Level of neutrophil chemotactic factor CINC/gro, a member of the interleukin-8 family, associated with lipopolysaccharide-induced inflammation in rats.

Rat cytokine-induced neutrophil chemoattractant (CINC), which is a counterpart of human gro and belongs to the interleukin-8 family, has been quantified by a new sandwich enzyme-linked immunosorbent assay. Administration of lipopolysaccharide (LPS) into an air pouch performed by subcutaneous injection of air caused inflammation and severe neutrophil infiltration. After the LPS injection, changes in the concentration of CINC/gro, chemotactic activity, and the number of neutrophils in the air pouch exudate were determined. The chemotactic activity of neutrophils was augmented before practical neutrophil infiltration. More than half of the chemotactic activity was neutralized by the antisera. The time kinetics of the level of CINC/gro coincided with the changes in chemotactic activity. The maximal level of rat CINC/gro was 85 ng/ml, which is sufficient to cause neutrophil migration in vitro and in vivo as described previously. These data suggest that rat CINC/gro is a functional chemoattractant for neutrophils in LPS-induced inflammation in rats.     

Rat CINC, a member of the interleukin-8 family, is a neutrophil-specific chemoattractant in vivo

Rat cytokine-induced neutrophil chemoattractant (CINC) is a member of the IL-8 family and its human counterpart is MGSA/gro. Rat neutrophil responses in vitro to rat CINC, human IL-8, and human MGSA/gro were studied. CINC concentrations as low as 1 nM induced apparent chemotaxis of rat neutrophils, but human IL-8 and MGSA/gro required concentrations one or two orders higher than that of CINC to attract neutrophils. These data indicate that human IL-8 and MGSA/gro cannot sufficiently substitute for rat counterparts such as CINC in rats. Therefore, the effect of rat CINC on rats was studied. Intradermally injected 10(-10)-10(-7) M CINC dose-dependently caused infiltration of neutrophils. Significant migration of neutrophils appeared by 30 min, and maximum infiltration was observed around 1-2 hr after the injection. CINC induced quick and transient neutrophil accumulation without lymphocyte and monocyte migration or edema formation. CINC, a member of the IL-8 family but a counterpart of human MGSA/gro-related proteins, is a specific neutrophil chemoattractant and can be distinguished from IL-8, which is a chemotactic factor for lymphocytes and neutrophils.     

Characterization of methylated bovine serum albumin-induced allergic inflammation in rats.

An air pouch type allergic inflammation in rats was induced using an insoluble cationic protein, methylated bovine serum albumin (MeBSA), as an antigen. Changes in vascular permeability, local tissue edema, histamine contents in the pouch fluid, and number of infiltrated leukocytes and chemotactic activity in the pouch fluid were analyzed during an 8-hour period after injecting the antigen solution into the air pouch of the immunized and nonimmunized rats. Vascular permeability during the first 30-min interval in the immunized rats was higher than that in the nonimmunized rats, reflecting a higher histamine level in the pouch fluid. However, both the increase in vascular permeability and histamine level in the immunized rats in this period were much lower than those induced by a soluble, noncationic antigen, azobenzenearsonate-conjugated acetyl bovine serum albumin. In the MeBSA-induced allergic inflammation model, a second peak of vascular permeability was induced at 2 h, and local tissue edema formation became apparent at 2 h, reaching a plateau at 4 h. A prominent increase in leukocyte infiltration, especially neutrophils, into the pouch fluid was induced at 4 h in accordance with an increase in chemotactic activity in the pouch fluid. These observations indicate that the acute phase of MeBSA-induced allergic inflammation is characterized by a weak anaphylactic response and a prominent neutrophil infiltration.     

Effect of rat CINC/gro, a member of the interleukin-8 family, on leukocytes in microcirculation of the rat mesentery.

Rat cytokine-induced neutrophil chemoattractant (CINC) is a member of the IL-8 family, and its human counterpart is gro/MGSA but not IL-8. We ascertained that chemically synthesized CINC was comparable to native CINC/gro with regard to chemotactic activity for rat neutrophils and studied the effect of synthesized CINC/gro on circulating leukocytes in microvascular vessels of rat mesentery. Exposure of rat mesentery to 10(-8)M authentic CINC/gro induced neutrophil adherence to and extravasation from postcapillary venules (PCVs) but not from capillaries or arterioles. CINC/gro concentrations as low as 10(-10) M were effective in causing neutrophil adherence. Neutrophils adhered to thin PCVs (mean diameter, approximately 25 microns) after exposure to CINC/gro for 15 min. The mean diameters of the PCV with adherence of neutrophils after exposure to CINC/gro for 30 and 60 min were 37 and 43 microns, respectively. The diameters of PCV with extravasation of neutrophils also increased in a time-dependent manner. The starting position of adherence of neutrophils was approximately 25-50 microns away from the upper junction of two vessels and remained virtually unchanged during exposure to CINC/gro for 60 min. However, the distance from the start to the end of neutrophil adherence increased in a time-dependent manner. The effect of CINC/gro on adherence and extravasation of leukocytes was neutrophil specific since other leukocytes such as lymphocytes and monocytes were not identified among the adherent and extravasated leukocytes.     

Leukocyte-Derived Neutrophil Chemotactic Factor-2 Produced by Infiltrated Leukocytes in Allergic Inflammation Model in Rats is Macrophage Inflammatory Protein-2

In the air pouch-type allergic inflammation model in rats, leukocytes collected from the pouch fluid 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils. The leukocytes from the immunized rats produced significantly higher amount of the chemotactic factors than that from the non-immunized rats. The major chemotactic factor, leukocyte-derived neutrophil chemotactic factor (LDNCF)-2, was purified and found to be identical with rat macrophage inflammatory protein (MIP)-2 by N-terminal amino acid sequence analysis. Expression of MIP-2 mRNA was higher in the leukocytes from the immunized rats than that from the non-immunized rats. Possible roles of LDNCF-2 (MIP-2) in neutrophil infiltration in the allergic inflammation is discussed.     

Leukocyte-Derived Neutrophil Chemotactic Factor-2 Produced by Infiltrated Leukocytes in Allergic Inflammation Model in Rats is Macrophage Inflammatory Protein-2

In the air pouch-type allergic inflammation model in rats, leukocytes collected from the pouch fluid 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils. The leukocytes from the immunized rats produced significantly higher amount of the chemotactic factors than that from the non-immunized rats. The major chemotactic factor, leukocyte-derived neutrophil chemotactic factor (LDNCF)-2, was purified and found to be identical with rat macrophage inflammatory protein (MIP)-2 by N-terminal amino acid sequence analysis. Expression of MIP-2 mRNA was higher in the leukocytes from the immunized rats than that from the non-immunized rats. Possible roles of LDNCF-2 (MIP-2) in neutrophil infiltration in the allergic inflammation is discussed.     

Inhibition of PAF-, LPS-, and cytokine-induced granulocyte accumulation in guinea pig lung by dexamethasone: Evidence that inhibition of IL-5 release is responsible for the selective inhibition of eosinophilia by glucocorticoids in guinea-pigs

The potency of dexamethasone has been determined as an inhibitor of intratracheally administered platelet activating factor- (PAF) or interleukin (IL)-5-induced eosinophilia, and of lipopolysaccharide-(LPS), tumour necrosis factor -(TNF) or cytokine-induced neutrophil chemoattractant- (CINC) induced neutrophilia in guinea-pig lungs. Dexamethasone was a potent inhibitor of PAF- induced eosinophil accumulation, but higher doses of dexamethasone were required to inhibit IL-5-induced eosinophilia. LPS-induced neutrophilia was less sensitive to the inhibitory effects of dexamethasone, than PAF-induced eosinophilia. Both LPS- and TNF-induced neutrophilia were inhibited by the same doses of dexamethasone. In contrast, higher doses of dexamethasone were required to inhibit CINC-induced neutrophilia. Since data in the literature show that PAF-induced eosinophilia in guinea-pig lungs is dependent on the generation of IL-5, it is concluded that inhibition of this response, by dexamethasone, is due to inhibition of release of IL-5. Similarly, although data in the literature show that LPS-induced neutrophilia is dependent on the generation of TNF, it is concluded that inhibition of this response, by glucocorticoids, is due to an action on an event which occurs after the release of TNF, possibly through inhibition of chemokine release.accepted by G. Bowen     

Downward regulation of neutrophil infiltration by endogenous histamine without affecting vascular permeability responses in air-pouch-type carrageenin inflammation in rats

The role of histamine in neutrophil infiltration and vascular permeability response in carrageenin air pouch inflammation in rats was examined. Injection of carrageenin solution into an air pouch induced a gradual increase in histamine content in the pouch fluid and histidine decarboxylase activity of pouch wall tissues, with a maximum attained at 24 h. Local administration of the H₂ antagonists cimetidine and famotidine, but not the H₁ antagonist pyrilamine, induced an increase in neutrophil infiltration at 24 h. Both types of histamine antagonists failed to suppress the vascular permeability response. In addition, H₂ antagonists attenuated the inhibitory effect of indomethacin on neutrophil infiltration without affecting the indomethacin-induced suppression of vascular permeability response. These results suggest that histamine produced in the inflammatory locus exerts a downward regulation of neutrophil infiltration through H₂ receptors but does not play any significant role in the vascular permeability response. Furthermore, the inhibition by indomethacin of neutrophil infiltration might be ascribed to the increase in histamine level in the pouch fluid.     

Contribution of cytokine-induced neutrophil chemoattractant CINC-2 and CINC-3 to neutrophil recruitment in lipopolysaccharide-induced inflammation in rats

OBJECTIVE: We have estimated the contribution of three types of cytokine-induced neutrophil chemoattractants (CINC-1, -2 and -3) and rat macrophage inflammatory protein (MIP)-1alpha to neutrophil recruitment in the air pouch/ lipopolysaccharide (LPS)-induced inflammation in rats. MATERIALS AND METHODS: Excess amounts of anti-CINC-2, CINC-3 and MIP-1alpha antibodies (Abs), together with LPS (1 microg/ml), were injected into the preformed air pouch of rats. Chemokine levels and chemotactic activity in the pouch fluid were measured using enzyme-linked immunosorbent assay and multiwell-type Boyden chambers in vitro, respectively. RESULTS: Excess amounts of the Abs significantly neutralized CINC-1 and almost completely neutralized CINC-2, CINC-3 and MIP-1 alpha in the pouch fluid, and a significant suppression (about 60% inhibition) of neutrophil infiltration by the Abs was found. In agreement with the in vivo results, in vitro neutralization experiments demonstrated that complete neutralization of CINCs and MIP-1alpha by the Abs resulted in a marked suppression (73% inhibition) of chemotactic potency of 8-h pouch fluid (exudate) from LPS-treated rats. On the other hand, treatment with anti-CINC-2, CINC-3 or MIP-1alpha Ab alone resulted in 48%, 34% or 10% inhibition, respectively, of chemotactic activity of the 8-h exudate. CONCLUSIONS: Our results suggest that CINC-2, a novel rat CXC chemokine and CINC-3 play an important role in neutrophil recruitment in the rat air pouch/LPS-induced inflammation.     

Rat Macrophage Inflammatory Protein-1α, a CC Chemokine, Acts as a Neutrophil Chemoattractant in Vitro and in Vivo

Recombinant rat macrophage inflammatory protein-1 (rMIP-1) at a concentration of 3 × 10^–8 M had strong neutrophil chemotactic activity, though the potency of rMIP-1 was less than that of cytokine-induced neutrophil chemoattractant (CINC)-1 at lower concentrations. In addition, rMIP-1 induced neutrophil chemotaxis in vivo when rMIP-1 was injected into the preformed air-pouch on the back of rats. The adhesion of rMIP-1-treated neutrophils to fibrinogen significantly increased, reaching a maximum adhesion at 10^–8 M. Stimulation of neutrophils with rMIP-1 induced a transient increase in intracellular free [Ca²⁺] dose-dependently. rMIP-1 still induced an increase in the intracellular [Ca²⁺] of rat neutrophils stimulated first with CINC-1, CINC-3 or C5a, suggesting that rat neutrophils have a specific receptor for rMIP-1. Supporting these findings, an additive increase in chemotactic potency was found when both rMIP-1 and CINC-1 were added to the lower wells of Boyden chamber in vitro. In addition, high levels of rMIP-1 were detected in the inflammatory site of air-pouch/carrageenan-induced inflammation in rats. Our results suggest that rMIP-1 acts as a neutrophil chemoattractant and, together with CINCs, plays an important role in infiltration of neutrophils into inflammatory sites in rats.     

Interleukin-1 stimulates rapid release of cytokine-induced neutrophil chemoattractant (CINC) in rat lungs

We found that intratracheal insufflation of interleukin-1 (IL-1) in rats rapidly increased lung lavage cytokine-induced neutrophil chemoattractant (CINC) concentrations, lung tissue myeloperoxidase (MPO) activity, and lung lavage neutrophil counts, and that CINC elevation preceded the migration of neutrophils into the lung. Further, we found that bolus CINC insufflation increased CINC concentrations in plasma, and we found that alveolar macrophages (AM) in lung tissue selections or AM recovered by lavage from rats given IL-1 intratracheally stained positively for CINC by immunohistochemistry. In addition, incubating rat AM with increasing doses of IL-1 in vitro progressively increased CINC concentrations in the culture medium. Our results suggest that the potent neutrophil chemoattractant CINC is rapidly produced and released by rat AM following challenge with IL-1 in vivo or in vitro, and support the hypothesis that CINC is an important mediator in the development of pulmonary inflammation in the rat.Parker B. Francis Fellow in Pulmonary Research.     

Possible role for platelet-activating factor in neutrophil infiltration in allergic inflammation in rats

Allergic inflammation was induced by injecting an antigen solution into an air pouch made on the dorsum of immunized rats with the antigen azobenzene-arsonate-conjugated acetyl bovine serum albumin. In this model, leukocyte infiltration into the pouch fluid was prominent 4-8 h after the antigen challenge. Most of the infiltrated leukocytes were neutrophils. Administration of the platelet-activating factor (PAF) antagonists such as CV-3988 and L-652,731 into the air pouch 15 min before and at the time of the antigen challenge failed to suppress leukocyte infiltration at 8 h. However, when the PAF antagonist was injected into an air pouch 4 h after the antigen challenge, neutrophil infiltration at 8 h was suppressed in a dose-dependent manner. Combined treatment with the 5-lipoxygenase inhibitor AA861 and the PAF antagonist did not potentiate the effect of the PAF antagonist, suggesting that participation of leukotriene B4 in neutrophil infiltration in this model is negligible. Eosinophil infiltration was very weak at 8 h, and the PAF antagonist showed no significant effect. At 8 h, the PAF level in the serum of the immunized rats was significantly higher than that of the nonimmunized rats. Intravenous administration of the PAF antagonist 15 min before the antigen challenge suppressed leukocyte infiltration more effectively than local administration into the pouch. These results indicate that PAF plays a significant role in neutrophil infiltration in allergic inflammation.     

In-vivo blockage of neutrophil migration by LPS is mimicked by a factor released from LPS-stimulated macrophages.

The present study was performed to determine the effect of an intravenous injection of the macrophage-derived neutrophil chemotactic factor (MNCF) (Cunha & Ferreira 1986) on neutrophil migration to rat peritoneal cavities, which were challenged with chemotactic stimuli. Macrophage monolayers stimulated by LPS release a factor (MW greater than 10,000 D) which, when injected intravenously, blocked neutrophil migration in carrageenin-induced peritonitis. This inhibition was dependent on dose and lasted more than 2 h. It was not due to neutropaenia, hypotension or LPS contamination. Neutrophil migration induced by LPS, MNCF, the Gram-negative bacterium Pseudomonas aeruginosa was also blocked by intravenous administration of the factor. Intravenous injection of recombinant interleukin 1 beta or tumour necrosis factor-alpha, present in the samples of the factor, failed to reproduce the described inhibitory effect on neutrophil migration. The release of this factor by LPS-stimulated macrophage monolayers was inhibited by dexamethasone but not by indomethacin. It is suggested that the failure of neutrophils to migrate during septicaemia may be the result of a continuous release of chemotactic factors in the circulation, particularly of the macrophage-derived neutrophil chemotactic factor(s).     

In-vivo blockage of neutrophil migration by LPS is mimicked by a factor released from LPS-stimulated macrophages

The present study was performed to determine the effect of an intravenous injection of the macrophage-derived neutrophil chemotactic factor (MNCF) (Cunha & Ferreira 1986) on neutrophil migration to rat peritoneal cavities, which were challenged with chemotactic stimuli. Macrophage monolayers stimulated by LPS release a factor (MW greater than 10,000 D) which, when injected intravenously, blocked neutrophil migration in carrageenin-induced peritonitis. This inhibition was dependent on dose and lasted more than 2 h. It was not due to neutropaenia, hypotension or LPS contamination. Neutrophil migration induced by LPS, MNCF, the Gram-negative bacterium Pseudomonas aeruginosa was also blocked by intravenous administration of the factor. Intravenous injection of recombinant interleukin 1 beta or tumour necrosis factor-alpha, present in the samples of the factor, failed to reproduce the described inhibitory effect on neutrophil migration. The release of this factor by LPS-stimulated macrophage monolayers was inhibited by dexamethasone but not by indomethacin. It is suggested that the failure of neutrophils to migrate during septicaemia may be the result of a continuous release of chemotactic factors in the circulation, particularly of the macrophage-derived neutrophil chemotactic factor(s).     

Aging Reduces the Expression of Lung CINC and MCP-1 mRNA in a P. aeruginosa Rat Model of Infection

We investigated dynamic changes of inflammatory cell infiltration and expression of cytokine-induced neutrophil chemoattractant (CINC) and monocyte chemoattractant protein-1 (MCP-1) mRNA in aged rats with Pseudomonas aeruginosa pulmonary infection. Disease manifestation and lung tissue pathology (lesion dispersion, inflammatory reactions, tissue edema and bleeding) were more severe in aged rats than young rats. At various time points, lung tissue polymorphonuclear neutrophil and mononuclear macrophage numbers were lower in the aged group than the young group (P < 0.05), and at 24 h there was no difference in mononuclear macrophage numbers. After inoculation with P. aeruginosa, CINC and MCP-1 mRNA expression increased in both groups, but the peak lagged in old rats compared with young. Thus, aging can reduce the expression of CINC and MCP-1 mRNA in lung tissues, and reduce the infiltration of neutrophils and monocyte–macrophages induced by CINC and MCP-1. This might lead to increased risk of pneumonia in elderly patients.     

Inhibition of neutrophil infiltration by a malleable protein matrix of lactic acid bacteria-fermented whey proteins in vivo

Objective:  A novel nutraceutical ingredient, the Malleable Protein Matrix (MPM), has previously demonstrated a significant anti-inflammatory effect in a systemic inflammatory disease model, comparable to conventional drugs. The objective of this study was to investigate the potential anti-inflammatory effects of MPM on neutrophil infiltration in vivo, phagocytosis activity as well as cytokine and chemokine production. Methods:  Groups of ten C57BL\6J mice received water or MPM per os for a period of 2 weeks prior to the creation of a murine air pouch. The subsequent neutrophil recruitment and activities were characterized following lipopolysaccharide injection. Results:  In the water control group, the number of recruited cells was 1.8X10⁷ cells/pouch, which was reduced to 9X10⁶ cells/pouch with oral MPM consumption, representing an inhibition of 50% of infiltrating leukocytes. A considerable reduction in the cytokine and chemokine production, mostly TNFα, IL-1β and IL-6 production in the MPM-treated group, suggested an inhibition of the mediators responsible for leukocyte extravasation. On the other hand, MPM consumption had no effect on neutrophil phagocytosis activity. Conclusion:  MPM administration demonstrates a significant reduction of neutrophil infiltration associated with an inhibition of cytokine and chemokine production. The air pouch model shares similarities with in vivo characteristics of rheumatoid arthritis and neutrophilic diseases, both of which would benefit from this 50% inhibition of neutrophil infiltration induced by MPM. Received 14 May 2007; returned for revision 7 October 2007; received from final revision 25 April 2008; accepted by S. Stimpson 27 June 2008     

Inflammation induced by bacterial cell wall fragments in the rat air pouch. Comparison of rat strains and measurement of arachidonic acid metabolites.

Streptococcal cell wall fragments, suspended in phosphate-buffered saline, were injected into a preformed subcutaneous air pouch in rats. The advantage of the air pouch model is the capacity for quantitation of exudative, cellular, and proliferative responses and soluble mediators. Accumulation of pouch fluid containing many leukocytes occurred during the first 3 days. Granulation tissue separable from the surrounding subcutaneous tissue developed by 6 days. Immunofluorescence and immunoperoxidase staining showed the presence of cell walls in inflammatory cells both in pouch fluid and in pouch tissue. Histologic features of this inflammation included an acute exudative phase with a predominantly neutrophil infiltration followed by a chronic phase characterized by fibroblast proliferation, formation of blood vessels, and infiltration with mononuclear cells. The lining of the pouch before injection of cell wall developed morphologic features of synovial membrane, which became more evident during the chronic phase of induced inflammation. Outbred Sprague-Dawley and inbred Lewis rats developed more pouch fluid, cell numbers in the pouch fluid, and granulation tissue than inbred Buffalo rats. The arachidonic acid metabolites, prostaglandin E2 and leukotriene B4, were measured in the pouch fluid, and more of each was produced in the Lewis than in the Buffalo strain. These measurements of inflammation are consistent with the relative susceptibility of these strains to cell-wall-induced arthritis. This model of inflammation can be used in the examination of the regulatory mechanisms of evolving chronic inflammation.     

Beneficial effects of troglitazone on neutrophil dysfunction in multiple low-dose streptozotocin-induced diabetic mice

Patients with poorly controlled diabetes are at high risk of acquiring bacterial infections. However, conflicting results have been reported on neutrophil function in diabetes. We periodically evaluated neutrophil dysfunction in multiple low-dose streptozotocin (STZ)-induced diabetic mice, and then evaluated the effects of troglitazone and other thiazolidinediones (TZDs) on the decline of neutrophil function. Zymosan was injected intraperitoneally and neutrophil infiltration and phagocytosis were evaluated. While phagocytosis of zymosan by peritoneal neutrophils was consistently reduced in diabetic mice, neutrophil infiltration was decreased on day 30, but increased on day 40 after STZ injection. The in vitro chemotactic and phagocytic activities of blood neutrophils in mice that did not receive zymosan were consistently reduced in diabetic mice. Phorbol myristate acetate (PMA)-stimulated superoxide production by zymosan-induced peritoneal neutrophils and the levels of zymosan-induced tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta in peritoneal exudate fluids were also reduced in the diabetic mice. Treatment of the diabetic mice with troglitazone beginning 2 weeks after STZ injection did not improve hyperglycaemia but did prevent the decline of zymosan-induced neutrophil infiltration on day 30, and additionally promoted the increased infiltration on day 40. Troglitazone also promoted the chemotactic activity of blood neutrophils isolated from normal mice in vitro. Rosiglitazone but not pioglitazone induced a similar effect. Neutrophil phagocytosis was not enhanced by troglitazone either in vivo or in vitro. Taken together, neutrophil function is impaired by STZ-induced diabetes, but inflammatory infiltration does not always vary with the chemotactic disability or cytokine levels. Furthermore, troglitazone and rosiglitazone were suggested to improve at least neutrophil chemotactic activity in these animals.     

Recombinant gamma interferon causes neutrophil migration mediated by the release of a macrophage neutrophil chemotactic factor

A dose-dependent neutrophil migration was observed following the injection of purified (Hu IFN-gamma) or recombinant (rIFN-gamma) human gamma interferon into rat peritoneal cavities. This finding contrasts with their inability to cause chemotaxis in vitro in the Boyden chamber. Neutrophil migration into peritoneal cavities and subcutaneous air pouches induced by both preparations of interferon was abolished by pretreatment of the animals with dexamethasone. IFN-gamma-induced neutrophil migration was enhanced when the macrophage population of the peritoneal cavities was increased by previous injection of thioglycollate and reduced by peritoneal lavage. Macrophage monolayers pretreated either with rIFN-gamma or with lipopolysaccharide from E. coli release into the supernatant a factor that stimulates neutrophil recruitment in animals treated with dexamethasone. Dexamethasone blocked this release but did not affect the neutrophil recruitment induced by this factor. These results suggest that IFN-gamma-induced neutrophil migration in vivo may be mediated by the release from resident macrophages of a neutrophil chemotactic factor and that dexamethasone blockade of neutrophil recruitment by IFN-gamma is due to inhibition of the release of this factor.     

Neutrophil-mediated tumor cell destruction in cancer ascites. II. A OK-432 attracts killer neutrophils through activation of complement C5

When a streptococcal preparation, OK-432, was administered intraperitoneally to patients with malignant ascites, the number of neutrophils with cytotoxic activity against tumor cells was increased in the peritoneal cavity immediately after the OK-432 injection. In order to investigate the underlying mechanisms of such neutrophil accumulation, a possible neutrophil chemotactic activity in ascitic fluid was assayed by a modified Boyden method. The chemotactic activity for neutrophils was found significantly higher 6 hr after the OK-432 injection. OK-432 along had no direct chemotactic activity for neutrophils. The chemotactic activity was generated in vitro when ascitic fluid from patients without OK-432 treatment was incubated with OK-432 for 30 min at 37 degrees C. However, preheating of the fluid at 56 degrees C for 30 min or the addition of EDTA to the fluid resulted in the failure of generation of the chemotactic activity after the incubation with OK-432. The addition of EGTA did not show a significant effect. The chemotactic activity in ascitic fluid was found near cytochrome c marker (MW 12,400 D), when fractionated by Sephadex G-200 gel chromatography. The chemotactic activity was heat stable, nondialyzable, and neutralized completely with anti-human complement C5 antibodies. These results suggest that C5a generated via the alternative pathway activated by OK-432 may be responsible for the infiltration of killer neutrophils in the peritoneal cavity in patients with malignant ascites when they are treated by the intraperitoneal injection of OK-432.