Mark H. Ginsberg's research while affiliated with Medical College of Wisconsin and other places

Low density lipoprotein (LDL) has previously been demonstrated to be a potent inhibitor of human inflammatory cell activation by monosodium urate (MSU) crystals in vitro. As suppression of cellular responses to crystals by LDL is known to be dependent on the binding of LDL to urate crystals we further evaluated the mechanism and regulation of LDL binding to urate crystals in vitro. Using nonlinear, least squares methodology to analyze binding, we found LDL to saturably and reversibly bind with high affinity (Kd 9.3 x 10(-9) M) to MSU crystals. LDL binding was competitively inhibited by LDL but not by a variety of other positively charged moieties. Glycosaminoglycans (GAG) (heparin, heparan sulfate, hyaluronate and chondroitin sulfate), in diminishing order of potency, inhibited the binding of LDL to crystals. This inhibitory activity was dose dependent, sensitive to digestion with glycosidases and appeared to be specific for polymerized GAG, as glucuronic acid, dextran, dextran sulfate, as well as isolated amino sugar constituents of GAG were either weakly inhibitory or inactive. GAG also promoted dissociation of bound LDL from the urate crystal surface. The results indicate that LDL binds saturably and reversibly to urate crystals and that polymerized, but not depolymerized, hyaluronate and other GAG inhibit LDL binding to urate crystals. This suggests that the amount of LDL coating intraarticular urate crystals could vary during the course of a gouty paroxysm.

Low density lipoprotein (LDL) inhibits phagocytosis of certain negatively charged particulates and also inhibits subsequent cellular secretory and oxidative responses to these particulates. In the present work, we have defined the structural features of LDL involved in this activity. Starch-heptane extraction depleted greater than 95% of neutral lipids but had little effect on the capacity of LDL to inhibit monosodium urate crystal- or polystyrene latex bead-induced neutrophil chemiluminescence (CL). Liposomes containing gamma-palmitoyl-beta-oleoylphosphatidylcholine (PC) with unesterified cholesterol (PC:cholesterol = 2:1), PC and sphingomyelin (PC:sphingomyelin = 2.3:1), or PC alone lacked the capacity to inhibit urate-induced CL. However, incorporation of apoB-100 into liposomes via cholate dialysis rendered them nearly as inhibitory for urate-induced neutrophil CL as LDL on a protein weight basis. Moreover, delipidated apoB-100, containing less than 3% residual phospholipid, inhibited neutrophil responses to urate crystals or latex beads (degranulation and superoxide anion release) in a stimulus-specific manner. Modifications of the lysine residues of apoB (e.g. acetylation) reduced both the capacity of LDL to inhibit urate crystal-induced CL and to bind to urate crystals. The effects of apoB lysine residue modification were reversible, proportional to the extent of modification, and were not attributable to alteration of the net charge of apoB. Thus, the apoB-100 of LDL both mediates and shares the capacity of native LDL to inhibit certain neutrophil responses to particulates.

The inflammatory response to intraarticular urate crystals is known to be variable in gouty arthritis. One source of variability may be the modulation of cellular responses by crystal-bound proteins. We have identified three apolipoproteins among the polypeptides bound to urate crystals exposed to plasma. Identification was first based on their coelectrophoresis with polypeptides from isolated lipoproteins and diminution in the protein coat of crystals exposed to lipoprotein-depleted plasma. The apoproteins were immunochemically identified by the Western blotting technique as apoprotein A-I, apoprotein B (apo B), and apoprotein E. Because neutrophils play a central role in acute gout, we investigated the potential effects of lipoproteins on neutrophil-urate crystal interactions. Plasma profoundly inhibited urate crystal-induced neutrophil luminol-dependent chemiluminescence (CL). Lipoprotein depletion by KBr density gradient centrifugation completely abrogated the inhibitory effect of plasma on urate-induced CL. The inhibitory activity of lipoprotein-depleted plasma was restored by adding back the d less than or equal to 1.25 g/cm3 lipoprotein fraction. Plasma also inhibited urate crystal-induced neutrophil superoxide generation and cytolysis (lactic dehydrogenase loss). This inhibition was significantly diminished by lipoprotein depletion, indicating that the lipoprotein effect was not limited to CL. Lipoprotein-depleted plasma reconstituted with very low, intermediate, and low density lipoproteins (LDL) inhibited crystal-induced CL. High density lipoprotein reconstitution was without effect. Immunodepletion from plasma of all apo B lipoproteins by agarose-bound apo B-specific antibody also removed all inhibitory activity for urate-induced CL. Thus, apo B lipoproteins were shown to be the inhibitory species in plasma. Binding of apo B lipoproteins to urate crystals and inhibition of CL was also seen in the absence of other plasma proteins. In addition, the binding of whole lipoprotein particles to the crystals was verified by detection of crystal-associated cholesterol in addition to the apoprotein. The effects of LDL on urate crystal-induced CL were stimulus specific. Coincubation of urate crystals and neutrophils in the presence of 10 micrograms/ml LDL resulted in 83% inhibition. In contrast, CL responses to a chemotactic hexapeptide, opsonized zymosan, and Staphylococcus aureus were not inhibited by LDL. The effects of depletion of apo B lipoproteins on plasma suppression of urate crystal-induced CL appeared to be unique. Plasma or sera depleted of other urate crystal-binding proteins including fibrinogen, fibronectin, C1q, and IgG retained virtually all their CL inhibitory activity. Lipoproteins containing apo B are thus a major regulator of neutrophil responses to urate crystals. These lipoproteins are present in variable concentration in synovial fluid and may exert an important influence on the course of gout.

Using 2-dimensional O'Farrell gel electrophoresis, we have mapped the proteins from undiluted plasma and serum which bind to monosodium urate (MSU) crystals. More than 30 crystal-associated polypeptides were visualized, including anionic and cationic species. Proteins increased on the crystals relative to plasma included C1q, C1-r, C1-s, fibronectin, fibrinogen, and kininogen. Crystal-bound polypeptides derived from IgG, albumin, and transferrin were recovered in decreased amounts relative to plasma. Direct evidence for activation of the complement and coagulation systems in plasma was provided by the identification of crystal-associated activation fragments of C1 and kininogen. Plasmas deficient in selected proteins (e.g., C1q and IgG) were used to define the role of these proteins in such activation events and confirmed activation of C1 in immunoglobulin-deficient plasma by MSU crystals. In summary, we have described a high resolution, semiquantitative approach to analyze protein binding to crystals, have documented the complexity of crystal-plasma protein interaction, and have provided direct evidence for the binding of coagulation system proteins and binding and activation of complement by MSU crystals, in whole plasma and IgG-deficient plasma.

The effects of protein absorption to monosodium urate monohydrate (MSU) crystals on crystal-induced lysosomal enzyme release from human neutrophils were studied. Native crystals produced prompt release of both lysosomal and cytoplasmic enzymes, suggesting that they are directly membranolytic for the neutrophil. When albumin, IgM, or beta-lactoglobulin were adsorbed to MSU crystals, lysis was blocked and lysosomal enzyme release was negligible. Prior incubation of crystals with 20% human serum or adsorption of IgG resulted in inhibition of lysis but enhancement of lysosomal enzyme liberation. This non-cytolytic lysosomal enzyme release induced by IgG-coated crystals may be important in the pathogenesis of acute gouty arthritis.

The mechanisms of urate-crystal-induced release of platelet constituents has been studied morphologically and biochemically. Urate crystals provoked an early energy-dependent release of the dense-body constituents serotonin, ADP, and ATP from washed platelets. Concurrently, platelet ultrastructure showed evidence of shape change, contractile wave, and aggregation. These are typical morphologic concomitants of platelet secretion. By 30 minutes' incubation, urate-induced platelet lysis occurred, as shown by loss of the cytoplasmic enzyme lactic dehydrogenase (LDH) and ultrastructurally by disruption of platelet membrane integrity. Cytochalasin B inhibited the urate-crystal-induced shape change, aggregation, and disruption of cell membranes. Platelet degranulation was not inhibited and the initial component of serotonin release was not affected. Cytochalasin B also abrogated crystal-induced LDH loss. Thus, the initial crystal-induced serotonin release does not depend on platelet lysis. It is concluded that urate-crystal--induced release of serotonin, ATP, and ADP represents an example of platelet secretion.

Monosodium urate crystals (MSU) stimulate suspensions of washed platelets or neutrophils. When MSU crystals are coated with IgG, as occurs in plasma, stimulation is markedly enhanced. These studies which use MSU-induced human platelet serotonin secretion as a model examine the nature of cellular recognition mechanisms for MSU crystals and IgG-coated MSU crystals. F(ab')2 fragments of specific anti-Fc antibody blocked and the lipopolysaccharide of Salmonella minnesota R595 enhanced human platelet secretion induced by IgG–coated urate crystals. These agents had little effect on stimulation by uncoated crystals. This indicated that urate crystals stimulate platelets independently of fluid phase IgG. Urate crystals directly stimulated suspensions of washed rabbit platelets which lack Fc receptors. In contrast to human cells, stimulation was blocked by IgG. This again demonstrated IgG-independent cell stimulation by urate crystals. Calcium pyrophosphate dihydrate crystals could trigger human platelet secretion only when coated with IgG. This suggests that when crystals are coated with IgG, the surface-bound IgG alone may be the stimulus to the cell. This was supported by the finding that polyvinylpyridine-N-oxide, a hydrogen acceptor, blocked human platelet stimulation by uncoated, but not IgG-coated, urate crystals. These data indicate that urate crystals (and potentially other surface or particles) can stimulate a mediator cell by at least two mechanisms: by direct stimulation without the mediation of adsorbed IgG or, when coated with IgG, by triggering the cell via immunoglobulin receptors.

The release of human platelet constituents by the etiologic agent of gout, the monosodium urate crystal, is described here. In suspensions of washed platelets, response to urate crystals proceeded in two phases: A secretory phase involved the rapid active release of serotonin, ATP, and ADP with little loss of lactic dehydrogenase or beta-glucuronidase. A lytic phase involved the slower loss of all platelet constituents. Both phases were inhibited by iodoacetate plus dinitrophenol, suggesting an energy requirement. In ultrastructural studies, lysis of washed platelets which appeared to contain crystals was seen. Urate crystals were also shown to induce serotonin release and platelet lysis in citrated platelet-rich plasma. Since urate crystals are deposited at a variety of sites, urate crystal-platelet interaction in vivo is a possibility. Such interactions, leading to release of platelet constituents, might contribute to gouty inflammation or to enhanced atherogenesis.