, J.J., Welte, K., Rubin, B.Y., Caniero,
S.M. and Nathan, CF. (1987) Experimental visceral leishmaniasis: production of interleukin 2 and interferon-y.
J. Immunol. 138, 2290-2296.