A phospholipase A2-activating protein (PLAP) stimulates human neutrophil aggregation and release of lysosomal enzymes, superoxide, and eicosanoids.

We have recently isolated a human phospholipase A2-activating protein (PLAP) that shares antigenic and biochemical similarities with melittin, a well characterized bee venom phospholipase-stimulatory peptide. To explore the potential mechanisms of action of PLAP that extend beyond its effects on eicosanoid synthesis, we examined its effects on the release of human neutrophil lysosomal enzymes and superoxide, and on RBC hemolysis. These results were compared to the effects of melittin, which has been reported to induce enzyme release and hemolysis. We also examined the effects of PLAP on neutrophil aggregation and chemotaxis. PLAP induced neutrophils to release beta-glucuronidase and metalloproteinase enzyme activities as well as produce superoxide ion in both a dose- and time-dependent manner. Eicosanoid synthesis inhibitors did not abrogate these responses. PLAP induced release of arachidonic acid metabolites, but this response could be abrogated by eicosanoid synthesis inhibitors. PLAP also induced neutrophil aggregation and chemokinesis, but not chemotaxis. Concentrations of PLAP that induced these responses did not induce cellular toxicity as determined by light and electron microscopy, lactic dehydrogenase release, trypan blue dye exclusion, and RBC hemolysis. In contrast, prolonged incubation with higher concentrations of PLAP induced cell death that was similar to that observed with melittin. These findings suggest that the mechanisms of action of PLAP extend beyond the eicosanoid synthetic pathway, and that disordered regulation of PLAP may be responsible, at least in part, for chronic immune and inflammatory states.

Monoclonal antibody to the crystal induced chemotactic factor from human neutrophils detects similar immunoreactive and biologically active protein in promyelocytic HL-60 cells.

The crystal induced chemotactic factor (CCF) is a protein released by human neutrophils (PMN-CCF) that phagocytize monosodium urate or calcium pyrophosphate crystals. This protein has been thought to play an important role in crystal induced arthritis. In the present report, a mouse hybridoma cell line A2900C2.12 which specifically secretes monoclonal antibody (MAb) to CCF has been developed. The antibody secreted by the cell line is mostly IgG2b and positive for both the light chains kappa and lambda. The MAb has been purified by affinity chromatography using a protein-A column. The purified MAb was found to be over 95% pure based on the results from SDS-PAGE. The MAb has been used to demonstrate that a leukotactic protein is released from polymorphonuclear leukocytes (PMN) and promyelocytic HL-60 cells in response to a phagocytic or degranulating stimuli such as monosodium urate crystals or N-formyl-Met-Leu-Phe (FMLP), respectively. "Western blots" and silver nitrate staining of the purified leukotactic protein from PMNs and HL-60 cells show that these proteins have the same immunodeterminant site(s) and both appear to have a molecular weight (m.w.) of about 15,000. A simple purification protocol including immunoaffinity high pressure liquid chromatography (HPLC) has been described for purifying chemotactic proteins with the same antigenic determinant as the PMN-CCF.

Purification of crystal induced chemotactic factor from human neutrophils.

The crystal induced chemotactic factor is a protein that is released by neutrophils in response to a phagocytic stimulus like monosodium urate or calcium pyrophosphate crystals. This protein has been postulated to play an important role in the development of crystal-induced arthritis. In this report, we present a protocol for its purification which involved ammonium sulfate fractionation, affinity chromatography, lectin chromatography, and high pressure chromatography (HPLC). The purified protein migrated as a single band on SDS-PAGE. Based on its electrophoretic and chromatographic (HPLC) properties, it appears to have a m.w. of 15,000. It has a blocked N-terminal since our attempt to sequence the chemotactic protein by established procedures failed. The protein contains one methionine residue which appears to be essential for its chemotactic activity since cyanogen bromide treatment abolished its chemotactic activity. To our knowledge this is the first report on purification of this autocrine leukoattractant from human neutrophils.

Effect of tunicamycin C2 on the induction of crystal induced chemotactic factor in human neutrophils.

We have investigated the effect of tunicamycin-C2, which specifically inhibits glycosylation of proteins but not their synthesis, in the induction of crystal-induced chemotactic factor (CCF) in human neutrophils by monosodium urate crystals. Tunicamycin C2 appears to impair transport of the CCF to the lysosomal fraction of the cell and its subsequent secretion into the extracellular fluid, resulting in accumulation of nonglycosylated chemotactic proteins in the cytosol. Based on the results of affinity chromatography studies with various lectins, the carbohydrate moiety(ies) of the glycosylated chemotactic peptide contain mannose, N-acetyl-D-galactosamine, galactose, and fucose.

Colchicine prophylaxis in pseudogout.

Ten patients with recurrent attacks of pseudogout were followed for 1 year before and 1 year after receiving oral colchicine 0.6 mg BID. Thirty-two episodes of acute arthritis were recorded in the year (3.2/patient/year) before the start of the drug and only 10 while taking the drug (1/patient/year) (p less than 0.001). Ninety percent of the patients benefitted from the drug. Our study supports the effectiveness of oral colchicine as a prophylactic agent in recurring pseudogout.

Chemical modification of human neutrophil membrane proteins: effect on fMet-Leu-Phe binding and function.

[3H]fMet-Leu-Phe binding to human neutrophil membrane proteins was shown to be inhibited by pretreatment of membranes with the histidine-preferring reagent diethylpyrocarbonate in a concentration- and time-dependent fashion. The inhibition was partially reversed by hydroxylamine and was affected by pH. The pH profile for inhibition and the partial reversibility of the inhibition by hydroxylamine are consistent with a modification of the histidine residue by diethylpyrocarbonate. The addition of unlabeled fMet-Leu-Phe to the membrane preparation prior to diethylpyrocarbonate treatment provided protection from the binding inhibition following washout of unlabeled fMet-Leu-Phe and unreacted reagent. Cells treated with diethylpyrocarbonate were inhibited in their ability to produce superoxide anions in response to fMet-Leu-Phe, but the concentration of the chemotactic factor required to obtain 50% of the response was alike for treated or untreated cells. These results suggest that a histidine residue at or near the receptor binding site for fMet-Leu-Phe is required for binding and cell activation. Neither N-acetylimidazole, an agent that preferentially reacts with tyrosine, nor acetic anhydride, which reacts with lysyl groups, affected [3H] fMet-Leu-Phe binding to plasma membrane proteins or superoxide production by intact cells. Scatchard analysis of the binding inhibition owing to diethylpyrocarbonate was consistent with a loss of receptor number rather than a change in affinity.

Opera-glass deformity and tendon rupture in a patient with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a disease entity that frequently becomes clinically evident as a generalized arthritis. Despite this common presentation, it is widely accepted that the arthritis of SLE rarely produces severe joint deformity with barely 10% of the patients developing swan neck, boutumiere deformity or a Jaccoud-like arthropathy. An even lesser proportion of patients develop joint erosions and there have been several clinical reports of tendon rupture in patients suffering from the above condition. However, there is still a great deal of controversy concerning the aetiology of the tendon rupture in these patients and while some authors suggest that corticosteroid therapy influences the outcome of this syndrome, pathology studies were included in a small number of these case reports. The subject of the present case report is a patient with SLE and resorptive arthropathy who suffered repeated episodes of tendon ruptures. The local pathologic findings in this patient suggest that the basic disease process was the cause of the tendon ruptures.

Deactivation of guinea pig pulmonary alveolar macrophage responses to N-formyl-methionyl-leucyl-phenylalanine: chemotaxis, superoxide generation, and binding.

Incubation of pulmonary alveolar macrophages (PAM) with the synthetic chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in deactivation of PAM chemotaxis. The chemotactic response to 10(-8) M FMLP was inhibited 85% after 30 min of preincubation with 10(-6) M FMLP and 48% by 10(-8) M FMLP. Only the higher dose of FMLP (10(-6) M) caused deactivation of the chemotactic response to C5a (20%). Preincubation with partially purified C5a at a concentration of 100 microliter/ml produced a 32% inhibition of the PAM response to 10(-8) M FMLP. In contrast, preincubation with FMLP had no significant effect on superoxide generation, either at baseline or after stimulation. Levels of intracellular cyclic adenosine-3',5'-monophosphate (cAMP) increased in response to PGE1 in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, but FMLP failed to induce a change in cAMP levels. Studies of 3H-FMLP binding were consistent with two populations of membrane receptors with different affinities. Preincubation of PAM with FMLP did not result in a reduction of maximal binding. We conclude that FMLP induces deactivation of PAM chemotaxis, but cross-deactivation occurs only after high dose treatment. Unlike the PMN, macrophage chemotactic activation is not accompanied by an elevation in cAMP levels. These observations suggest that PAM chemotaxis is influenced by prior exposure to chemotactic stimuli, but other aspects of the PAM response diverge from that of PMN. The mechanism of deactivation of PAM does not appear to result from a shift in the dose-response curve or decreased availability of membrane receptors, but may involve uncoupling of post-receptor cellular responses.

Binding characteristics of radioiodinated crystal-induced chemotactic factor to human neutrophils.

The binding of radiolabeled crystal-induced chemotactic factor (CCF) to human neutrophils is characterized. Binding of 125I-CCF to the cells was higher at 4 degrees C than at 24 degrees or 37 degrees C and was found to be independent of CA2+ and Mg2+ ion concentration. The binding showed a pH optimum of 6.0. Tosylamido phenylethyl chloromethyl ketone at 100 mumol/L concentration inhibited 20% of 125I-CCF binding, but phenylmethylsulfonyl fluoride at 200 mumol/L had no effect. Approximately 50% of cell-associated 125I-CCF was released after treatment with proteases. The nonspecific uptake by the cells, as measured by the uptake of 3H-sucrose and 14C-inulin in the presence of CCF, was negligible. After the steady-state binding of 125I-CCF to the cells, approximately 15% of the cell-associated radioactivity at 4 degrees C and 40% to 50% at 24 degrees and 37 degrees C was released into the medium after 60 minutes of incubation in medium alone. Dissociation of the radioactive material was not affected by the presence of tosylamido phenylethyl chloromethyl ketone or phenanthroline in the media. The dissociated material was determined to be degraded 125I-CCF, suggesting that degradation of 125I-CCF occurs after binding to its specific receptor on the human neutrophil.

Receptor blockade as a mechanism of deactivation of human neutrophils by pepstatin and formyl-Met-Leu-Phe.

The pentapeptide pepstatin was shown to be chemotactic for human neutrophils by two techniques: ED50 for chemotaxis was found to be 3 microM by the agarose method and 0.2 microM by the Boyden chamber technique. Pepstatin also induced superoxide radical generation, release of lysosomal enzymes, and a transient increase in intercellular adenosine-3',5'-cyclic monophosphate (cAMP) levels in a dose-dependent manner. Carbobenzoxy-phenylalanyl-methionine (CBZ-PM), which competitively inhibits formyl-methionyl-leucyl-phenylalanine (FMLP) -induced neutrophil functions, also inhibited pepstatin-induced neutrophil function of superoxide generation in a dose-dependent fashion. Likewise, pepstatin inhibited the binding of [3H]FMLP to the cells. Furthermore, preincubation of neutrophils with suboptimal concentrations of FMLP or pepstatin diminished the cellular response toward either factor when tested for their chemotactic activity and for their ability to induce superoxide generation, to release granule enzymes, and to induce a transient increase in intracellular cAMP levels. The concentrations of pepstatin or FMLP tested had no effect on superoxide generation, granule enzyme release, or intracellular levels of cAMP on subsequent challenge with C5a; both of these factors, however, cross-deactivated the chemotactic response of the cells towards C5a. Similar results were observed when cells were preincubated with C5a and subsequently challenged with pepstatin or FMLP. These results suggest that FMLP and pepstatin interact with the same receptor molecules to activate human neutrophil functions. Furthermore, our data indicate that the deactivation of the neutrophil functions of superoxide production and granule enzyme release are receptor specific, but the heterologous deactivation of chemotaxis involves a postreceptor mechanism(s).

Sulfasalazine inhibition of binding of N-formyl-methionyl-leucyl-phenylalanine (FMLP) to its receptor on human neutrophils.

Sulfasalazine, a drug useful in the therapy of inflammatory bowel disease, was found to block N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced arthritis in rabbits as well as FMLP-induced superoxide production and chemotaxis in human neutrophils in vitro. Sulfasalazine was also found to block FMLP binding to human neutrophils with an I50 of 10 microM. The dose-response curve for the inhibition of binding was very similar to the dose-response curves for the inhibition of FMLP-induced neutrophil activation.

Benign asbestos pleurisy in the rabbit. A model for the study of pathogenesis.

Benign asbestos pleurisy is a manifestation of asbestos-induced disease that is not uncommon but often ignored. We developed an animal model to study the pathogenesis of this syndrome. Crocidolite asbestos injected into the rabbit pleural space resulted in the appearance of chemotactic activity in an exudative effusion, characterized by a polymorphonuclear leukocyte (PMN) response that peaked 4 h after the injection. The chemotactic activity and the PMN responses occurred equally in normal animals and in animals that had been depleted of complement with cobra venom. Neutropenia, induced with vinblastine, did not alter the appearance of chemotactic activity but abrogated the PMN response. Treatment with colchicine failed to block the release of chemotactic activity into the pleural fluid, but decreased the PMN response without affecting the ability of peripheral blood PMN to respond to pleural fluid chemotactic activity. When pleural fluid containing chemotactic activity was injected into the pleural space of another rabbit, a PMN response was observed. These studies suggest that the acute exudative response seen in the rabbit model of benign asbestos pleurisy results from the release of chemotactic activity from sources other than complement. The response probably depends on interaction between the asbestos and the pleural tissue. Asbestos-PMN interaction may release chemotactic factors that amplify the response secondarily. Colchicine blocked the acute response to intrapleural asbestos by a mechanism yet to be understood.

Heterologous receptor population for a chemotactic factor F-Met-Leu-Phe on the human neutrophil. Effect of pH and temperature.

Association kinetics of a chemotactic factor [3H]FMLP to its specific receptors on human neutrophils and the modulation of kinetic constants by pH and temperature were studied. The half-time for [3H]FMLP association to its specific receptors as calculated by a least-square fit equation was 27 min at 4 degrees C, 13 min at 24 degrees C, and 5 min at 37 degrees C. The Scatchard plot of [3H]FMLP binding to its specific receptors was found to be nonlinear, suggesting a heterogeneity of [3H]FMLP receptors and/or functional negative cooperativity. The computer analysis of the data showed two populations of FMLP receptors on human neutrophils with Kd (dissociation constant) values of 0.17 +/- SD 0.1 nM and 0.94 +/- SD 0.1 nM at 4 degrees C, and 0.17 +/- SD 0.02 nM and 1.1 +/- SD 0.2 nM at 37 degrees C. The optimum [3H]FMLP binding at 37 degrees C was observed at pH 7.5. There was essentially no difference in Kd values at pH 6.0, 7.5, or 8.5. Our results suggest the presence of two populations of [3H]FMLP receptors on human neutrophils in support of previous observations of other investigators. Our data also demonstrate that association constants between [3H]FMLP and its specific receptors on human neutrophils are not affected by changes in pH or temperature; maximal binding, however, is dependent on pH.

Evidence that the functional responses of human neutrophils occur independently of transient elevations in cyclic AMP levels.

Exposure of human neutrophils to the tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP) leads to a transient, 2-3 fold elevation of adenosine-3',5'-cyclic monophosphate (cAMP) that peaks at 5-15 seconds. This cAMP transient has been hypothesized as constituting an early activation event that may be responsible for subsequent functional responses. In order to evaluate the dependence of several FMLP-stimulated functional responses on elevated cAMP levels, we utilized 9-(tetrahydro-2-furyl)adenine (SQ 22,536), a putative inhibitor of adenylate cyclase. Pretreatment of cells with SQ 22,536 (1-1000 microM) caused dose-dependent inhibition of the FMLP (0.1 microM)-induced cAMP elevation (ID50 approximately 5 microM). Similar results were observed when cells were activated by the divalent cation ionophore A23187 (20 microM). At 1000 microM, a drug concentration which completely abolished the cAMP transient, SQ 22,536 had no effect on FMLP-stimulated superoxide radical (O2-) generation, granule enzyme release, or chemotaxis and only a modest inhibitory effect on A23187-induced O2- production. These studies strongly suggest that these FMLP- and A23187-induced responses occur independently of a transient elevation of cAMP and that, in intact human neutrophils, SQ 22,536 is a non-toxic inhibitor of adenylate cyclase.

Induction of a chemotactic factor from human neutrophils by diverse crystals.

The purpose of this study was to isolate and compare the chemotactic factor generated by human neutrophils after phagocytosis of three structurally different crystals. An apparently identical chemotactic factor for neutrophils was isolated from the granular fraction of human neutrophils allowed to phagocytose MSU, CPPD, or amorphous DC. The isolated chemotactic factors migrated identically on SDS polyacrylamide gel electrophoresis, with a relative motility of 8.05, and competed with MSU-induced chemotactic factor for binding sites on the human neutrophils in an identical fashion. The data support the concept that the generation of the chemotactic factor represents a general response of the neutrophil to the phagocytosis of foreign particles.

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump.

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.

Stimulation of the respiratory burst in human neutrophils by crystal phagocytosis.

Exposure to monosodium urate crystals (MSU) stimulated the respiratory burst of human neutrophils as measured by increased O2 consumption and the generation of superoxide radicals (O(2)). From the comparison of data derived from nitroblue tetrazolium and cytochrome C reduction (two methods of detecting O(2) release), it appears tht O(2) production in response to MSU may be compartmentalized, i.e., occur predominantly in the intracellular space. After exposure to MSU, neutrophils from patients with chronic granulomatous disease lost viability at the normal rate; thus products of the respiratory burst are not likely to be responsible for cell death.