Inhibition of the activity of pro-inflammatory secretory phospholipase A2 by acute phase proteins

Pro-Inflammatory non-pancreatic phospholipase A2 (sPLA2) is markedly over-expressed in acute systemic and chronic local inflammatory processes. Since in acute phase reaction sPLA2 is often over-expressed simultaneously with acute phase proteins (APP), it is important to determine whether APP interacts with sPLA2. We tested ten APPs for interaction with sPLA2 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 sPLA2 activity with an IC50 of 25 μg/ml, 7.5 μg/ml and 50 μg/ml, respectively, corresponding to 0.21 μM, 0.1 μM and 0.21 μM respectively. Using PE:Lyso PE substrate only SAP was inhibitory, with an IC50 of 10 μg/ml (0.04 μM). 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, β2-microglobulin, α2-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 sPLA2 activity. Inhibition of pro-inflammatory sPLA2 by APP may be one of the protective mechanisms of the acute phase reaction.


Introduction
Secretory non-pancreatic phospholipase A2 (sPLA2) belongs to the group of low-molecularweight, calcium-dependent, lipolytic enzymes. It plays an important physiological role in host defence participating in the destruction of Gramnegative microorganisms. 2'3 sPLA was also found to exert modulatory activity on the cellular proliferation and tumour formation in the intestinal tract. 4'5 Excessive activity of circulating sPLA 2 was discovered in several systemic inflammatory response syndromes (SIRS) such as clinical and experimental sepsi6s,9multiorgan failure and salicylate intoxication,-and in more localized processes such as peritonitis and inflammatory arthritis.'A pathogenetic role of sPLA 2 was implicated by the observation that enzymatic activity and immunoreactivity of sPLA 2 in SIRS correlated with the severity and outcome of the disease, [6][7][8][9] and by the fact that hypotension induced either by Gram-negative microorganisms 7 or by infusion of sPLA26 could be attenuated by inhibition of the enzyme. Acute inflammatory processes induced by sPLA 2 administered intracutaneously, 2 into subcutaneous air 14 15 pouches 13 or intraarticularly, could be blocked by inhibitors of PEA2 . [12][13][14][15] In general terms inhibition of sPLA 2 can be induced by three mechanisms: inhibition of synthesis, competition for substrate, or direct inhibition of sPLA 2 enzymatic activity. The best characterized inducers of its synthesis are cytokines, such as IL-1 and TNF. 6 IL-6 and oncostatin M induce sPLA 2 in cells of hepatic origin such as Hep G217'18 and normal human liver cells (W. Pruzanski et al., unpublished). Endotoxin was also found to be a strong inducer of sPLA2 .18 The above observations were consistent with the postulate that sPLA2 plays an important pathogenetic role in inflammatory processes and led to an extensive search for exogenous inhibitors. However, very little is known about endogenous modulation of sPLA2. Two endogenous inhibitors, glucocorticoid-inducible lipocortin 9 and complement fragments 2 have been found, but neither has evolved into a useful therapeutic agent.
During the acute phase reaction the synthesis phosphatidylethanolamine (PE) with or without and release of sPLA 2 occurs simultaneously with LysoPE (2:1) were prepared in chloroform and that of a large number of acute phase pro-evaporated to dryness. Multilamellar liposomes teins. 1'2 Experimental studies have shown that were made by dispersing the resulting lipid endotoxin, IL-1, TNF and IL-6 are the main indumixture in 100 mM Tris HC1 buffer, pH 8.0, cers of such release. 21 '22 Therefore the process followed by heating for 2 min at 41C and vorof induction of acute phase proteins by liver cells texing for 2 min before use. Only freshly preseems to be similar to that of sPLA2. Little is pared liposomes were used. Assays were carried known about the possible interactions of acute out in a total volume of 0.2 ml of 100 mM Tris phase proteins. We reported recently that CRP, HC1, pH 8.0 containing 2.5 or 10 mM CaCl2, 0.1% one of the classical acute phase proteins is a bovine serum albumin and 5-30 nmoles of PC strong inhibitor of sPLA2, binding competitively (or PE) vesicles (containing 2-16 nCi of [4C] to the phosphorylcholine-containing substrates 2 dipalmitoyl PC per assay). The optimal conwhereas SAA, another acute phase protein, centration was found to be 20 nmoles/assay and enhances sPLA 2 activity. 24 Herein we report that this was used in all experiments. If acute phase two other acute phase proteins, serum amyloid P proteins were included, they were preincubated (SAP) and lactoferrin suppress sPLA2 activity, with 20 nmoles of liposomes for 1 h at 41C These, previously unrecognized, interactions of before the assay. The reaction was then started acute phase proteins with proinflammatory by addition of 20 l.tl of recombinant human sPLA2, may add a new aspect to the under-sPLA2 stock solution with final sPLA2 concentrastanding of the complex role of acute phase tions ranging between 10 and 200 ng/200 l.tl reaction, assay volume unless otherwise stated. The reaction mixture was then incubated for 30 min at Materials and Methods 41C. The reaction was stopped by the addition of 1.32 ml isopropanol/heptane/0.5 M H2SO4 1,2-Dipalmitoyl-phosphatidylcholine (dipalmi-40:10:1 (v/v/v). The mixture was heated for 1 toyl PC) was obtained from Avanti Polar Lipids min at 60C before addition of 0.66 ml H20 and (Birmingham, AL). Phosphatidylcholine I-a-dipal-0.8 ml heptane. The two phases were allowed to mitoyl [2-palmitoyl-l-4C] (55.5 mCi/mmol) and separate and after centrifugation for 10 min at oleic acid [1-14C] (40-60 mCi/mmol) were pur-1 500 rpm, 0.8 ml of the upper phase was added chased from DuPont NEN Products. t-3-phosphato 1.0 ml heptane containing 100 mg silica gel. tidylethanolamine 1-palmitoyl [2-14C linoleoyl] The mixture was spun again for 10 min at 1 500 (50-60 mCi/mmol) was obtained from Amerrpm and 1.0 ml of the supernatant was used for sham (Arlington Heights, IL). Recombinant scintillation counting of 42-1abelled free palmitic human sPLA 2 (rh-sPtA2) was a generous gift of acid. All assays were done in triplicate. Dr Jeffrey Browning, Biogen Corporation (Cambridge, MA). Bio-Rad protein assay reagent was Immunologic assays: Anti-human (mouse IgG2) purchased from Bio-Rad (Richmond, CA). t-t-CRP/SAP antibody (clone CRP-20) C6552 was lysophosphatidylcholine palmitoyl, -a-phosphaobtained from Sigma (St Louis, MO). It reacted tidylethanolamine-]-linoleoyl-y-palmitoyl, -a-lysowith an epitope located on the 24 kDa subunit phosphatidylethanolamine palmitoyl, bovine of denatured and reduced CRP and it recognized serum albumin and silica gel were purchased CRP independently of the Ca 2+ binding site. from Sigma Chemical Corporation. All reagents This antibody did not recognize the calcium were analytical grade or better, dependent phosphorylcholine binding site of Recombinant human lysozyme, bovine serum CRP. It cross-reacted with human SAP, but not albumin (BSA), human serum albumin (HSA), with CRP from Limulus polyphemus. Working lactoferrin purified from human milk, transferrin, dilutions were at least 1:4 000 per btg of antigen [2-microglobulin purified from human urine, in indirect EI.ISA assay. In antibody assays, the limulin from Limulus polyphemus and ai-macro-antigens were preincubated with appropriately globulin were obtained from Sigma Chemical diluted antibodies for 60 min at room tempera-Company (St Louis, MO). CRP purified from rare. Then the liposomal substrate was added human plasma was obtained from Helix Biotech and the mixture was further incubated for 60 Corporation, Richmond, BC and SAP purified min at 41C. The PEA 2 was finally added and the from human serum, from Calbiochem, San incubation was carried on for an additional 30 Diego

Results
Ten acute phase proteins were tested for modulation of sPLA 2 activity, using as a substrate multilamellar liposomes composed either of PC:Lyso PC or PE:Lyso PE in the ratio of 2:1. Using the former substrate, lactoferrin (Fig. 1) CRP (Fig. 2) and SAP (Fig. 3) were inhibitory, with an IC50 of 7.5 l.tg/ml, 25 I.tg/ml and 50 Ig/ml respectively. These concentrations corresponded to 0.10 I.tM, 0.21 l.tM and 0.21 l.tM respectively (Fig. 4). Using the latter substrate, only SAP was inhibitory, with an IC50 of 10 btg/ml (0.04 btM) (Fig. 3). Using mixed substrate (PE:Lyso PC), no inhibition of sPLA 2 activity by CRP was noted ( Table 1)  had no effect on inhibitory activity of SAP, CRP or lactoferrin.
Anti CRP/SAP monoclonal antibody alone had no effect on sPLA 2 activity. This antibody preincubated with CRP or SAP did not alter their inhibitory activity in the range of concentrations used in the assays (CRP or SAP 40 l.tg/ml, sPLA2 100 ng).
To test whether chelation may have an effect on activity of sPLA2,, various concentrations of calcium were tested using either PC:Lyso PC or Since the increase in circulatory sPLA 2 activity parallels temporally that of acute phase proteins, it was of interest to investigate whether there is an interaction between APPs and sPLA 2. Of ten APPs tested, three were found to inhibit sPLA 2 activity. These included CRP, SAP and lactoferrin. CRP is one of the best studied acute phase proteins, increasing rapidly up to 1000-fold in APR. 34 Both sPLA 2 and CRP bind to the phospholipids of perturbed membranes of living cells 2v and to PC:Lyso PC substrate. 27'35 It was found that CRP is a strong inhibitor of sPLA 2 PE:Lyso PE as a substrate. Maximum activity of activity, acting most probably as a competitor sPLA 2 was observed at the level of 0.5-1.0 l.tM for the substrate. 2 The substrates used in the Ca 2+ In some experiments with CRP, SAP, or former 2 and present study form vesicles of diflactoferrin, Ca 2+ concentrations were increased ferent phospholipid composition. It was found up to 20 I.tM. Excess of Ca 2+ had no effect on that binding of CRP to such vesicles is preinhibitory activity of these proteins, ferential when they are composed of PC:Lyso PC Human and bovine serum albumin, transferrin, in the proportion of 2:1. Most probably it is lysozyme, 2-microglobulin, ai-macroglobulin related to optimally altered surface packing and limulin had no effect on the activity of sPLA 2. density of the substrate. PE:Lyso PE and PE:Lyso PC substrates were not susceptible to hydrolytic Discussion activity of sPLA 2. Since CRP belongs to the family of pentrax-An insult to an organism's homeostasis caused ins (PEP), two other pentraxins, SAP and by injury, infection or inflammation, leads to a limulin were also tested. The former was found swift systemic response called the acute phase to inhibit sPLA 2 whereas the latter did not. SAP reaction (APR) (reviewed in References 21, 25is a major APP in mice 27 but a minor one in 27). In the case of chronic or recurrent man, 6 increasing in APR only three-fold from inflammation, APR may become quite pro-the physiological level of 30-40 I.tg/ml to no longed. 25 An integral part of APR is a rapid more than 90 l.tg/ml. 27'36 It shares 60% homosynthesis and extracellular release of a large logy in amino acid sequence with CRP. 2 SAP number of acute phase proteins (APP), and circulates in the form of two pentameric molesimultaneous decrease in some other proteins, cules bound 'face to face' and, in contrast to the so called negative APP. Liver is the major CRP, is glycosylated. v The gene for SAP is source of APP synthesis, but other cells partici-located on the long arm of chromosome 1 pate in the synthesis of APP as well. 22'26'28 APR is (1q12-1q23), close to the gene coding for CRP. orchestrated by a group of inflammatory media-It was suggested that both are products of an tors, including glucocorticoids, cytokines, ana-ancestral duplication event. 8 Whereas CRP 21 25 phylotoxins and growth factors. The group binds mainly to phosphorylcholine, 6'9 SAP has of cytokines which induce APP synthesis high affinity to phosphorylethanolamine. 6 In includes, but is not limited to, IL-1, TNF, IL-6 and our study, SAP inhibited sPLA 2 activity when oncostatin. 21 '22'26'29'3 either PE:Lyso PE or PC:Lyso PC were used as The same group of cytokines was also found substrates; however, much lesser concentrations to induce the synthesis and release of the pro-of SAP were needed to achieve IC50 when inflammatory enzyme, secretory non-pancreatic PE:Lyso PE was employed. In human serum, 16 18 phospholipase A2 (sPLA2). sPLA2, first dis-SAP binds to C4-binding protein (C4BP) and covered in experimental peritonitis and in to various types of phospholipid vesicles. These fluids draining inflammatory sites, 2 was found reactions were found to be calcium dependent to raise rapidly in the circulation in systemic and can be disrupted by phosphorylethanolinflammatory response syndromes such as septic amine. In our study phosphorylethanolamine shock, 7 salicylate poisoning 9 and malaria and did not block the inhibitory activity of SAP.
in the milieu of more localized inflammatory Limulin in Limulus polyphemus is analogous 11 sites such as arthritis. In the former group, cir-to CRP in humans. It shares only 25-30% amino culating sPLA 2 correlated with both complicaacid homology with CRP 40'41 and SAP 41 and, tions and the outcome of the disease, 7 whereas similarly to CRP, binds avidly to phosphorylchoin the latter it correlated with the disease line. 34 (Table 2). Of three sPLA 2 inhibitors, lactoferrin was the most active. Lactoferrin is one of the iron-carrying proteins, produced mainly by polymorphonuclear cells. 45 '46 Its synthesis is induced by TNF 45'46 and in sepsis it behaves like a classical acute phase protein 46'47 with the potential to increase in the circulation ten or more fold from 46 48 its physiological level of 0.2-2.8 mg(.ml.-Infusions of LPS to healthy volunteers 46 or to piglets, 48 or of Escherichia coli to piglets 47 lead to a marked increase in circulating lactoferrin. In turn, lactoferrin interacts with LPS, preventin. iron-catalysed formation of hydroxyl radicals. Lactoferrin protects mice against a lethal dose of E. coli in vivo, 47 acting as bacteriostatic glycoprotein and inducing damage to the outer membrane of Gram-negative bacteria. 49 It seems, therefore, that lactoferrin acts upon Gram-negative bacteria similarly to bacterial permeability increasing protein (BPI). 2 Since sPLA 2 hydrolyses membrane phospholipids of killed by BPI, the fact that microorganisms 2 lactoferrin inhibits sPLA 2 activity may mean that it acts as a limiting factor in hydrolytic activity of the latter. The property of iron chelation by lactoferrin was not related to inhibitory anti-sPLA2 activity, since transferrin, another iron chelator did not inhibit sPLA 2. The fact that during the acute phase there is simultaneous co-induction and over-expression of both inhibitors such as CRP, SAP and lacto-