Major histocompatibility complex class II (DR) antigen and costimulatory molecules on in vitro and in vivo activated human polymorphonuclear neutrophils

We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic 'stores' of three key molecules normally associated with antigen presentation and T-cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7-1) and CD86 (B7-2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross-linking (X-L) of Mac-1: an early neutrophil activation signal. In this study we have compared X-L of Mac -1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N-formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G-Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow-cytometry measurements of size, granularity and phenotype. Significant up-regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual-staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy.     

Neutrophil expression of tumour necrosis factor receptors (TNF-R) and of activation markers (CD11b, CD43, CD63) in rheumatoid arthritis.

In vitro analysis of polymorphonuclear neutrophils (PMN) has allowed various stages of cell activation to be distinguished, characterized by the expression level of specific membrane markers and of functional receptors. Among those, TNF-alpha receptors (TNF-R) are modulated by various PMN activators, a mechanism which may be important to control cell responses to TNF in inflammatory reactions such as rheumatoid arthritis (RA). PMN, isolated from the blood of 36 RA patients and from the synovial fluid of 23 of them, were analysed for membrane expression of the two TNF-R (p55 and p75). Soluble p55 and p75 (sTNF-R) and TNF concentrations were measured in the plasma and synovial fluid by specific ELISA assays. Our results show that PMN from the blood of RA patients bear a normal number of TNF-R, with a normal p55/p75 ratio, compared with PMN from normal controls. Soluble TNF-R levels were similar in patients and normal plasma. In spite of high endogenous TNF concentration, patients' circulating PMN were not activated, as shown by a CD11b/CD18 expression similar to that of control resting cells. In contrast with blood neutrophils, PMN from RA patients' synovial fluids had an activated phenotype, characterized by increased expression of CD11b, decreased expression of leukosialin, CD43, and the appearance on the plasma membrane of an azurophil granule protein, CD63. High levels of soluble TNF-R were measured in RA synovial fluids. Nevertheless, membrane TNF-R levels and p55 and p75 proportions were similar to those of PMN from normal blood. These results suggest the existence of regulatory mechanisms which maintain a stable neutrophil expression of TNF-R as well as a balance between both types of receptors in inflammatory situations where neutrophils are strongly activated.     

Human neutrophils release their major membrane sialoprotein, leukosialin (CD43), during cell activation.

Leukosialin (CD43) is a sialic acid-rich molecule with a relative molecular mass (M(r)) of 140,000 highly represented on polymorphonuclear neutrophils (PMN) and on most leukocytes. One of its functions may be to prevent nonspecific cell-to-cell interactions through negative charge repulsions. As tested by immunofluorescence, neutrophil CD43 membrane expression was shown to decrease by up to 80% upon cell activation by phorbol myristate acetate (10 ng/ml) or by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 10(-6) M) in the presence of cytochalasin B. The kinetic of this decrease paralleled that of CD11b up-regulation. FMLP alone, tumor necrosis factor (TNF-alpha), lipopolysaccharide and granulocyte macrophage colony-stimulating factor had moderate or insignificant effects, while inducing striking CD11b up-regulation. Cell priming with TNF-alpha followed by FMLP stimulation resulted in up to 40% decrease of CD43 expression. Anti-CD43 mAb immunoprecipitated three fragments of M(r) 130,000, 49,000 and 34,000 from the cell-free supernatant of activated neutrophils, suggesting that CD43 is released from the membrane by proteolysis. Indeed, the decrease in CD43 expression was inhibited by phenylmethanesulfonylfluoride (PMSF). Homotypic aggregation of activated PMN was also inhibited by PMSF and could result, at least in part, from the shedding of CD43. The shedding of such a strongly anionic and major membrane protein should drastically modify PMN surface charge and may allow previously hindered interactions by exposing new adhesion molecules.     

In vivo expression of CD69 on lung eosinophils in eosinophilic pneumonia: CD69 as a possible activation marker for eosinophils.

The antigen, CD69, has been demonstrated to be expressed on activated T cells and natural killer cells. There have been no studies concerning the expression of CD69 on eosinophils. In this article, we demonstrate that lung eosinophils obtained from the bronchoalveolar lavage fluid of patients with eosinophilic pneumonia expressed significant levels of CD69, whereas peripheral blood (PB) eosinophils did not express CD69. We also activated PB eosinophils in vitro using phorbol myristate acetate and cytokines to determine whether CD69 was expressed. PB eosinophils expressed CD69 after short-term culture with phorbol myristate acetate and eosinophil hemopoietic cytokines (interleukin-3, granulocyte-macrophage--colony-stimulating factor, and interleukin-5). These findings suggest that CD69 may be a useful marker for activated eosinophils at inflammatory sites.     

Rabbit polymorphonuclear granulocyte function during ethanol administration--migration and oxidative responses in a joint with immune complex synovitis.

Functional impairments of polymorphonuclear granulocytes (PMN) are believed to contribute to hampered inflammation and host defence in alcoholics. We studied effects of i.v. ethanol administration on PMN responses in rabbits during induction of a knee-joint synovitis. The synovitis conferred systemic effects, since chemiluminescent responses of peripheral blood PMN to opsonized zymosan and phorpbol myristate acetate (PMA) increased 6.4- and 17.9-fold, respectively. Chemiluminescent responses of synovial PMN were further amplified. This up-regulation was reduced to 33% in rabbits treated with ethanol when opsonized zymosan was used as the PMN stimulus; in contrast, PMA responses were unaffected. The appearance and migration of PMN to the synovitis joint were normal despite a blood ethanol concentration of 0.5%. Thus, ethanol impaired release of oxygen metabolites from PMN, but not the delivery of cells at an inflammatory site.     

Increased expression of surface activation markers on neutrophils following migration into the nasal lumen

BACKGROUND : The sequence of events following the recruitment of a free-flowing neutrophil in the peripheral circulation, via adhesion, migration and release of mediators, to a neutrophil on the surface of the nasal epithelium is a co-ordinated process. Little is known about the state of neutrophil activation following this course of events. OBJECTIVES : To investigate the expression of surface activation markers on neutrophils, reflecting activation during their recruitment to the nose, and to see whether the inflammatory process during allergic rhinitis influences this process. METHOD : Nine healthy controls and 12 patients with grass pollen-induced intermittent allergic rhinitis were investigated during the peak of the pollen season. The expression of CD11b, CD66b and CD63 on the neutrophil cell surface, as a reflection of activation, was analysed using flow cytometry. Neutrophils were derived from peripheral blood and nasal lavage fluid. In addition, eosinophil cationic protein (ECP) and myeloperoxidase (MPO) as well as L-, P- and E-selectins in the nasal lavage fluid were analysed using RIA and ELISA, respectively. RESULTS : A marked increase in the expression of all three CD markers on the neutrophil cell surface was noticed following migration from the bloodstream to the surface of the nasal mucosa. At the peak of the grass pollen season, the MPO levels increased, reflecting an increase in the total number of nasal fluid neutrophils. In parallel, the expression of CD11b was further augmented. The expression of the CDb11b was reduced on neutrophils remaining in the circulation. In addition, the level of L-selectin was reduced on neutrophils derived from the blood during allergic inflammation. CONCLUSION : Neutrophils might become activated during their transfer from the blood to the surface of the nasal mucosa, but these changes may also be due to depletion of activated neutrophils in the blood via activated endothelial/epithelial adhesion and chemoattractant measures. The increased expression of surface activation markers during allergic rhinitis suggests roles for neutrophils in the inflammatory process.     

Increased fMet-Leu-Phe receptor expression and altered superoxide production of neutrophil granulocytes in septic and posttraumatic patients

Summary Activation of neutrophils by various inflammatory stimuli has been shown to play a pivotal role in septic and posttraumatic tissue injury. To further elucidate the mechanisms modulating the oxidative metabolism, we assessed superoxide production induced by N-formylmethionyl-leucylphenylalanine (FMLP) and phorbol myristate acetate and the expression of FMLP receptors of human neutrophils on several days during sepsis and after trauma. Neutrophils of septic patients isolated on days 0–4 after the diagnosis of sepsis showed a significant, more than twofold increase in specific binding of [³H]FMLP at 1, 120, and 240 nM. Scatchard plot analyses revealed that this increase in specific binding was due to an increase in the number of low- and high-affinity FMLP receptors with no changes in receptor affinity. On days 5–10 after the onset of sepsis the up-regulation of FMLP receptors on circulating neutrophils was followed by receptor down-regulation. Likewise, neutrophils from patients with trauma that was not complicated by sepsis bound significantly more [³H]FMLP than neutrophils from volunteers. However, the increase in FMLP receptors was less than that in septic neutrophils and returned earlier to normal. In accordance with the up-regulation of FMLP receptors, neutrophils obtained from patients with sepsis or after trauma on days 1–4 and days 1–2, respectively, produced significantly more superoxide anion upon stimulation with FMLP. However, after stimulation with phorbol myristate acetate, a receptor-independent activator of protein kinase C, these cells released less superoxide anion than controls. Our findings suggest that during sepsis and trauma circulating neutrophils become transiently primed for an enhanced oxidative metabolism upon stimulation with FMLP but desensitized to protein kinase C dependent stimulation.Abbreviations PMN neutrophilic granulocytes - FMLP N-formylmethionyl-leucyl-phenylalanine - PMA phorbol myristate acetate - SO oxygen superoxide - PKC protein kinase C - TNF tumor necrosis factor - HBSS Hank's balanced salt solution     

Retinoids Activate Superoxide Production by Polymorphonuclear Leucocytes

Retinol and retinoic acid were effective activators of oxygen consumption by human polymorphonuclear leucocytes (PMN) in micromolar concentrations. In contrast, retinyl acetate was ineffective as an activator. Retinol caused activation only after a lag time, the length of which depended on retinol concentration. Oxygen consumption was due to superoxide production by PMN. Superoxide production was observed as superoxide dismutase-inhibitable cytochrome c reduction. Previously, retinoids have been reported to inhibit PMN activation by phorbol myristate acetate, a tumour promoter. This retinoid-induced inhibition of PMN activation has been suggested to be a mechanism by which retinoids may protect against carcinogenesis in animals. However, the retinoid concentrations at which PMN inhibition was reported were much higher than those found to cause activation in this study. We found that retinoic acid slightly inhibited phorbol myristate acetate-activated superoxide production, but only at concentrations that caused activation. In contrast, activation by formyl-Met-Leu-Phe was effectively inhibited at a retinoic acid concentration that did not cause activation by itself.     

Polymorphonuclear Leucocytes Defective in Oxidative Metabolism Inhibit in Vitro Growth of Plasmodium falciparum

This report presents evidence that polymorphonuclear leucocytes (PMN) from chronic granulomatous disease (CGD) patients, who are defective in oxidative metabolism, are capable of inhibiting in vitro multiplication of Plasmodium falciparum . Using a microtitre in vitro inhibition assay, we incubated various numbers of peripheral blood neutrophils from CGD patients and from normal individuals with P. falciparum isolate F32 in the in vitro culture system. Inhibition of parasite growth by neutrophils was determined after 48 h of culture. At PMN to erythrocyte ratio of 1:50 there was an inhibition of parasite growth of 57% by normal neutrophils and 39% to 68% by CGD cells. When the neutrophils were stimulated by phorbol myristate acetate, both cell types enhanced inhibition of parasite growth. These findings indicate that the oxygen-independent systems of human neutrophils are involved in parasite destruction. Constituents of neutrophil granules such as acid hydrolases, lactoferrin, and cationic proteins could be regarded as potential mediators of parasite destruction.     

Neutrophil membrane sulfhydryl groups are involved in stimulated neutrophil adherence to endothelium

We investigated the role of membrane sulfhydryl groups in adherence of stimulated polymorphonuclear neutrophils to cultured endothelial cells. Treatment of neutrophils with p-chloromercuriphenyl sulfonate (PCMPS), a slowly penetrating sulfhydryl reagent, inhibited phorbol myristate acetate (PMA)- or calcium ionophore A23187-stimulated adherence to cultured human or bovine endothelial cells. At concentrations which completely blocked PMA-stimulated adherence, PCMPS did not cause release of lactic dehydrogenase, inhibit PMA-mediated degranulation or hydrogen peroxide production, or prevent the PMA-induced increased surface expression of CD11b/CD 18 (Mac-1). Coincubation with a competing reduced sulfhydryl compound protected neutrophils from inhibition of PMA-stimulated adherence by PCMPS, whereas coincubation with an oxidized sulfhydryl compound did not. Monobromotrimethylammoniobimane, a nonpenetrating sulfhydryl reagent that is structurally unrelated to PCMPS, also inhibited stimulated neutrophil adherence to endothelium cultures. We conclude that stimulated neutrophil adherence to endothelium involves neutrophil membrane protein sulfhydryl groups.     

Inhibitory effects of human neutrophil functions by the 45-kD glycoprotein derived from the parasitic nematode Trichinella spiralis

AIM: We evaluated the effect of the 45-kD protein of Trichinella spiralis (gp45), purified by affinity chromatography, on random migration and chemotaxis, the oxidative metabolism of human neutrophils and on the CD11b upregulation induced by formyl-methionyl-leucyl-phenylalanine (f-MLP). METHODS: Donor neutrophils incubated with different amounts of gp45 (0.5, 1, 1.5, 2 microg/ml) or buffer and the random migration and chemotaxis, evaluated by means of a special technique of image analysis, and the chemiluminescence response to f-MLP or phorbol myristate acetate (PMA) were analyzed. The effect on CD11b upregulation was assessed incubating cells with the protein, when activating them with f-MLP. RESULTS: The results showed that gp45 inhibited both random and stimulated migrations, and reduced the response to f-MLP and PMA. Furthermore, gp45 significantly reduced the upregulation of the CD11b induced by f-MLP. CONCLUSION: The results show that gp45 inhibits PMN in different functions, suggesting an anti-inflammatory action.     

Enhanced Expression of Integrins and CD66b on Peripheral Blood Neutrophils and Eosinophils in Patients with Rheumatoid Arthritis, and the Effect of Glucocorticoids

The aim of this study was to elucidate signs of granulocyte activation by studying adhesion and phagocytosis receptors on peripheral blood granulocytes from patients with rheumatoid arthritis (RA), and to observe the effect of glucocorticoids. Analyses by flow cytometry showed elevation of the neutrophil and eosinophil expression of the alpha- and beta-chains of the beta2-integrin Mac-1 (CD11b/CD18) and of the CEA-gene family member 6 (CGM6, CD66b). Expression of the adhesion receptor antigens CD11a, CD29, CD49d, CD49f and CD44, and the Fcgamma receptors II and III, was unaffected. Treatment with low-dose prednisolone reduced the expression of CD11b on neutrophils and of CD11b, CD18 and CD66b on eosinophils to the same level as that found in healthy controls. Metyrapone treatment increased the surface expression of CD35 and CD49f on eosinophils, but did not affect surface expression on neutrophils. Activation of blood granulocytes may be important for the increased recruitment of neutrophils and eosinophils to the synovial cavity in RA. Treatment with low doses of glucocorticoids in RA normalizes the enhanced expression of the studied adhesion molecules in eosinophils but has minor impact on neutrophil activation. Endogenous glucocorticoid production seems to have minimal or no effect on the expression of adhesion and phagocytosis receptors on circulating granulocytes.     

Analysis of function-associated receptor molecules on peripheral blood and synovial fluid granulocytes from patients with rheumatoid and reactive arthritis

In this study we report the expression pattern of 13 different function-associated surface molecules on synovial fluid and peripheral blood granulocytes from rheumatoid and reactive arthritis patients. We found increased expression of the complement receptors 1 (CD35) and 3 (CD11b) and of the activation-associated antigens CD67, CD24, and M5 on synovial fluid granulocytes from rheumatoid and/or reactive arthritis patients compared to autologous peripheral blood granulocytes. In addition, synovial fluid granulocytes expressed IgG Fc receptor 1 (CD64) and complement receptor 4 (CD11c), neither of which can be found on peripheral blood granulocytes. Peripheral blood granulocytes from rheumatoid and reactive arthritis patients expressed higher levels of leucocyte function-associated antigen 1 (CD11a) and of the membrane proteins CD31, CD24, M5, and M6 compared to peripheral blood granulocytes from healthy controls and patients with degenerative joint disease. No significant differences in the expression of any of the molecules studied could be observed between cells from rheumatoid and cells from reactive arthritis patients, suggesting a similar activation process for granulocytes in these two diseases.     

Human eosinophils are more toxic than neutrophils in antibody-independent killing

Eosinophils (EOSs) are implicated in damaging host tissues in diseases such as asthma and eosinophilic gastroenteritis. In the present study, we assessed the cytotoxicity of human EOSs from peripheral blood of patients with eosinophilia and from peritoneal fluid of patients undergoing continuous peritoneal dialysis and compared them to normal neutrophils. Cytotoxicity was measured by the release of 51chromium from cultured tumor cells and chicken erythrocytes. Both EOSs and neutrophils were separated on discontinuous Percoll gradients with greater than 95% purity. The granulocytes were activated by preincubation in an ice bath with phorbol myristate acetate and washed before incubation with the target cells. The EOSs lysed significantly more tumor cells (K562, Raji, and CEM lines) in an 18-hour assay than did neutrophils, and no significant difference was found between the peritoneal and blood EOSs. The EOSs were also much more efficient than neutrophils in lysing chicken erythrocytes when they were activated by granulocyte-macrophage colony-stimulating factor instead of phorbol myristate acetate. Cytolysis by EOSs is mediated by both oxidative and nonoxidative mechanisms, as indicated by experiments with cells from patients with chronic granulomatous disease. Thus, EOSs are much more cytotoxic than neutrophils and potentially much more damaging to patients with eosinophilia.     

Enhanced inhibition of in vitro multiplication of Plasmodium falciparum by stimulated human polymorphonuclear leucocytes

The effect of normal human peripheral blood polymorphonuclear leucocytes on in vitro multiplication of Plasmodium falciparum malaria parasites was investigated. It was shown that normal neutrophils were able to phagocytose parasitized erythrocytes and free parasites and thus inhibit in vitro multiplication of the parasite. Stimulation of the neutrophils by phorbol myristate acetate, a potent stimulus of leucocyte oxidative metabolism, resulted in enhanced inhibition of parasite growth. Superoxide dismutase, scavenger of superoxide anion, catalase, inhibitor of hydrogen peroxide, and sodium azide, inhibitor of myeloperoxidase, did not abrogate the inhibitory ability of the neutrophils. The results indicate that polymorphonuclear leucocytes play an important role in the defence against P. falciparum malaria.     

Identification of an IgA inhibitor of neutrophil chemotaxis and its membrane target for the metabolic burst.

Affinity-purified IgA from the serum of an 8-year-old boy with a 5-year history of recurrent facial nodules, intermittent neutropenia and elevated immunoglobulin levels, inhibited the chemotaxis of polymorphonuclear neutrophils (PMN) from both patient and normal adults. Preincubation of normal PMN with IgA from the patient's serum (0.5 mg/ml) inhibited chemotaxis to C5a and to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) by 80%, while IgA or IgG from pooled human serum and IgG from the patient were without effect. Normal PMN chemotaxis was restored after IgA depletion of the patient's serum by affinity chromatography. The patient's IgA, but not IgA from pooled human serum, bound specifically to normal PMN by its antigen-binding sites and recognized a 62,000 MW membrane protein on normal neutrophils, which was distinct from the FMLP receptor, the C5a receptor, or the Fca receptor. Attachment of the patient's IgA to the 62,000 MW protein activated intracellular oxidative metabolism on a parity with phorbol myristate acetate (PMA) and resulted in a significant up-regulation of membrane receptors for FMLP. After the binding of patient (Pt) IgA, normal neutrophils were rendered significantly less responsive to subsequent stimulation with phorbol esters. These results characterize a novel mechanism of chemotactic inhibition by serum IgA and also identify a neutrophil membrane protein that is linked to intracellular oxidative metabolism.     

Dysregulation of PMN antigen expression in Type 2 diabetes may reflect a generalized defect of exocytosis: influence of hypertension and microalbuminuria.

Defective exocytosis could underlie clinical and metabolic abnormalities in Type 2 diabetes. Because many SNARE proteins appear to be common mediators of exocytosis, we examined phorbol myristate acetate-stimulated expression of CD11b and CD69 on polymorphonuclear leukocytes (PMN) from Type 2 diabetic subjects with hypertension and microalbuminuria (D-htma), hypertension only (D-ht) or uncomplicated (D-uc), and normal controls (NC) by flow cytometry. CD11b expression was rapid (half maximal by 7 min), initially on all PMN. CD69 expression took place subsequently but on PMN that did not express CD11b. The proportion of CD11b-positive PMN at 30 min was higher in all diabetic groups than in NC. Expression of CD11b was higher and CD69 lower in D-uc and D-htma but were similar in NC and D-ht. In Type 2 diabetes the transition from the CD11b-positive to CD69-positive state is impaired. The defect in the process of CD69 expression appeared most marked in diabetic subjects with hypertension and microalbuminuria.     

Down-regulation of the T cell receptor CD3 zeta chain in rheumatoid arthritis (RA) and its influence on T cell responsiveness

T cells implicated in chronic inflammatory diseases such as RA respond weakly when stimulated in vitro with mitogen or antigen. The mechanism behind this hyporesponsiveness is unclear, but a depressed expression of the T cell receptor (TCR)-associated CD3zeta chain has been suggested. In the present work we describe a low expression of CD3zeta in synovial fluid (SF) T cells from RA patients compared with peripheral blood (PB) T cells, but no difference in CD3zeta expression between RA and healthy control PB T cells. In vitro studies demonstrated that granulocytes but not SF macrophages are able to down-regulate the expression of CD3zeta. Through stimulation with anti-CD3 antibodies we demonstrated that the TCR-dependent proliferative response was decreased in SF T cells compared with PB T cells. Stimulation with phorbol ester and ionomycin also resulted in a low proliferative response of SF T cells, indicating that both signal transduction through the TCR (stimulation with anti-CD3) and events further downstream in the signalling pathways (stimulation with phorbol ester and ionomycin) are affected. A similar depression of T cell activity was observed when induction of IL-2 and IL-4 was measured. However, SF T cells were not defective in the induction of interferon-gamma (IFN-gamma) when stimulated with phorbol myristate acetate (PMA)/ionomycin, in contrast to the diminished IFN-gamma response observed after stimulation with anti-CD3. This indicates that the hyporesponsiveness of SF T cells can not be generalized to all T cell functions. The differential response to external stimuli is likely to be of importance for the capacity of SF T cells to influence inflammatory reactions.     

Selective inhibition of polymorphonuclear leukocytes by immunosuppressive concentration of prostaglandin E2.

Prostaglandin E2(PGE2) has been implicated as an immunosuppressive agent and plasma levels of PGE2 are elevated in patients sustaining thermal injury. We examined the effect of 10(-7) M prostaglandin E2(PGE2) on human polymorphonuclear leukocytes (PMN) to determine whether it directly inhibits stimulated responses of these cells. At this concentration, PGE2 alone was incapable of stimulating PMN intracellular hydrogen peroxide production (indirectly assayed by fluorescence of 2'',7''-dichlorofluorescin) or expression of the PMN CD11b/CD16 surface glycoproteins. PMN incubated in the presence of the soluble stimuli phorbol myristate acetate(PMA, 100 ng/ml) or recombinant human C5a(rHC5a, 10(-8) M) generated significant amounts of hydrogen peroxide, increased their CD11b expression and decreased their CD16 expression. Pre-incubation of cells with PGE2 caused significant inhibition of all the observed changes stimulated by rHC5a. In contrast, events stimulated by PMA were not affected by preincubation of cells with PGE2. We conclude that PGE2, in concentrations identical to those found in the plasma of patients with burn injuries, is capable of selectively inhibiting some stimulated events and phenotypic expression of PMN in vitro study.     

Expression and function of AIM, an activation inducer molecule of human lymphocytes, is dependent on the activation of protein kinase C

AIM is an activation inducer molecule selectively expressed by activated lymphocytes through which agonistic proliferative signals can be triggered. The relationship between the expression of AIM with the activation of protein kinase C (PKC) has been studied. Different activators of PKC such as the active phorbol esters, phorbol myristate acetate and phorbol dibutyrate, or the phorbol-related ester mezerein were able to induce AIM expression on peripheral blood lymphocytes as assessed by immunofluorescence flow cytometry. Moreover, the expression of this activation antigen was also induced by treatment of peripheral blood lymphocytes either with dioctanoyl-rac-glycerol, a synthetic analogue of diacylglycerol, the physiological mediator of PKC activation. Further indirect evidence that AIM expression was dependent on the activation of PKC was obtained by blockade of the induction of its expression in cells treated with H7, an inhibitor of PKC. The AIM expression can be detected as early as 3 h after addition of phorbol esters and it requires active RNA and protein synthesis. The activation of PKC appears to be also required in the proliferative response induced by anti-AIM monoclonal antibody (mAb) in conjunction with phorbol esters. Agents such as phorbol myristate acetate, phorbol dibutyrate or mezerein but not the inactive phorbol ester methyl-phorbol myristate acetate induced a high proliferation of peripheral blood lymphocytes in the presence of anti-AIM mAb. In addition, we have demonstrated that the anti-AIM mAb is not sufficient by itself to induce cellular proliferation once the AIM antigen is expressed at the cell surface, requiring the simultaneous stimulation of the PKC to trigger high proliferative responses. Furthermore, the anti-AIM mAb did not appear to exert its effect on proliferation by rapidly increasing the intracytoplasmic Ca2+ levels. Taken together all these results indicate that the expression and function of AIM antigen is dependent on the activation of PKC.