Ecotin protects Salmonella Typhimurium against the microbicidal activity of host proteases.

Salmonella enterica serovar Typhimurium causes acute diarrhea upon oral infection in humans. The harsh and proteolytic environment found in the gastrointestinal tract is the first obstacle that these bacteria face after infection. However, the mechanisms that allow Salmonella to survive the hostile conditions of the gut are poorly understood. The ecotin gene is found in an extensive range of known phyla of bacteria and it encodes a protein that has been shown to inhibit serine proteases. Thus, in the present work we studied the role of ecotin of Salmonella Typhimurium in host-pathogen interactions. We found that Salmonella Typhimurium Δ ecotin strain exhibited lower inflammation in a murine model of Salmonella induced colitis. The Δ ecotin mutant was more susceptible to the action of pancreatin and purified pancreatic elastase. In addition, the lack of ecotin led to impaired adhesion to Caco-2 and HT-29 cell lines, related to the proteolytic activity of brush border enzymes. Besides, Δ ecotin showed higher susceptibility to lysosomal proteolytic content and intracellular replication defects in macrophages. In addition, we found Ecotin to have a crucial role in Salmonella against the microbicide action of granules released and neutrophil extracellular traps from human polymorphonuclear leukocytes. Thus, the work presented here highlights the importance of ecotin in Salmonella as countermeasures against the host proteolytic defense system. IMPORTANCE: The gastrointestinal tract is a very complex and harsh environment. Salmonella is a successful food borne pathogen, but little is known about its capacity to survive against the proteolysis of the gut lumen and intracellular proteases. Here, we show that Ecotin, a serine protease inhibitor, plays an important role in protecting Salmonella against proteases present at different sites encountered during oral infection. Our results indicate that Ecotin is an important virulence factor in Salmonella , adding another tool to the wide range of features this pathogen uses during oral infection.

Desiccating stress-induced disruption of ocular surface immune tolerance drives dry eye disease.

Dry eye is an allegedly autoimmune disorder for which the initiating mechanisms and the targeted antigens in the ocular surface are not known, yet there is extensive evidence that a localized T helper type 1 (Th1)/Th17 effector T cell response is responsible for its pathogenesis. In this work, we explore the reconciling hypothesis that desiccating stress, which is usually considered an exacerbating factor, could actually be sufficient to skew the ocular surface's mucosal response to any antigen and therefore drive the disease. Using a mouse model of dry eye, we found that desiccating stress causes a nuclear factor kappa B (NF-κB)- and time-dependent disruption of the ocular surface's immune tolerance to exogenous ovalbumin. This pathogenic event is mediated by increased Th1 and Th17 T cells and reduced regulatory T cells in the draining lymph nodes. Conversely, topical NF-κB inhibitors reduced corneal epithelial damage and interleukin (IL)-1ß and IL-6 levels in the ocular surface of mice under desiccating stress. The observed effect was mediated by an augmented regulatory T cell response, a finding that highlights the role of mucosal tolerance disruption in dry eye pathogenesis. Remarkably, the NF-κB pathway is also involved in mucosal tolerance disruption in other ocular surface disorders. Together, these results suggest that targeting of mucosal NF-κB activation could have therapeutic potential in dry eye.

Benzalkonium chloride breaks down conjunctival immunological tolerance in a murine model.

The impact of topical eye drops with benzalkonium chloride (BAK) as a preservative could involve more than the reported toxic effects on the ocular surface epithelium and ultimately affect the immune balance of the conjunctiva. We found that BAK not only impairs tolerance induction in a murine model, but leads to mild systemic immunization. Contrasting with antigen only-treated mice, there was no induction of interleukin 10-producing antigen-specific CD4(+) cells in BAK-treated animals. Moreover, the tolerogenic capacity of migrating dendritic cells (DCs) was reduced, apparently involving differential conditioning by soluble epithelial factors. Accordingly, epithelial cells exposed in vitro to BAK were less suppressive and failed to induce tolerogenic DCs in culture. As this effect of BAK was dependent on epithelial nuclear factor κB pathway activation, our findings may provide new therapeutic targets. Thus, tolerance breakdown by BAK should be considered an important factor in the management of glaucoma and immune-mediated ocular surface disorders.

Downregulation of mac-1 expression in monocytes by surface-bound IgG.

Physical and functional association between the beta2-integrin Mac-1 (CD11b/CD18) and receptors of immunoglobulin G (IgG) (FcgammaRs) has been previously reported. In this study, we examined the modulation of Mac-1 expression by IgG in different leucocyte populations. Our data show that human monocytes, but not neutrophils, macrophages, dendritic or natural killer cells, downregulate the expression of Mac-1 after overnight exposure to surface-bound IgG. This effect, which requires at least 6 h of incubation, is not associated with a general downmodulation of membrane antigens, and is selectively induced by immobilized IgG (iIgG), as the stimulation of monocytes with N-formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide, tumour necrosis factor-alpha (TNF-alpha) or soluble IgG did not modify the Mac-1 expression after 18 h in culture. The loss of Mac-1 was completely prevented by blocking antibodies (Abs) directed to FcgammaRII (CD32) or CD18. On the other hand, the serine protease inhibitor, phenyl methyl sulphonyl fluoride, but not inhibitors of cysteine proteases or neutral endopeptidases, partially prevented the downregulation of Mac-1 by iIgG. Monocytes cultured overnight on iIgG exhibited a dramatic decrease in their capacity to ingest zymosan particles that could be attributed to the reduced expression of Mac-1. However, there was no inhibition of TNF-alpha production induced by zymosan, suggesting that Mac-1-dependent responses require different levels of its expression to be fully activated.

Non-malignant leukocytes delay spontaneous B-CLL cell apoptosis.

Malignant B cells from chronic lymphocytic leukemia (B-CLL) patients have a long survival in vivo, although, in culture, they spontaneously die by apoptosis. Here, we analyzed the capacity of accessory leukocytes to modulate apoptosis of B-CLL cells in vitro. To this end, we performed long-term cultures using total mononuclear cells (TMC) from B-CLL patients and TMC depleted from monocytes, NK cells and T lymphocytes (B-CLL cells). In all the patients studied (n = 25) the presence of accessory leukocytes markedly prolonged the survival of B-CLL cells. The anti-apoptotic effect was exerted by monocytes and, to a lesser degree, NK cells, partially through the release of soluble factors. Indeed, accessory leukocytes separated from leukemic cells by semipermeable membranes were still able to prolong B-CLL cell survival. By flow cytometric analysis we found that the protective effect of non-malignant cells was associated with delayed down-regulation of Bcl-2 expression on leukemic cells. By contrast, the expression of Fas and Fas ligand proteins was unchanged in most samples. Our findings suggest that monocytes and NK cells, by delaying leukemic cell apoptosis, may play a role in B-CLL cell accumulation in vivo.

Promotion of neutrophil apoptosis by TNF-alpha.

We examined the ability of TNF-alpha to modulate human neutrophil apoptosis. Neutrophils cultured with TNF-alpha alone undergo a low but significant increase in the number of apoptotic cells. More interestingly, when neutrophils were pretreated with TNF-alpha for 1-2 min at 37 degrees C and then were exposed to a variety of agents such as immobilized IgG, IgG-coated erythrocytes, complement-treated erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP, Escherichia coli, and GM-CSF for 3 h at 37 degrees C, a marked stimulation of apoptosis was observed. Similar results were obtained in neutrophils pretreated with TNF-alpha for 30 min, 1 h, 3 h, and 18 h. Dose-dependent studies showed that TNF-alpha enhances neutrophil apoptosis at concentrations ranging from 1 to 100 ng/ml. In contrast to the observations made in neutrophils pretreated with TNF-alpha, there was no stimulation of apoptosis when TNF-alpha was added to neutrophils previously activated by conventional agonists. Experiments performed to establish the mechanism through which TNF-alpha promotes neutrophil apoptosis showed that neither reactive oxygen intermediates nor the Fas/Fas ligand system appear to be involved. Our results suggest that TNF-alpha plays a critical role in the control of neutrophil survival by virtue of its ability to induce an apoptotic death program which could be triggered by a variety of conventional agonists.

Immune complexes inhibit apoptosis of chronic lymphocytic leukaemia B cells.

In the present study we examined the effect of immune complexes (IC) on the survival of chronic lymphocytic leukaemia (B-CLL) B cells. Our results showed that either precipitating IC (pIC), Ab-coated erythrocytes (E-IgG) or heat-aggregated IgG (aIgG) significantly inhibited spontaneous apoptosis of B-CLL cells, as well as that induced by fludarabine, chlorambucil or dexamethasone. After depletion of T lymphocytes, monocytes and NK cells, incubation with IC was no longer able to delay B-CLL cells apoptosis, suggesting that prevention of apoptosis depends on IC interaction with accessory leucocytes. The release of IFNgamma by non-malignant cells upon activation with IC was responsible, to some extent, for IC effects as shown by the fact that neutralizing anti-IFNgamma MoAb partially prevented their ability to inhibit B-CLL cells apoptosis. The observation that treatment with IC resulted in increased expression of HLA-DR on B-CLL cells suggests that inhibition of apoptosis is associated with cellular activation.

Acidic pH increases the avidity of FcgammaR for immune complexes.

The interaction of immunoglobulin G (IgG) antibodies with FcgammaR constitutes a critical mechanism through which IgG antibody effector functions are mediated. In the current work we have examined whether human neutrophil FcgammaR exhibit pH dependence in their association with IgG. Binding assays were performed in culture medium adjusted to different pH values. It was found that the binding of either heat-aggregated human IgG (AIgG), soluble immune complexes (sIC) or IgG-coated erythrocytes (IgG-E) was markedly higher at pH 6.5 than at pH 7.3. This effect was not observed when saturation of FcgammaR was achieved, suggesting that acidic pH increases the avidity of FcgammaR for IC without modifying the total binding capacity. Similar results were observed for the binding of AIgG to either monocytes, natural killer (NK) or K562 cells, suggesting that acidic pH increases the avidity of both, FcgammaRII and FcgammaRIII. Additional experiments were performed to analyse whether the binding of IgG to FcgammaRI also showed pH dependence. To this aim, we employed interferon-gamma-treated human neutrophils and mouse inflammatory macrophages, previously incubated with blocking antibodies directed to FcgammaRII and FcgammaRIII. Acidic pH did not enhance the binding of AIgG nor monomeric IgG under these experimental conditions. Further studies are required to determine whether the enhancement of FcgammaR avidity for IC could be attributed to titration of histidine(s) residues on the Fc fragment of IgG.

Extracellular acidification induces human neutrophil activation.

In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5-7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.

Effect of nitric oxide donors on oxygen-dependent cytotoxic responses mediated by neutrophils.

We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1-2 min at 37 degrees C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions.

Modulation of human neutrophil apoptosis by immune complexes.

In the present study we examined whether immune complexes (IC) are able to modulate human neutrophil apoptosis. We observed different effects depending on the type of IC employed. Precipitating IC (pIC) and Ab-coated erythrocytes (E-IgG) triggered a marked stimulation of apoptosis, while heat-aggregated IgG and soluble IC, significantly delayed spontaneous apoptosis. Blocking Abs directed to Fcgamma receptor type II (FcgammaRII), but not to FcgammaRIII, markedly diminished the acceleration of apoptosis triggered by either pIC or E-IgG, supporting a critical role for FcgammaRII in apoptosis stimulation. This phenomenon, on the other hand, does not appear to involve IC phagocytosis or the participation of CR3. Acceleration of neutrophil apoptosis triggered by either pIC or E-IgG seems to require the activation of the respiratory burst, as suggested by 1) the ability of catalase to prevent apoptosis stimulation; 2) the effect of azide, an heme enzyme inhibitor, which dramatically enhanced apoptosis induced by pIC or E-IgG; and 3) the inability of pIC or E-IgG to accelerate apoptosis of neutrophils isolated from CGD patients. It is well established that IC affect the course of inflammation by inducing the release of inflammatory cytokines, proteolytic enzymes, oxidative agents, and other toxic molecules. Our results suggest that IC may also affect the course of inflammation by virtue of their ability to modulate neutrophil apoptosis.

Losartan, a selective inhibitor of subtype AT1 receptors for angiotensin II, inhibits the binding of N-formylmethionyl-leucyl-phenylalanine to neutrophil receptors.

Losartan, a selective antagonist of AT1 receptors for angiotensin II, is widely used clinically to manage hypertension. We report here that losartan markedly inhibits neutrophil shape change, adherence and chemiluminescence responses triggered by N-formylmethionyl-leucyl-phenylalanine (fMLP), without affecting responses induced by immune complexes, zymosan or concanavalin A. Neither saralasin, another antagonist of angiotensin II receptors, nor captopril, an angiotensin-converting enzyme inhibitor, reproduced the effects of losartan. It was also observed that neutrophil responses triggered by fMLP were not affected by exogenously added angiotensin II. The effect of losartan on the binding of fMLP was measured using [3H]fMLP. It was found that losartan inhibits the binding of [3H]fMLP to neutrophil receptors. As observed for neutrophils, studies performed with monocytes showed that losartan inhibits chemiluminescence emission triggered by fMLP, without affecting chemiluminescence responses triggered by immune complexes, zymosan or concanavalin A.

Inhibition of human neutrophil apoptosis by platelets.

In the absence of appropriate stimuli, polymorphonuclear neutrophils rapidly undergo characteristic changes indicative of programmed cell death or apoptosis. We report here that neutrophils cultured in the presence of platelets (neutrophil:platelet ratios of 1:50, 1:25, and 1:10) show a dramatic inhibition of apoptosis compared with neutrophils cultured alone. Similar degrees of apoptosis delay were induced by viable unstimulated platelets, fixed unstimulated platelets, or fixed activated (1 U/ml thrombin) platelets. Inhibition of apoptosis was associated with prolongation of the functional lifespan of the neutrophil, as indicated by the higher capacity of platelet-treated neutrophils to display chemiluminescence responses triggered by FMLP, immune complexes, and zymosan. The mechanism responsible for the inhibition of neutrophil apoptosis by platelets has not yet been defined. However, it seems that classical recognition systems such as those mediated by the interaction between platelet P-selectin (CD62) or glycoprotein IIb/IIIa complex and their counter-receptors expressed by neutrophils are not involved.

Nitric oxide synthase inhibitors enhance plasma levels of corticosterone and ACTH.

The role of nitric oxide in the regulation of adrenal steroidogenesis was examined in BALB/c mice by employing the nitric oxide synthase inhibitors NG-nitro L-arginine methyl ester (L-NAME) and NG-nitro L-arginine (L-NNA). The administration of a single dose of nitric oxide inhibitors (50 mg kg-1 body wt. i.p.) induced a fourfold increase in plasma corticosterone. Treatment with L-arginine (750 mg kg-1 body wt. s.c.), but not D-arginine, completely prevented corticosterone increases induced by L-NAME. To analyse whether the activation of adrenal steroidogenesis induced by nitric oxide synthase inhibitors involved the stimulation of the hypothalamo-pituitary-adrenal axis. ACTH levels were assessed. It was found that L-NAME significantly enhanced plasma ACTH concentrations. Genetic variations in this regulatory pathway are suggested by the fact that L-NAME increased corticosterone levels in BALB/c. C3H/He and DBA-2 mice, but not in C57BI/c mice, a strain characterized by a low steroid response to stress.

Neutrophil apoptosis induced by proteolytic enzymes.

In this study, we show that three proteolytic enzymes of different specificity-pronase, chymotrypsin, and trypsin-induced a dramatic stimulation of neutrophil apoptosis as shown by morphologic characteristics, analysis of cell DNA content, and presence of a characteristic "ladder" pattern of DNA fragmentation. The action of either chymotrypsin or trypsin was completely prevented by the serine protease inhibitor aprotinin, indicating that the proteolytic activity of the enzymes accounts for apoptosis induction. Stimulation of neutrophil apoptosis by proteases was observed in culture medium supplemented with either inactivated fetal calf serum (0.1-50%), autologous serum (0.1-50%), bovine serum albumin (0.1%), or in protein-free medium. Other cell types such as human peripheral blood monocytes and lymphocytes, human leukemic cells from THP-1, HL-60 and K562 lines, murine L929 fibroblasts, and unstimulated murine macrophages harvested from the peritoneal cavity were not induced to undergo apoptosis after the treatment with proteases. In an attempt to determine whether neutrophil serine proteases could induce apoptosis as chymotrypsin and trypsin do, the effect of elastase was assessed. A significant increase in the percentage of apoptotic cells was observed in elastase-treated neutrophils. We propose that the selective stimulation of neutrophil apoptosis by proteolytic enzymes may play an important role in the normal resolution of inflammation by limiting the autotoxic potential of the neutrophil.

Activation of human neutrophils induced by immune complexes prepared with cationic and anionic fractions of normal IgG antibodies.

The authors have recently shown that the ability of immune complexes (IC) to trigger Fc gamma R-dependent cell responses can be dramatically enhanced when the isoelectric point (pI) of normal IgG antibodies is increased from 5.8-8.5 to 8.5-9.8 by treatment with 1-ethyl-3-2(3-dimethylaminopropyl) carbodiimide HCl and ethylene diamine. In the current work the authors analyse whether differences in the charge of normal IgG antibodies may also affect IC activity. Soluble IC (sIC) were prepared with (a) rabbit IgG antibodies to human IgG and anionic or cationic fractions of human IgG; and (b) bovine serum albumin (BSA) and anionic or cationic fractions of rabbit IgG anti-BSA antibodies. Similar abilities to bind to neutrophil surface were observed for sIC prepared with both anionic (anIC) and cationic fractions of IgG (catIC). Moreover, no differences were found when neutrophil shape change, chemiluminescence (CL) emission and elastase release were induced by either anIC or catIC. As in the case of sIC, particulate IC prepared with erythrocytes (E) and anionic or cationic fractions of specific IgG antibodies (IgG-E) showed no differences in their abilities to trigger either CL emission or ADCC. Taken together, these results suggest that the pI of normal IgG antibodies do not affect the ability of IC to trigger neutrophil responses mediated by receptors for the Fc portion of IgG antibodies (Fc gamma R).

Effect of nitric oxide donors on neutrophil responses induced by immune complexes.

The present study characterizes the effect of two nitric oxide (NO) donors, S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the ability of neutrophils to perform different responses triggered by immune complexes (IC). Pretreatment of neutrophils with either GSNO or SNP exerted a biphasic action on antibody-dependent cellular cytotoxicity (ADCC) performed against erythrocytes (E) coated with IgG antibodies (IgG-E), depending on the amount of IgG employed. While with high amounts of antibodies ADCC was markedly inhibited, at low amounts of antibodies it was significantly increased. Both effects were prevented by haemoglobin, a NO scavenger. Moreover, these effects were reproduced by the cell-permeable analogue of cGMP, dibutyryl cGMP (Bt2cGMP). Other neutrophil functions triggered by IgG-E were also examined. It was found that NO donors did not affect either the phagocytosis of IgG-E or the emission of chemiluminescence (CL). Finally, neutrophil functions triggered by soluble IC (sIC) and precipitating IC (pIC) were analysed. It was observed that NO donors did not modify either cytotoxicity performed towards non-sensitized target cells or CL emission. The significance of these results is discussed.

Involvement of nitric oxide in the regulation of peripheral blood leukocyte counts.

A role for nitric oxide (NO) in the regulation of blood leukocyte numbers was examined in BALB/c mice by employing the NO synthase inhibitor NG-nitro L-arginine methyl ester (L-NAME). Treatment of animals with a single dose of 50 mg/kg body wt caused a dramatic increase in the number of circulating neutrophils and a moderate decrease in the number of circulating lymphocytes. These effects were partially reversed by the simultaneous inoculation of L-arginine (250 mg/kg body wt.) but not by D-arginine. A second NO synthase inhibitor, NG-nitro L-arginine, induced changes comparable to those elicited by L-NAME. Because catecholamines and glucocorticoids are well-known modulators of blood leukocyte counts, experiments were carried out in adrenalectomized mice. It was found that adrenalectomy did not modify the increase in the number of circulating neutrophils induced by L-NAME but completely prevented the decrease of circulating lymphocytes. Taken together, these findings support the hypothesis that NO plays an important role in the regulation of the peripheral blood number of neutrophils and lymphocytes, and that this function involves, in each case, the participation of different mechanisms.

Neutrophil chemotaxis induced by immune complexes.

In the current work we have analyzed the ability of different soluble immune complexes (IC) prepared with IgG antibodies to induce neutrophil chemotactic responses in vitro. While, in all cases, IC were able to induce neutrophil migration in a concentration-dependent fashion, IgG antibodies alone were completely unable to induce locomotor responses. Checkerboard analysis indicated the chemotactic nature of motility. On the other hand, chemotaxis induced by IC was markedly inhibited by IV. 3, a monoclonal antibody (mAb) to Fc gamma RII, slightly reduced by 3G8 F(ab')2, a mAb to Fc gamma RIII, and nearly abrogated by both mAbs. The impact of IC on neutrophil migration induced by FMLP was also studied. We found that when a suboptimal concentration of FMLP was employed, the simultaneous addition of IC increased the migration acting in additive form. The significance of these results is discussed.

Effect of proteolytic enzymes on neutrophil Fc gamma RII activity.

Previous studies have demonstrated that the treatment of neutrophils with proteolytic enzymes markedly reduces the expression of receptor III for the Fc portion of IgG (Fc gamma RIII), but it does not affect the number of Fc gamma RII on the cell surface. In the present study, we analysed the effect of proteolytic enzymes on functional responses of neutrophils induced by immune complexes (IC). Our results showed that treatment with pronase or chymotrypsin markedly increased the binding of IgG-coated erythrocytes (IgG-E) to neutrophils, as well as their capability to display IgG-mediated functions such as antibody-dependent cellular cytotoxicity (ADCC) and chemiluminescence (CL) induced by IgG-E, responses that have been shown to be completely dependent on Fc gamma RII. A similar enhancing effect was observed, in all cases, after neutrophil treatment with neuroaminidase. We also studied the effect of proteolysis on neutrophil activation induced by other types of IC. It was found that pronase and chymotrypsin significantly enhanced CL responses induced by soluble IC (sIC) but did not modify the responses induced by either precipitating IC (pIC) or soluble IC prepared with cationized antibodies (catIC). On the other hand, neuroaminidase significantly enhanced CL induced by either sIC, pIC or catIC. Taken together, our data suggest that the activity of Fc gamma RII can be up-regulated by proteolysis. However, this effect appears to be strongly dependent on the characteristics of the IC employed as stimulus.