Diverse activity of human secretory phospholipases A2 on the migration of human vascular smooth muscle cells.

OBJECTIVE: Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC). MATERIAL: We investigated the impact of sPLA2 IIA, V, and X and of oleic acid, linoleic acid and lysophosphatidylcholine on the migration of human VSMC. METHODS: Recombinant human sPLA2's and Boyden's chamber method were applied. RESULTS: sPLA2, IIA but not V or X enhanced migration of VSMC in a dose/time dependent manner. Oleic and linoleic acids, and lysophosphatidylcholine markedly enhanced migration. CONCLUSIONS: These results imply that sPLA2 IIA, which is known to be present in the arterial wall in the vicinity of VSMC, as well as products of lipid hydrolysis induced by sPLA2, enhance the migration of VSMC, and thus may contribute to atherogenic process.

Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2.

We investigated the hydrolysis of the minor glycerophospholipids of human HDL(3), total HDL and LDL using human group IIA, V and X secretory phospholipases A(2) (sPLA(2)s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA(2)s, and to a lesser extent by group IIA sPLA(2), the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA(2)s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA(2)s, although the group V sPLA(2) attacked it more readily than group X sPLA(2) (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA(2) hydrolysis remained minimal at 10-15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA(2) at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA(2)s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA(2) digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA(2)s. Since these sPLA(2)s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function.

Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2.

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.

Mitogenic effect of lipoproteins on human vascular smooth muscle cells: the impact of hydrolysis by gr II A phospholipase A(2).

Multifactorial interaction among lipoproteins, vascular wall cells, and inflammatory mediators has been recognized as the basis of atherogenesis. In the arterial wall high-density lipoprotein (HDL) and human secretory phospholipase A(2) (sPLA(2)) colocalize with vascular smooth muscle cells and concentrate in the atherosclerotic lesions. It has been shown that gr IIA sPLA(2) hydrolyzes lipoproteins, altering their structure and releasing active agents such as lyso-phosphatidylcholine (PtdCho) and free fatty acids. We investigated the impact of normal HDL(3) (NHDL(3)), acute phase HDL(3) (APHDL(3)), and low-density lipoprotein (LDL), both unhydrolyzed and sPLA(2)-hydrolyzed, and some products of hydrolysis, such as lyso-PtdCho, oleic and linoleic acid, on [(3)H] thymidine incorporation by DNA of cultured human vascular smooth muscle cells (VSMC). NHDL(3) markedly enhanced mitogenic activity of VSMC in a dose- and time-dependent manner. Doubling of thymidine incorporation was usually achieved by 40 microg/ml of NHDL(3) after 4 hours of incubation. APHDL(3) had invariably a stronger inducing effect on the mitogenic activity than NHDL(3); 40 microg/ml more than tripled [(3)H] thymidine incorporation after 4 hours of incubation. NHDL(3) preincubated with human apo serum amyloid A apolipoprotein-induced higher mitogenic activity in VSMC than NHDL(3) alone. Hydrolysis of NHDL(3), APHDL(3), or LDL by gr IIA sPLA(2) markedly enhanced mitogenic activity of VSMC as compared with unhydrolyzed lipoproteins. sPLA(2) concentrations that can be found in atherosclerotic vascular walls markedly enhanced lipoprotein-induced mitogenic activity of VSMC. sPLA(2) per se did not affect thymidine incorporation and VSMC did not release sPLA(2) into the medium. There was no evidence for hydrolysis of the wall of VSMC by gr IIA sPLA(2). The presence of the products of hydrolysis of lipoproteins such as oleic and linoleic acids and lyso-PtdCho or their combinations with NHDL(3) explains in part markedly enhanced mitogenic activity of VSMC. It is conceivable that sPLA(2,) which is known to colocalize with lipoproteins in the vascular wall in the domain of VSMC, is capable of induction of the mitogenic activity in these cells in vivo and should be considered as a proatherogenic enzyme.

Comparative analysis of lipid composition of normal and acute-phase high density lipoproteins.

In the acute-phase response and in diseases with prolonged acute phases, normal HDL (NHDL) is converted into acute-phase HDL (APHDL) and becomes proinflammatory and unable to protect LDL against oxidative modification. Earlier work had demonstrated that these changes are associated with alterations in apolipoprotein composition and enzymatic activity of APHDL, but the effect of the acute-phase condition on the lipid composition of APHDL had remained obscure. The present study shows marked quantitative differences in lipid composition between NHDL and APHDL. Specifically, APHDL contained 25% less total lipid per milligram of protein. Up to 50% of cholesteryl ester in the lipid core of APHDL was replaced by triacylglycerol; however, the total phospholipid/total neutral lipid ratios were the same as in NHDL, both lipoproteins giving similar calculated lipid core radii. Furthermore, the phosphatidylcholine/sphingomyelin ratio in APHDL was nearly double that in NHDL, indicating a relative loss of sphingomyelin. A decrease was also seen in diacyl and alkenylacyl glycerophosphatidylethanolamine as well as in phosphatidylinositol of APHDL when compared with NHDL. APHDL contained proportionally more saturated and less polyunsaturated and isoprostane-containing species of phosphatidylcholine, as well as more saturated than unsaturated cholesteryl esters. APHDL also contained significantly more free fatty acids, lysophosphatidylcholine, and free cholesterol. These changes in the lipid composition of HDL are consistent with the alterations in the apoprotein composition and enzymatic activity of APHDL and indicate proinflammatory and proatherogenic roles for APHDL.

Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL.

Group IIA secretory phospholipase A2 is an acute phase enzyme, co-expressed with serum amyloid A protein. Both are present in atherosclerotic lesions. We report that human normal and acute phase high density lipoproteins and low density lipoprotein are effective substrates for human group IIA phospholipase A2. The enzyme hydrolyzed choline and ethanolamine glycerophospholipids at the sn -2 position resulting in an accumulation of the corresponding lysophospholipids, including the unhydrolyzed alkyl and alkenyl ether derivatives. The hydrolysis of acute phase high density lipoprotein was 2- to 3-fold more rapid and intensive than of normal high density lipoprotein. The hydrolysis of lipoproteins was noted at enzyme concentration as low as 0.05 microgram/mg protein, which was within the range observed in the circulation in acute and chronic inflammatory diseases. The enzyme hydrolyzed the different molecular species of the residual glycerophospholipids in proportion to their mass, showing no preference for the release of arachidonic acid. Group IIA phospholipase A2 preferentially attacked the hydroxy and hydroperoxy linoleates and possibly other oxygenated fatty acids, which were released from the glycerophospholipids at early times of incubation. There was no effect on the content or molecular species composition of the sphingomyelins or neutral lipids of the lipoproteins. In conclusion, human plasma lipoproteins are the first reported natural biological substrates for human group IIA phospholipase A2. The enhanced hydrolysis of acute phase high density lipoproteins is probably due to its association with serum amyloid A protein, which enhances the activity of the enzyme and may promote its penetration to the lipid monolayer. As sPLA2-induced hydrolysis of the lipoproteins leads to accumulation of lysophosphatidylcholine and potentially toxic oxygenated fatty acids, overexpression of this enzyme may be proatherogenic.

Chemically modified non-antimicrobial tetracyclines inhibit activity of phospholipases A2.

OBJECTIVE: Tetracyclines have been recognized as useful agents for therapy of inflammatory arthritides. However, prolonged use of tetracyclines is limited by their detrimental antimicrobial properties. Recently, a group of chemically modified tetracyclines (CMT) devoid of antimicrobial properties has been synthesized. Some CMT were found to inhibit various matrix metalloproteinases (MMP). We reported previously that antimicrobial tetracyclines inhibit the activity of proinflammatory secretory group II phospholipase A2 (sPLA2). The objective of this study was to detect whether non-antimicrobial CMT also inhibit sPLA2 and other phospholipases A2. METHODS: Ten synthetic CMT were tested for inhibition of sPLA2 human and porcine PLA2, and Naja naja PLA2. PLA2 activity was assessed by radiolabeled Escherichia coli assay using standard and high calcium concentrations. RESULTS: Six of 10 CMT inhibited sPLA2 activity at concentrations close to or lower than 50 microg/ml. All 6 CMT had identical C1-3 and C10-12a positions in the 4-ringed nucleus of the tetracycline molecule. Calcium concentrations up to 20 mM did not eliminate the inhibitory activity of CMT. Inhibition of other PLA2 was induced by some CMT, all but one (CMT-9) belonging to the group of strong inhibitors of sPLA2. Thus, inhibition of PLA2 different from sPLA2 does not necessarily require identical C1-3/C10-12a residues. CONCLUSION: Since CMT, which inhibit proinflammatory sPLA2, are also inhibitors of some MMP, they may be useful for therapy of inflammatory diseases in which both MMP and sPLA2 are overexpressed.

Regulation of the cellular expression of secretory and cytosolic phospholipases A2, and cyclooxygenase-2 by peptide growth factors.

Secretory group II (sPLA2) and cytosolic (cPLA2) phospholipases A2 and cyclooxygenase-2 (Cox-2) play a pivotal role in release of proinflammatory eicosanoids. Excessive activity of sPLA2 per se can also propagate inflammation. Endogenous control of the above enzymes has not been completely elucidated. We investigated the combined impact of promoting cytokines and inhibitory peptide growth factors on the expression of mRNA of the above enzymes, on protein content and extracellular release of sPLA2 and on PGE2 production in osteoblasts (FRCO). The synthesis and release of sPLA2 were enhanced by about 20-fold by 0.5 ng/ml IL-1beta or by 50 ng/ml of TNFalpha. Coaddition of both cytokines resulted in synergistic 150-fold increase in the release of sPLA2 implying the existence of two paths of induction. IL-1beta and TNFalpha markedly enhanced the transcription of sPLA2 mRNA. Kinetic study showed that IL-1/TNF initiated sPLA2 release after 12 h, reaching maximum at 48 h. IL-1alpha was a weak stimulator of sPLA2 release, whereas IL-6, IL-8, IGF, IFN-gamma, growth hormone, insulin and GM-CSF were not stimulatory. Peptide growth hormones TGFbeta, PDGF-BB, EGF and bFGF markedly inhibited the extracellular release of sPLA2. TGFbeta and PDGF-BB significantly reduced the level of sPLA2 mRNA, thus acting upon transcription whereas EGF and bFGF were not inhibitory, acting rather upon the translational or posttranslational steps. IL-1/TNF and growth factors had no significant effect on cPLA2 mRNA expression. Cox-2 mRNA expression was markedly enhanced by IL-1/TNF and suppressed by all growth factors tested. Cytokines enhanced the extracellular release of PGE2 and further enhancement was induced by growth factors with the exception of TGFbeta. Cycloheximide abolished completely the release of sPLA2 and markedly reduced the release of PGE2 from cytokine-stimulated FRCO, regardless of whether growth factors were present or not. NS-398, a specific inhibitor of Cox-2 abolished almost completely the release of PGE2 from cytokine-stimulated cells, regardless of the presence of growth factors. Thus, different signalling mechanisms are involved in the impact of growth factors on mRNA expression of sPLA2, cPLA2 and Cox-2. The differences between the impact on FRCO sPLA2 and that reported in other cells, imply that endogenous control of arachidonic acid cascade is cell-specific.

Inhibition of type I and type II phospholipase A2 by phosphatidyl-ethanolamine linked to polymeric carriers.

We have previously shown that cell surface proteoglycans protect the cell membrane from the action of extracellular phospholipase A2 (PLA2) enzymes [Dan, P., Nitzan, D. W., Dagan, A., Ginsburg, I., and Yedgar, S. (1996) FEBS Lett. 383, 75-78]. Cell-impermeable PLA2 inhibitors (ExPLIs) were prepared by linking phosphatidylethanolamine (PE) to polymeric carriers, specifically, carboxymethylcellulose, heparin, or hyaluronic acid. The structure of these inhibitors enables the incorporation of their PE moiety into the membrane while the polymer remains at the membrane surface. In the present study, we show that the ExPLIs are effective inhibitors of the hydrolysis of different phospholipids in biological (Escherichia coli) and model (phospholipid vesicle) membranes, by diverse types of PLA2 enzymes, specifically human recombinant synovial fluid and C. atrox (type II), as well as Naja mocambique and porcine pancreatic (type I) PLA2. It is proposed that the external polymers of the ExPLIs, which are anchored to the membrane by the PE, mimic the naturally occurring cell surface proteoglycans and similarly protect membranes from the action of exogenous PLA2.

Hepatitis G and hepatitis C RNA viruses coexisting in cryoglobulinemia.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Induction of secretory phospholipase A2 confirms the systemic inflammatory nature of adjuvant arthritis.

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.

Inhibition of extracellular release of proinflammatory secretory phospholipase A2 (sPLA2) by sulfasalazine: a novel mechanism of anti-inflammatory activity.

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 (P < 0.001) and maximal at 400 microM, and that of CL 42A at 10 microM (P < 0.001) 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.

Anaphylactic shock induced by intraarticular injection of methylprednisolone acetate.

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.

Coordinate expression of group II phospholipase A2 and the acute-phase proteins haptoglobin (HP) and alpha1-anti-chymotrypsin (ACH) by HepG2 cells.

The early response to inflammation is characterized by the synthesis of a variety of proteins under cytokine and glucocorticoid control. During episodes of infection or inflammation, a secretory phospholipase A2 (sPLA2) appears in the circulation along with a variety of acute-phase proteins (APP), suggesting possible common regulatory elements amongst sPLA2 and APP. Using the human hepatoma line, HepG2, regulation of sPLA2 expression was examined in relation to synthesis of HP and ACH. The patterns of induction of sPLA2, HP and ACH were distinct for each of IL- 1, tumour necrosis factor (TNF) and IL-6, oncostatin M, IL-11 and leukaemia inhibitory factor. Dexamethasone had an enhancing effect on IL-6-induced expression of HP and ACH, but inhibited sPLA2 expression by 50%. Both 8-bromo-cAMP and dibutyryl cAMP increased sPLA2 expression (48.8-fold and 64.2-fold, respectively), whereas KT5720, an inhibitor of protein kinase A, down-regulated cytokine-induced sPLA2 synthesis by 51%. These data show that a panel of cytokines induced varying patterns of up-regulation of sPLA2, ACH and HP. Although dexamethasone potentiated IL-6-induced APP expression in HepG2 cells, it suppressed sPLA2 expression in a dose-dependent manner. In several respects, sPLA2 regulation is similar to that of HP and ACH, but a notable difference is the reciprocal effect of glucocorticoids on sPLA2 expression compared with that of ACH and HP.

Microtubule depolymerization selectively down-regulates the synthesis of proinflammatory secretory nonpancreatic phospholipase A2.

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.

Relationship of the dose of intravenous gammaglobulin to the prevention of infections in adults with common variable immunodeficiency.

The objective was to assess clinical efficacy of 3 dosages of intravenous gammaglobulins to prevent infectious episodes in adult common variable immunodeficiency. We designed a randomized, double blind, dose-assessing study. The setting was at University Hospital, Out-patient Clinic. Our patients were twenty-one adult patients with common variable immunodeficiency. The measurements were comparative study of the number and severity of infections using 3 various dosages of intravenous gammaglobulins, each given monthly for M least 6 months. Results indicated four hundred and eighty-four infectious episodes occurred while giving 305 infusions of IVIG 200 mg/kg; 205 infectious episodes while giving 170 infusions of 400 mg/kg and 436 infectious episodes while giving 247 infusions of 600 mg/kg. The morbidity scores (infection/infusion) were 1.59, 1.21 and 1.77 respectively (p - N/S). There was no significant difference in the severity of infections on the above 3 dosages, and no difference in the duration of infection-free intervals. The conclusions resulted in no significant differences in morbidity in adult patients with common variable immunodeficiency treated in cross-over pattern with IVIG 200 mg/kg, 400 mg/kg and 600 mg/kg. Thus, high dosages of IVIG are not conferring better protection against infections in such patients.

Secretory phospholipase A2 as an index of disease activity in rheumatoid arthritis. Prospective double blind study of 212 patients.

OBJECTIVE: 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. METHODS: 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. RESULTS: sPLA2 activity assessed as a dichotomous variable (less or more than mean +/- 2 SD) correlated highly (p < 0.005) 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. CONCLUSION: 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.

Inhibition of the activity of pro-inflammatory secretory phospholipase A(2) by acute phase proteins.

Pro-Inflammatory non-pancreatic phospholipase A(2) (sPLA(2)) is markedly over-expressed in acute systemic and chronic local inflammatory processes. Since in acute phase reaction sPLA(2) is often over-expressed simultaneously with acute phase proteins (APP), it is important to determine whether APP interacts with sPLA(2). We tested ten APPs for interaction with sPLA(2) using as a substrate multilamellar Hposomes composed either of PC:Lyso PC or PE:Lyso PE. Using PC:Lyso PC substrate, CRP, lactoferrin and SAP were found to inhibit sPLA(2) activity with an IC(50) of 25 mug/ml, 7.5 mug/ml and 50 mug/ml, respectively, corresponding to 0.21 muM, 0.1 muM and 0.21 muM respectively. Using PE:Lyso PE substrate only SAP was inhibitory, with an IC(50) of 10 mug/ml (0.04 muM). Phosphorylcholine abolished the inhibitory activity of CRP but not of SAP or lactoferrin. Addition of phosphorylethanolamine or of excess calcium had no effect on the inhibitory activity of APP. Limulin, lysozyme, transferrin, beta(2)-microglobulin, alpha(2)-macroglobulin, human and bovine albumins had no effect on sPLA2 activity. Therefore neither the structure of pentraxins, or ironbinding, bacteriostatic property or amyloidogenic property preclude whether APP modulates sPLA(2) activity. Inhibition of pro-inflammatory sPLA(2) by APP may be one of the protective mechanisms of the acute phase reaction.