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Adjuvant arthritis (AA) is an accepted model of inflammatory arthritis. Until now, however, there is little information about inflammatory mediators, specifically in relation to the arachidonic acid cascade in AA. Our objective was to study the expression of secretory (sPLA2) and cytosolic (cPLA2) phospholipases A2 in various organs during the course of AA. AA was induced in Lewis rats which were sacrificed at days 0, 7, 14, 21, 28 and 42. Expression of sPLA2 mRNA and protein and mRNA of cPLA2 in paws, regional lymph nodes, spleen, liver, lungs and aorta was investigated. Serum sPLA2 activity increased from 15213 ± 1131 to a maximum of 32455 ± 4109 nmol/30′ on day 21. Maximal increase in sPLA2 mRNA in paws, lung and aorta was observed on day 14, and in the lymph nodes and spleen on day 28. In the liver, trace levels were found with no corresponding protein expression. In paws, lung, aorta and lymph nodes maximum increase in sPLA2 protein was noted on day 14 whereas the spleen showed constant sPLA2 protein level during AA. cPLA2 mRNA detected in all organs, did not significantly change during the course of AA, with the exception of regional lymph nodes where the message increased between 14 and 28 day. Induction of mRNA and protein of sPLA2 in several organs is an evidence that AA is a systemic inflammatory process. The parallelity of the sPLA2 expression to the severity of inflammatory process, implies that sPLA2 may play pathogenic role in AA. Lack of enhancement of cPLA2 mRNA may mean that this enzyme is either not induced in AA, or it increases earlier in the course of the inflammatory process.

Some clinical manifestations of severe malaria resemble those of sepsis and there may be mediators of the host response that are common to both sepsis and malaria. Phospholipase A2 (PLA2), a proinflammatory enzyme whose expression is induced by tumor necrosis factor (TNF), has been implicated in the pathogenesis of complications of the sepsis syndrome. We examined levels of circulating PLA2 in Plasmodium falciparum malaria and studied the association of PLA2 with disease severity. Plasma PLA2 and TNF were measured in 75 Malawian children with P. falciparum malaria. The mean (SD) plasma PLA2 activity in children with acute malaria was 53,804 (37,256) units/ml as compared with 424 (349) units/ml in 34 healthy controls (P < 0.00001). The mean PLA2 activity in 45 convalescent patients was 2,546 (7,372) units/ml (P < 0.00001). In 48 patients with pretreatment PLA2 activity less than 60,000 units/ml, mortality was 8.3%, while in 27 patients with pretreatment PLA2 levels greater than 60,000 units/ml, mortality was 33.3% (P = 0.008). There were significant correlations between PLA2 and TNF (r = 0.471, P < 0.01), density of parasitemia (r = 0.443, P < 0.0001) and a decrease in hematocrit (r = 0.352, P < 0.005). These data show that P. falciparum malaria is associated with a markedly increased circulating PLA2, especially in patients with severe disease, as manifested by high parasite burden, anemia, coma, and death.

Interleukin-1 (IL-1) may be an important mediator of bone remodeling, since it is a potent stimulator of bone resorption and has biphasic effects on bone formation. Continuous exposure of fetal rat calvaria (RC) cells to IL-la or IL-1β results in a dose-dependent inhibition of both bone nodule formation and mineralization of the organic matrix. In this study, the effects of recombinant human IL-1α on the mineralization process were examined by the addition of IL-la late in the culture period, after osteoid nodules had formed and when they were induced to mineralize by the addition of organic phosphate. By means of a quantitative 45calcium radiolabeling assay, it was shown that short-duration exposures of fully-formed bone nodules to IL-la also inhibited mineralization, and that the duration of treatment directly correlated with the degree of inhibition. Because our earlier studies had demonstrated that IL-1 stimulated the release of PLA2 and PGE2 from RC cells, the effects of PLA2 and of inhibition of PGE2 synthesis on mineralization were investigated. Exogenous Naja naja group I PLA2 had little effect on the mineralization of bone nodules; however, Crotalus adamanteus group II PLA2 inhibited mineralization at concentrations similar to those found in the media from IL-la-treated cultures. Although PLA2 is thought to stimulate PGE2 synthesis by releasing arachidonic acid from membrane phospholipids, PGE2 release by RC cells accounted for only part of the IL-la-mediated inhibition, suggesting the presence of other mechanisms of exogenous PLA2 action in inhibiting mineralization. These results suggest that the IL-1-induced synthesis and release of PLA 2 from RC cells is primarily responsible for the inhibition ofmineralization seen in vitro with exposure to IL-1. Thus, the presence of IL-la in vivo at sites of chronic inflammation or infection may compromise tissue repair by inhibiting bone formation and mineralization as well as stimulating bone resorption.

To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B- cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline- encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti- I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.

Human groups IIA, V and X secretory phospholipases A2 (sPLA2s) were incubated with human HDL3, total HDL and LDL over a range of enzyme and substrate concentrations and exposure times. The residual phosphatidylcholines (PtdChos) were assayed by high performance liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The enzymes varied markedly in their rates of hydrolysis of the different molecular species and in the production of lysoPtdCho. The sPLA2s were compared at a concentration of 1 microg/ml and an incubation time of 4 h, when all three enzymes showed significant activity. The groups V and X sPLA2 were up to 20 times more reactive than group IIA sPLA2. Group X sPLA2 hydrolyzed arachidonate and linoleate containing species preferentially, while group V hydrolyzed the linoleates in preference to polyunsaturates. In all instances, the arachidonoyl and linoleoyl palmitates were hydrolyzed in preference to the corresponding stearates by group X sPLA2. The group IIA enzyme appeared to hydrolyze randomly all diacyl molecular species. The minor alkylacyl and alkenylacyl glycerophosphocholines (GroPChos) were poor substrates for groups V and X sPLA2s and these phospholipids tended to accumulate. The present study demonstrates a preferential release of arachidonate from plasma lipoprotein PtdCho by group X sPLA2, as well as a relative resistance of polyunsaturated PtdChos to hydrolysis by group V enzyme, which had not been previously documented. The use of lipoprotein PtdCho as substrate with LC/ESI-MS identification of hydrolyzed molecular species eliminates much of the uncertainty about sPLA2 specificity arising from past analyses of fatty acid release from unknown or ill-defined sources.

Background Inflammatory vascular changes and enhanced atheromatosis are well known in rheumatic diseases. Mitogenesis of vascular smooth muscle cells (VSMC) in above conditions is of prime importance. Secretory phospholipase A 2 (sPLA 2 ), which is known to induce mitogenesis, was found in and cloned from the synovial fluid (1,2). High serum levels of sPLA 2 were found in various rheumatic diseases (3-6). sPLA 2 was found in synovial cells (7) human cartilage (8) and chondrocytes (9). Pro-inflammatory effect of sPLA 2 on joint tissue was reported (10). Objectives To investigate the role of sPLA 2 IIA, V and X on mitogenesis of VSMC, release of PGE 2 and LTB 4 and effect on lipoproteins. Furthermore, the effects of enzyme hydrolysis of pro-inflammatory products of Oxo-PtdCho were investigated. Methods Cultures of VSMC, study of mitogenesis, origins of sPLA 2 9s and lipoproteins were reported (11). The analyses of the products of PtdCho oxidation, such as core aldehydes, isoprostanes, hydroxides and hydroxyperoxides were done by LC/ESI-MS.PGE 2 and LTB 4 release were tested by EIA systems. Results Exposure of VSMC to sPLA 2 IIA or X did not increase mitogenesis, whereas exposure to grV markedly increased it in a dose/time fashion. Likewise, VSMC exposed to HDL or LDL hydrolysed by sPLA 2 V (and less by X) became mitogenic. Interaction of sPLA 2 with lipoproteins showed that hydrolysis of HDL and LDL by sPLA 2 9s and the impact on mitogenesis were invariably enhanced in order V>X>IIA. Release of PGE 2 was enhanced by sPLA 2 X and LTB 4 by gr X and V. Investigation of the products of lipoproteins hydrolysis showed that there is formation of core aldehydes, isoprostanes, hydroxides and hydroperoxides of PtdCho. sPLA 2 grV hydrolysed hydroxides and hydroxyperoxides of linoleolyl GroPCho in preference to arachidonoyl GroPCho while groups IIA and X did the opposite. Conclusions The above studies show diverse activity of sPLA 2 9s gr IIA, V and X on mitogenesis of VSMC (early activation of inflammation) and on release of PGE 2 and LTB 4 from VSMC (late phase of proinflammatory activity). Further study showed differences between the effects of sPLA 2 V as opposed to IIA and X on mitogenesis and on hydrolysis of lipoproteins, showing the complexity of mitogenic proinflammatory response. Further studies directed to block sPLA 2 9s especially V in inflammation and enhanced atherogenesis in rheumatic diseases is advisable. References Stefanski E. et. al. J. Biochem.100:1297, 1986. Seilhamer J.J. et al. J. Biol. Chem. 264:5335, 1981. Pruzanski W. et al. J. Rheumat. 15:1601, 1988. J. Rheumat. 18:117, 1991. J. Rheumat. 21:1951, 1994. J. Rheumat. 21:252, 1994. J. Rheumat. 15:791, 1988. J. Rheumat. 17:569, 1990. J. Rheumat. 17:1386, 1990. J. Rheumat. 13:1990, 1986. Lab. Invest. 81:757, 2001. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.1520

Hepatitis G virus (HGV) has been identified as a new member of the Flaviridae family, which includes the hepatitis C virus (HCV). There is a well established association between HCV and cryoglobulinemia; however, to date HGV has not been linked with various types of cryoglobulinemia. We investigated the association of HGV with cryoglobulinemia.We studied 10 patients with cryoglobulinemia. The cryoglobulins were purified and identified by immunofixation electrophoresis. HGV and HCV RNA were studied in the serum and in purified cryoglobulins by reverse transcriptase polymerase chain reaction.Nine of 10 patients were women, aged 37-74 years. These patients had combinations of hepatitis C (6), vasculitis (7), lymphoproliferative (3), and autoimmune and connective tissue diseases (3). Of the 10 patients, 3 were positive for both HGV RNA and HCV RNA. Two of three patients with dual infection of HGV and HCV had malignancies (Waldenstrom's macroglobulinemia, B cell lymphoma). In one of these 3 patients HGV RNA was positive in the cryoglobulin fraction, but not in serum. Three other patients were positive for HCV RNA alone.HGV may be associated with cryoglobulins. Our three patients were co-infected with HCV. Since our series is small, the pathogenetic role of hepatitis G and its relationship to malignancy remain to be elucidated.

Adjuvant arthritis (AA) is an accepted model of inflammatory arthritis. Until now, however, there is little information about inflammatory mediators, specifically in relation to the arachidonic acid cascade in AA. Our objective was to study the expression of secretory (sPLA2) and cytosolic (cPLA2) phospholipases A2 in various organs during the course of AA. AA was induced in Lewis rats which were sacrificed at days 0, 7, 14, 21, 28 and 42. Expression of sPLA2 mRNA and protein and mRNA of cPLA2 in paws, regional lymph nodes, spleen, liver, lungs and aorta was investigated. Serum sPLA2 activity increased from 15213 +/- 1131 to a maximum of 32,455 +/- 4109 nmol/30' on day 21. Maximal increase in sPLA2 mRNA in paws, lung and aorta was observed on day 14, and in the lymph nodes and spleen on day 28. In the liver, trace levels were found with no corresponding protein expression. In paws, lung, aorta and lymph nodes maximum increase in sPLA2 protein was noted on day 14 whereas the spleen showed constant sPLA2 protein level during AA. cPLA2 mRNA detected in all organs, did not significantly change during the course of AA, with the exception of regional lymph nodes where the message increased between 14 and 28 day. Induction of mRNA and protein of sPLA2 in several organs is an evidence that AA is a systemic inflammatory process. The parallelity of the sPLA2 expression to the severity of inflammatory process, implies that sPLA2 may play pathogenic role in AA. Lack of enhancement of cPLA2 mRNA may mean that this enzyme is either not induced in AA, or it increases earlier in the course of the inflammatory process.

Sulfasalazine is widely used in rheumatoid arthritis and inflammatory bowel diseases. The mechanisms of its activity have not been elucidated. In leukocytes, sulfasalazine and its analogue, CL 42A, inhibited the formation of leukotrienes and possibly of the second messenger compounds at the level of phospholipase C. Partial inhibition of interleukin-lbeta (IL-1beta), IL-6 and tumor necrosis factor-alpha (TNF-alpha) was also found. Since the synthesis of eicosanoids is induced by phospholipase A2 and since secretory phospholipase A2 (sPLA2) is proinflammatory, we investigated the impact of sulfasalazine and related compounds on mRNA, protein synthesis, and release of sPLA2 from osteoblasts. Sulfasalazine and CL 42A markedly inhibited extracellular release of sPLA2. The impact of sulfasalazine was evident at 50 microM (P0.001) and maximal at 400 microM, and that of CL 42A at 10 microM (P0.001) and 200 microM, respectively. Split products of sulfasalazine, 5-aminosalicylic acid (400 microM) and sulfapyridine (400 microM), had no impact. The effect of sulfasalazine and CL 42A was evident regardless of whether the cells were stimulated with IL-1beta/TNF-alpha, lipopolysaccharide/forskolin, or dibutyryl-cAMP. Sulfasalazine and CL 42A did not alter the level of sPLA2 mRNA. Exposure of stimulated fetal rat calvaria osteoblasts (FRCO) to sulfasalazine did not show accumulation of the intracellular sPLA2 protein as tested by western blot; however, enzymatic activity of PLA2 in disrupted cells was definitely increased. Thus, the impact is on the post-transcriptional release of sPLA2 rather than on the synthesis. There was also an increase in the extracellular release of prostaglandin E2 from FRCO exposed to sulfasalazine or to CL 42A. In contrast, sulfasalazine had no effect on the extracellular release of gelatinase from the cells or on mRNA of cytosolic PLA2 or cyclooxygenase 2. We conclude that the anti-inflammatory activity of sulfasalazine may be related, in part, to the selective inhibition of the extracellular release of proinflammatory sPLA2.

There are numerous reports of hypersensitivity reactions to corticosteroids. However, cases of anaphylactic shock after intraarticular injection of corticosteroids are exceedingly rare. We describe a case of anaphylaxis in a 31-year-old woman after intraarticular injection of synthetic methylprednisolone acetate. Immediately after injection she developed sneezing, angioedema, tachycardia, and marked hypotension. She responded promptly to treatment with subcutaneous epinephrine. She had received uneventfully one intraarticular injection of the same compound 4 years earlier. Intradermal skin testing showed strong reactivity to methylprednisolone acetate suspension, moderate reactivity to hydrocortisone, and weak reactivity to betamethasone. Tests with dexamethasone, triamcinolone, lidocaine, latex and nonsteroid constituents of the injected suspension including polyethylene glycol, polysorbate 80, mono and dibasic sodium phosphate, and myristyl-gamma-picolinium chloride were negative. This patient had developed anaphylaxis due to methylprednisolone acetate alone. Although such events are very rare, it is advisable to keep injectable epinephrine in the offices of rheumatologists.

Microtubule depolymerizing agents (MTD) diminish the expression of cell surface receptors for TNF-alpha. Because TNF-alpha along with IL-1 beta markedly enhance the gene expression and extracellular release of proinflammatory secretory nonpancreatic phospholipase A2 (sPLA2), we tested the impact of MTD on the expression of sPLA2. We report that MTD markedly inhibit the expression and release of sPLA2 by fetal rat calvarial osteoblasts (FRCO), which synthesize and release sPLA2. When FRCO were pretreated with colchicine and then stimulated with IL-1 beta 0.2 ng/ml and TNF-alpha 25 ng/ml (IL-1/TNF), minute quantities of colchicine (1.25 nM) reduced the released sPLA2 activity to 11% of that in controls. IC50 was 0.75 nM. When IL-1/TNF and colchicine were added simultaneously, similar inhibition (8% of that in controls) required higher concentrations of colchicine (0.125 microM). IC50 was 68.75 nM. When FRCO were prestimulated by IL-1/TNF, much higher concentrations of colchicine were required to reduce sPLA2 activity. MTD inhibited the expression of sPLA2 by a mechanism(s) different from the way in which they impact TNF surface receptors, because they inhibited sPLA2 expression in FRCO stimulated by IL-1 beta or by cell-permeable cAMP analogs. Colchicine (1 microM) reduced the expression of sPLA2 induced by dibutyryl cAMP (2 mM) and 8-bromo-cAMP (4 mM) to 38% and 58% of that n controls, respectively. Photoinactivated lumicolchicines beta and gamma were noninhibitory. Microtubular stabilizer taxol (5 microM) abolished inhibitory activity of colchicine, increasing the expression of sPLA2 3.2-fold compared with that in control cells cultured without taxol. Other MTD, such as vinblastine (0.01 microM), inhibited sPLA2 release to 27% of the controls, whereas nocodazole (10 microM) was less inhibitory. Northern blot analysis of FRCO showed that sPLA2 mRNA was greatly induced by IL-1/TNF. The induction of sPLA2 mRNA by IL-1/TNF was nearly completely abolished by colchicine in a dose-related manner. Western blot analysis of intra- and extracellular sPLA2 protein showed complete inhibition of the synthesis by MTD. To determine whether the inhibition of sPLA2 is selective, mRNA levels of cytosolic PLA2 and of inducible cyclooxygenase-2 were investigated. Colchicine had no effect on the mRNA levels of these two enzymes, which suggests that the inhibitory effect of MTD on sPLA2 expression is selective and occurs at the transcriptional level. Thus, the microtubular system plays a significant role in the synthesis of proinflammatory sPLA2, a fact that may explain in part the anti-inflammatory activity of microtubular disrupters.

A limited retrospective study of patients with rheumatoid arthritis (RA) found that serum secreted phospholipase A2 (sPLA2) activity correlates with disease activity (J Rheumatol 1988; 15:1351-5). To assess the strength of this relationship we investigated prospectively 212 patients with RA using a double blind approach.212 patients who fulfilled the 1987 ACR criteria for RA had 420 clinical and laboratory assessments. 65 patients were assessed on one occasion and 147 on multiple occasions (a mean of 2.41 visits/patient). sPLA2 was tested by an independent investigator.sPLA2 activity assessed as a dichotomous variable (less or more than mean +/- 2 SD) correlated highly (p0.005) with Lansbury index, number of effusions, number of damaged joints, erythrocyte sedimentation rate (ESR), platelet count, and low hemoglobin. Univariate and multivariate regression analyses showed significant correlations with Lansbury index, active and effused joints, hemoglobin, platelet count, and ESR. The best correlation was observed in a multivariate model that included Lansbury index, ESR, and platelet count (r = 0.60). Analysis of longitudinal changes in sPLA2 activity in 147 patients assessed more than once showed that sPLA2 correlates significantly with Lansbury index, active and effused joints, and hemoglobin.Serum sPLA2 activity correlates significantly with Lansbury index, active and effused joints, ESR, platelet court, and hemoglobin. Thus, sPLA2 can serve as an index of disease activity in RA.

To test the effect of infusions of CAMPATH-1H on levels of proinflammatory secretory phospholipase A2 (sPLA2) and tumor necrosis factor alpha (TNF-alpha) in patients with rheumatoid arthritis (RA).Two patients with RA were infused with CAMPATH-1H; extracellular nonpancreatic sPLA2 activity was tested using radiolabelled E. coli membrane phospholipid, and circulating TNF-alpha levels were tested by ultrasensitive immunoassay.Circulating TNF-alpha began to rise within the first 2 h after infusion, reaching1000-fold values compared to preinfusion levels. Circulating sPLA2 activity began to rise a few hours after the start of infusion and reached extremely high values in 12 h, concomitant with fever and hypotension. The activity of sPLA2 decreased to pretreatment values in 3-18 days after infusion.The mechanism leading to the increase of TNF-alpha and hyperphospholipasemia A2 has not been elucidated. It is possible that CAMPATH-1H activates cells that synthesize and release TNF-alpha and sPLA2, and/or that it induces interleukin 2 release, which in turn activates TNF-alpha, with subsequent release of sPLA2.

Secretory nonpancreatic phospholipase A2 (snpPLA2) is a proinflammatory enzyme and its activity in serum correlates with disease activity in adults with rheumatoid arthritis. Juvenile rheumatoid arthritis (JRA) may be stratified into 3 clinical types with differing degrees of disease activity. Since in JRA there are no reliable indices of disease activity, our objective was to find whether the level of circulating snpPLA2 correlates with the severity of inflammation and with JRA activity.PLA2 enzymatic activity was assayed using E. coli membranes labelled with (14C)-oleic acid. SnpPLA2 immunoreactivity was tested by ELISA technique using monoclonal antibodies against recombinant human (rh) snpPLA2. SnpPLA2 activity was determined in sera of 127 children including 25 with systemic (S-JRA), 50 with polyarticular (Po-JRA) and 52 with pauciarticular (Pa-JRA) types of JRA. Twenty-five patients with active disease, were subsequently restudied in an inactive phase.Markedly increased snpPLA2 (mean + 2 SD of normal mean, i.e.,575 U/ml) was found during the active disease in 100% S-JRA, 57% Po-JRA and 25% Pa-JRA patients. The differences in the mean and median PLA2 activity among these 3 subtypes of JRA were highly significant (p0.001) with the highest levels found in S-JRA and the lowest in Pa-JRA. Presence of rheumatoid factor and/or of antinuclear antibody had no relation to the level of snpPLA2. SnpPLA2 activity became markedly lower when active inflammation became quiescent. In the whole group, snpPLA2 activity correlated highly with the Lansbury index, number of involved joints and number of effusions. A significant positive correlation was also found between snpPLA2 and erythrocyte sedimentation rate (ESR) and neutrophil count, while a significant negative correlation was noted with the level of albumin and hemoglobin. With the exception of snpPLA2, other laboratory variables did not correlate with the number of effusions or the number of active joints. However a negative correlation was noted between both hemoglobin and albumin, and Lansbury index.Circulating snpPLA2 significantly correlates with JRA activity and may serve as an index of activity in JRA especially in patients with systemic type of disease.

Human non-pancreatic PLA2 has been the object of intense scrutiny for a relatively short period of time. Its role in physiology remains enigmatic. While PLA2 may serve to remodel or remove peroxidised or senescent phospholipids, the enormous magnitude of its upregulation during infectious or inflammatory episodes is consistent with a role in host defense. However, the nature of this role remains elusive. Attempts to relegate this enzyme to the genre of acute phase reactants have not been helpful in unravelling its role. Difficulty in obtaining adequate amounts of native snp-PLA2 prior to the availability of recombinant snp-PLA2 led to the widespread use of snake venom homologs, particularly in studies of the biology of PLA2. This review has underscored the pitfalls inherent in that approach given the major differences between some venom PLA2s as compared to snp-PLA2. In addition, it bears reiterating that the complex composition of venom allows for potentiation of PLA2 activity by other constituents present in venom. Whether human host defense networks employ this interactive strategy is largely unknown. Nonetheless, in spite of these reservations, some very compelling data have emerged in recent years implicating snp-PLA2 in the initiation or potentiation of local and systemic inflammatory processes. These include sepsis and associated acute lung injury as well as inflammatory arthritides, with rheumatoid arthritis as the prototype. The mechanisms of snp-PLA2 homeostasis are considerably better understood, and it has become apparent that snp-PLA2 is an integral part of a larger network of proinflammatory cytokines, growth factors and lipid mediators. The interrelationship between the functions of secretory and cytosolic PLA2s remains to be defined. A number of selective PLA2 inhibitors have been identified which will allow for discrimination between the actions of these classes of PLA2. The availability of synthetic inhibitors in conjunction with endogenous modulators of PLA2s will shift the biology of PLA2 from the realm of the inferential to that of the mechanistic.

Phospholipase A2 catalysed hydrolysis of phospholipid substrates is rate-limiting in the release of arachidonic acid for subsequent downstream metabolism to biologically active eicosanoids. Concomitant release of lysoplatelet activating factor (lyso PAF) serves as the precursor of PAF. Thus, phospholipase A2 (PLA2) is pivotal in the generation of a spectrum of biologically active lipids. Over the course of the past decade, studies have increasingly pointed to a pathogenetic association between endotoxin-induced PLA2 expression and ensuing circulatory shock and multisystem organ failure in animals with experimental endotoxemia as well as in patients with septic shock. The structural, functional, regulatory and biologic characteristics of PLA2 are reviewed in relation to septic shock and its complications and areas of controversy are highlighted.

Circulating phospholipase A2 (PLA2) has been recognized as a mediator of cardiovascular collapse in septic shock. Proximal mediators of endotoxemia, including tumor necrosis factor and interleukin 1, induce PLA2 synthesis and release, but the factors regulating PLA2 elimination are unknown. Similarly, the kinetics of PLA2 clearance during recovery from septic shock have not been examined. An autoregressive mathematical model was developed to describe the rate of PLA2 clearance during the recovery phase of septic shock. This model (which estimates that the current day's PLA2 level is 77% of the previous day's level), accounted for 89% of the variability seen in the data. The estimated circulating half-life of soluble PLA2 in septic shock in man was 32 hours. Since elevation in serum PLA2 activity is closely associated with bacteremia or endotoxemia, a significant deviation from predicted PLA2 values may denote impending relapse.

Opportunistic infections occur in patients with rheumatic diseases treated with low dose methotrexate (MTX) with or without other immunosuppressants. Our case report illustrates a fatal case of Pneumocystis carinii pneumonia in a patient with rheumatoid arthritis treated with low dose MTX and glucocorticoid. A review of the literature reveals other opportunistic infections such as Cryptococcus, Nocardia, and herpes zoster presenting in such patients. These occurrences suggest that MTX should be used cautiously in patients with rheumatic disease receiving concomitant medical therapy.

On the basis of the observation that serum levels of phospholipase A2 (PLA2) are elevated in pancreatitis and systemic sepsis, and the association of these conditions with the subsequent development of acute lung injury, the present investigation examined the structural and physiologic consequences of intratracheal administration of PLA2 to adult male rats. Rats received direct intratracheal instillation of either control vehicle or 40,000 units/kg of PLA2 repurified from Naja naja venom. Animals treated with PLA2 showed higher cumulative mortality (33% versus 0%, n = 79; p less than 0.01) than did their control littermates. The PLA2-treated animals showed histologic evidence of acute lung injury characterized by interstitial and alveolar edema, accumulation of inflammatory cells, and alveolar wall thickening, which reached maximal severity 48 h after enzyme instillation. Forty-eight hours after PLA2 administration experimental animals had lower arterial oxygen tensions (73.9 +/- 7.66 mm Hg versus 96.7 +/- 2.52 mm Hg, mean +/- SEM; p less than 0.01), higher alveolar-arterial oxygen gradients (35.3 +/- 6.3 mm Hg versus 18.8 +/- 1.42 mm Hg, p less than 0.01), and higher wet-dry lung weight ratios (5.08 +/- 0.26, mean +/- SEM, n = 7 versus 3.29 +/- 0.08, n = 3; p less than 0.002) than did control animals. Lung lavage from experimental animals 48 h after PLA2 instillation showed increased total cell counts [(26.6 +/- 5.04) x 10(6) cells versus (4.69 +/- 1.48) x 10(6) cells; p less than 0.01], an increased percentage of neutrophils (34.2 +/- 4.6% versus 1.25 +/- 0.25%, mean +/- SEM; p less than 0.01), and increased protein concentrations in lavage fluid (0.38 +/- 0.06 mg/ml, mean +/- SEM, n = 4 versus 0.27 +/- 0.02 mg/ml, n = 5; p less than 0.05). The histologic and physiologic abnormalities had largely resolved by 240 h. These results suggest that PLA2 may be a potent mediator of lung inflammation and that intratracheal administration of PLA2 to adult rats may provide a useful experimental model of acute lung injury.

High activity of phospholipase A2 (PLA2) was found in 58 synovial fluids of patients with osteoarthritis (OA), with a range of 2.2-75.1 nmol/min/ml and mean +/- SEM of 22.0 +/- 1.4 nmol/min/ml. This PLA2 was a calcium dependent enzyme composed of 2 isoforms called A and B. Twelve of 25 patients with OA (48%) were found to have high circulating PLA2, but no correlation to the numbers or size of affected joints was found. High concentrations of PLA2 were detected in human articular cartilage ranging from 33 to 4257 nmol/min/mg protein. The deep layers of the cartilage contained on average 3-fold more PLA2 than the superficial layers. Also synovial cells cultures and chondrocyte cultures that derived from OA joints synthesized and released PLA2 extracellularly. Since PLA2 is proinflammatory, its role in the inflammation that complicates OA process is highly probable. Further studies of PLA2 impact on OA joints and clinical trials with PLA2 inhibitors in OA are warranted.

High activity of proinflammatory, type II phospholipase A2 (PLA2) was found in synovial fluids (SF) in inflammatory arthritis. In search for the sources of this PLA2, we cultured human articular chondrocytes and cartilage explants from healthy, osteoarthritic and rheumatoid joints. All cultures, unstimulated by cytokines, released PLA2 extracellularly. Cultures obtained from the deep layers of the cartilage released more PLA2 than those obtained from the superficial layers. Deep layer explants released 0.38 to 18.16 pmol/min/mg protein PLA2/day, whereas superficial layer explants released 0.39-3.18 pmol/min/mg/day. Chondrocyte cell cultures continuously released PLA2, in the first day 909-46347 pmol/min/(10)6 cells and after 9-26 days of culture 166-2115 pmol/min/10(6) cells. PLA2 released from chondrocytes was calcium dependent and had optimum activity at pH 7.5. Cycloheximide markedly inhibited its release. Chondrocyte cultures also released muramidase (LZM) but there was no correlation between PLA2 and LZM release. It may be concluded that cytokine unstimulated human articular chondrocytes synthesize and release PLA2 extracellularly which is similar to that found in the SF. Thus, chondrocytes may possibly serve as one of the sources of intraarticular PLA2.

Phospholipase A2 (PLA2), total and pancreatic, were quantitated in 91 sera of patients with systemic sclerosis (SSc). The mean total PLA2 of 216 +/- 161 U/ml (SD) was significantly lower (p less than 0.001) than in controls (317 +/- 128 U/ml). In 55 of 91 patients (60%) PLA2 was more than 1 SD and in 6 (7%) more than 2 SD below the normal mean. Serum pancreatic PLA2 was also significantly lower in SSc. The prevalence of low serum total PLA2 was significantly greater (p less than 0.001) than in healthy adults, or in patients with rheumatoid arthritis, systemic lupus erythematosus or vasculitis. Repeat assays of PLA2 activity in 10 patients with SSc documented persistence of low PLA2. Among 76 patients with SSc with complete clinical and laboratory assessment, there were 48 with low and 28 with normal (or slightly elevated) PLA2. These 2 groups showed no differences in disease manifestation or therapy. The group with low serum PLA2 had lower erythrocyte sedimentation rates (p less than 0.0005) and lower neutrophil (p less than 0.05) and monocyte counts (p less than 0.025) in the peripheral blood. The finding of low serum PLA2 activity adds to the spectrum of arachidonic acid pathway abnormalities associated with SSc, and may in part be related to the paucity of inflammatory changes observed in this disease.

Our understanding of the mechanisms of initiation and propagation of local and systemic inflammatory processes is clearly imperfect if one uses the available therapeutic modalities as a yardstick. While glucocorticoids are potent anti-inflammatory drugs, the pharmacologic target of this class of agents has not been identified with certainty, and the use of steroids is fraught with the risk of considerable and potentially dangerous side effects. On the other hand, non-steroidal anti-inflammatory drugs (NSAIDS), while more specific, are relatively weak anti-inflammatory compounds and frequently require the addition of more potent agents. Cytotoxic drugs or anti-metabolites effectively suppress acute and chronic inflammatory reactions, but also predispose to infection and initiate the development of neoplasms following long-term exposure. The inadequacy and relative non-specificity of these approaches underscore the deficiencies in our understanding of the principles that govern these responses. A better understanding of these processes will be applicable to broad categories of human disease including autoimmunity, the collagen vascular diseases, aberrations in host defense and the response to trauma and infection.

Mammals are now known to contain at least two distinct classes of phospholipases A2, the progenitors of which can be seen in the venoms of snakes. Mammalian "Type I" PLA2, synthesized primarily by the pancreas, is also present in smaller amounts in other tissues including lung, spleen, and kidney. Recently, a mammalian "Type II" PLA2 has been sequenced, and shown to occur in platelets, synovial cells and fluid, cells of inflammatory peritoneal exudate, liver, intestine, kidney, and placenta. This form, referred to here as Type IIA PLA2, could play a key role in arachidonate release in both normal and pathologic inflammation. The genes encoding both forms have also been recently cloned. Here, the sites of synthesis and respective roles of the two known enzymes are discussed, along with an analysis of the evolutionary conservation of Type IIA PLA2 gene sequence. In addition, two related genetic elements containing sequences homologous to a portion of Type II PLA2 are described, which map to the same chromosome as the Type IIA PLA2 gene (chromosome 1). Either or both of these could also encode a portion of additional mammalian PLA2s.

One hundred and twenty sera with monoclonal immunoglobulin M's (IgM's) were tested for cryptic cold agglutinin activity against papain- and neuraminidase-treated adult and cord red blood cells (RBC). Fifteen sera (12.5%) agglutinated papain-treated RBC. Fourteen of these 15 sera also agglutinated neuraminidase-treated RBC. Ten additional sera agglutinated neuraminidase-treated but not papain-treated cells. Three patterns were observed among agglutinators of papain-treated RBC: either predominant agglutination of adult RBC, or of cord RBC or equal reaction, thus resembling I, i and non-Ii patterns of conventional cold agglutinins. We propose to call these patterns, tentatively, Icr (cr for cryptic), icr, and non-Iicr, respectively. Purified monoclonal IgM's retained the above patterns. Eluates from enzyme-treated cells contained monoclonal IgM's. Investigation of neuraminidase-treated RBC has shown that in distinction to T-anti-T system (Thomsen-Friedenreich receptor system), the majority of sera with monoclonal IgM's agglutinated more strongly adult RBC, and some agglutinated more strongly cord RBC. There was no loss of agglutinating activity after repeat freezing and thawing of the sera. Thus, it seems that monoclonal IgM's which react with neuraminidase-treated RBC detect antigenic determinants different from the T antigen. Cryptic CA activity of IgM's should be added to the list of antibody activities of these monoclonal immunoglobulins.

Bacteriolytic and bactericidal activity was evaluated in sera and synovial fluids of 28 patients with rheumatoid arthritis and 13 patients with osteoarthritis. An attempt was made to correlate the results with the concentrations of the total hemolytic complement, lysozyme, immunoglobulins, transferrin, and the titer of the Latex fixation. Bacteriolytic activity in all but 5 hypocomplementemic rheumatoid sera and in all osteoarthritic sera was normal. There was a correlation (P = 0.01) between bacteriolytic activity and the level of complement, but not the level of other factors or the titer of the Latex fixation. Bacteriolytic activity in the rheumatoid and osteoarthritic synovial fluids was low, being respectively four times and two times weaker than in the corresponding sera. It correlated well only with the level of complement (P < 0.01). Bactericidal activity of rheumatoid and osteoarthritic sera was normal but was very significantly decreased in the synovial fluids, being respectively 10 times weaker and four times weaker than in the corresponding sera. The only positive correlation was found between bactericidal activity and the complement level (P < 0.01). There was no correlation between the titer of the Latex fixation and the level of the total hemolytic complement either in serum or in synovial fluids. The influence of 6 antiarthritic drugs on antibacterial activity was tested by addition of drugs to the normal human serum. There was no change in bacteriolytic activity, however bacterial growth was inhibited when high concentrations of drugs were mixed with the culture medium.