Collagenolytic activity of carrageenin granuloma in rats.

Collagenolytic activity at various phases of the development of carrageenin granuloma was investigated by measuring the amount of dialysable hydroxyproline formed during incubation in vitro of minced granulation tissue. Collagenolytic activity reached a maximum at day 8 after carrageenin injection and then decreased gradually, while collagen synthetic activity was rapidly decreased from day 4 to day 11. The significance of dialysable hydroxyproline in native collagen breakdown of carrageenin granuloma is discussed.

The content of prostaglandin E and prostaglandin F2alpha in the exudate of carrageenin granuloma of rats.

1. Granuloma was made by the subcutaneous injection of 2% carrageenin solution on the dorsum of male rats. Eight, 16, 24 and 72 h after the injection. the exudate from each rat granuloma was withdrawn and extracted for rpstaglandins. 2. Extracted prostaglandins were separated prostaglandin E and prostaglandin F group by silicic acid mini-column chromatography. Then the amount of prostaglandin E and prostaglandin F2alpha were determined by the radioimmunoassay method. 3. The levels of prostaglandin E in the granuloma exudates were 4.6 ng/ml at 8 h after the carrageenin injection, then decreased 3.6 ng/ml and to 1.1 ng/ml at 16 h and 24 h, respectively. Seventy-two h after the injection, prostaglandin E level was increased to 8.1 ng/ml. 4. The levels of prostaglandin F2alpha in the exudate were as follows: At 8 h after the carrageenin injection, the level was 9.4 ng/ml, then decreased to 1.3 ng/ml and to 0.8 ng/ml at 16 h and 24 h, respectively. Seventy-two h after the carrageenin injection, it was again elevated to 4.7 ng/ml. 5. The exudate of granuloma, 24 and 72 h after the carrageenin injection, was incubated with [3H]prostaglandin E1 at 37 degrees C for 30 min. Then the acidic ether extract was subjected to reversed phase partition chromatography. It was found that the exudate of 24 h and 72 h granuloma had little activity of prostaglandin 15alpha-hydroxy dehydrogenase.

Stimulation of histamine release and arachidonic acid metabolism in rat peritoneal mast cells by thapsigargin, a non-TPA-type tumor promoter.

Thapsigargin, a non-TPA-type tumor promoter, releases histamine and stimulates arachidonic acid metabolism in rat peritoneal mast cells. In order to clarify the relationship between the histamine-releasing activity and the arachidonic acid metabolism-stimulating activity of thapsigargin in mast cells, the effects of cyclooxygenase inhibitors, indomethacin and ibuprofen, a lipoxygenase inhibitor, AA861, and dual inhibitors for cyclooxygenase and lipoxygenase, nordihydroguaiaretic acid and BW755C, on histamine release and arachidonic acid metabolism were examined. High-performance liquid chromatography analysis revealed that the peritoneal mast cells preferentially produce prostaglandin D2 by thapsigargin treatment. These inhibitors suppressed thapsigargin-induced prostaglandin D2 production in a dose-dependent manner, but failed to inhibit histamine release, suggesting that the mechanisms for stimulation of histamine release by thapsigargin is not dependent on increased arachidonic acid metabolism. Time-course experiments of histamine release and the release of radioactivity from [3H]arachidonic acid-labeled mast cells also provide evidence for a difference in mechanism.

Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages.

Rat peritoneal macrophages were prelabeled with [3H]arachidonic acid. The release of radioactivity into the medium was increased by treatment with TPA-type tumor promoters, such as TPA, teleocidin and aplysiatoxin, and the non-TPA-type tumor promoter, thapsigargin. Gossypol, at concentrations of 3 and 10 microM, inhibited the release of radioactivity stimulated by both types of tumor promoter, although the mechanism of stimulation of arachidonic acid metabolism is different in the two types of tumor promoter. Stimulation of prostaglandin E2 production by these tumor promoters was also inhibited by treatment with gossypol. Calcium ionophore A23187-stimulated release of radioactivity and prostaglandin E2 production were also inhibited by gossypol treatment. The mechanism of inhibition by gossypol of prostaglandin E2 production is discussed.

Okadaic acid and dinophysistoxin-1, non-TPA-type tumor promoters, stimulate prostaglandin E2 production in rat peritoneal macrophages.

Okadaic acid and dinophysistoxin-1 isolated from a black sponge, Halichondria okadai are non-12-O-tetrade-canoylphorbol 13-acetate (non-TPA)-type tumor promoters of mouse skin. Okadaic acid at concentrations of 10-100 ng/ml stimulated prostaglandin E2 production in rat peritoneal macrophages. Dinophysistoxin-1 (35-methylokadaic acid) stimulated prostaglandin E2 production as strong as okadaic acid, but okadaic acid tetramethyl ether, an inactive compound as a tumor promoter, did not. Okadaic acid at 10 ng/ml (12.4 nM) stimulated prostaglandin E2 production as strongly as TPA at 10 ng/ml (16.2 nM) 20 h after incubation. Unlike TPA-type tumor promoters, okadaic acid required a lag phase before stimulation. The duration of this lag phase was dependent on the concentration of okadaic acid. Indomethacin inhibited okadaic acid-induced preostaglandin E2 production in a dose-dependent manner, and its inhibition was more strongly observed in okadaic acid-induced prostaglandin E2 production. Cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages and prostaglandin E2 production dose dependently, suggesting that protein synthesis is a prerequisite for the stimulation of arachidonic acid metabolism. These results support our idea that tumor promoters, at very low concentrations, are able to stimulate arachidonic acid metabolism in rat peritoneal macrophages.

Analysis of tumor-promoter-induced inflammation in rats: participation of histamine and prostaglandin E2.

Inflammatory reactions induced by TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoters, including TPA, teleocidin and aplysiatoxin, and chemical mediators responsible for such inflammatory reactions were analyzed. The tumor promoter dissolved in a 0.8% sodium carboxymethyl cellulose solution was injected into a subcutaneous air pouch preformed on the dorsum of rats. Within 30 min after the injection, vascular permeability as measured by the leakage of labeled albumin into the pouch fluid was increased, with a concomitant increase in histamine level. This increase in vascular permeability was inhibited by a histamine antagonist, pyrilamine, and a serotonin antagonist, methysergide. Vascular permeability at 4 h was not inhibited by pyrilamine or methysergide but was inhibited by a cyclooxygenase inhibitor, indomethacin, with a parallel decrease in the prostaglandin E2 level in the pouch fluid. These results suggest that the TPA-type tumor promoters induce inflammation by the mechanism of mast cell degranulation within a short period, this being followed by the stimulation of arachidonic acid metabolism. The mechanism of the in vivo effect of the TPA-type tumor promoters is discussed and compared with in vitro effects that we have previously reported.

Stimulation of prostaglandin E2 production by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters in macrophages and its inhibition by cycloheximide.

The effects of TPA (12-O-tetradecanoylphorbol 13-acetate)-type and non-TPA-type tumor promoters on prostaglandin E2 production by peritoneal macrophages of rats were examined. Among the TPA-type tumor promoters, aplysiatoxin was most potent in stimulating prostaglandin E2 production followed by dihydroteleocidin B, teleocidin, TPA and debromoaplysiatoxin. Prostaglandin E2 production by aplysiatoxin treatment was stimulated at doses up to 0.1 ng/ml. Palytoxin, a non-TPA-type tumor promoter, also stimulated both prostaglandin E2 production and the release of radioactivity from [3H]arachidonic acid-labeled macrophages. However, the dose required for the expression of these effects by palytoxin was up to 3 pg/ml. It was suggested that the tumor promoters are associated with the activity to stimulate arachidonic acid metabolism, irrespective of their type. Cycloheximide, a protein synthesis inhibitor, inhibited both prostaglandin E2 production and the release of radioactivity from prelabeled macrophages stimulated either by the TPA-type tumor promoters or by the non-TPA-type tumor promoter. It is possible that the tumor promoters may induce the synthesis of some proteins responsible for the stimulation of arachidonate metabolism.

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages.

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.

Synergistic stimulation of histamine release from rat peritoneal mast cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters.

Thapsigargin, a non-TPA (12-O-tetradecanoylphorbol 13-acetate)-type tumor promoter, provoked histamine release from rat peritoneal mast cells at concentrations above 30 ng/ml, but not at 10 ng/ml. TPA-type tumor promoters such as TPA, teleocidin and aplysiatoxin released very little, if any, histamine even at 100 ng/ml. When mast cells were incubated in medium containing thapsigargin at 10 ng/ml and varying concentrations of TPA-type tumor promoters, histamine release was increased synergistically. Maximum synergistic effects were observed at 10 ng/ml of each TPA-type tumor promoter. Palytoxin, another non-TPA-type tumor promoter, having no effect on histamine release at up to 10 pg/ml, also induced histamine release in the presence of 10 ng/ml of each TPA-type tumor promoter. However, no synergistic effect on histamine release was observed when mast cells were incubated in medium containing two different non-TPA-type tumor promoters, e.g., 10 ng/ml thapsigargin and 10 pg/ml palytoxin, or in medium containing two different TPA-type tumor promoters, e.g., TPA and teleocidin, TPA and aplysiatoxin, or teleocidin and aplysiatoxin (all at 10 ng/ml). These results suggest that the release of histamine from mast cells is stimulated synergistically under the mutual influence of TPA-type tumor promoters and non-TPA-type tumor promoters.

Rat CINC/gro: a novel mediator for locomotive and secretagogue activation of neutrophils in vivo.

The effects of rat CINC/gro, a member of the interleukin-8 family, on the endothelium-neutrophil interaction and transendothelial macromolecular leakage were studied in rat mesenteric microcirculation. Thirty minutes after superfusion with 10 nM CINC/gro, the number of neutrophils adherent to the venular endothelium and those migrated across the venules were significantly increased with a concomitant elevation of luminol-dependent chemiluminescence at the site of adhesion. Transendothelial macromolecular leakage as assessed by the relative length of venular wall stained with monastral blue B was also increased at 30 min after the start of CINC/gro superfusion. Pretreatments with a CD18-directed monoclonal antibody, WT-3 (1 mg/kg), significantly attenuated the increase in number of adherent and migrated neutrophils, the increase in luminol-dependent chemiluminescence, and the venular macromolecular leakage after the application of CINC/gro. These data suggest that CINC/gro is a novel stimulator that evokes not only locomotive but also secretagogue activation of neutrophils via a CD18-dependent mechanism in vivo.

Structure of the gene encoding rat neutrophil chemo-attractant Gro.

A cloned rat gro gene encoding the neutrophil chemo-attractant Gro was isolated from a lambda Charon4A rat genomic library, and the nucleotide (nt) sequence of a 2500-bp fragment encompassing the coding region and 3'- and 5'-flanking regions was determined. The gene consisted of four exons separated by three introns. The transcription start point was determined by primer-extension analysis and found to be a G located 72 nt upstream from the start codon. The 5'-flanking region of the gene contained a 'TATA'-like structure and an NF-kappa B-binding sequence.

The three dimensional structure of rat cytokine CINC/Gro in solution by homonuclear 3D NMR.

The solution conformation of rat cytokine-induced neutrophil chemoattractant (CINC/Gro), a small protein consisting of 72 amino acid residues with proinflammatory activities, and a member of the interleukin 8 family corresponding to a counterpart of human Gro, was investigated with homonuclear 2D and 3D NMR spectroscopy. At each phase of the structural analysis, the homonuclear 3D NOESY-HOHAHA and HOHAHA-NOESY spectra afforded valuable data, removing ambiguities intractable by conventional 2D NMR techniques. CINC/Gro exists as a dimer in solution and contains a triple stranded anti-parallel beta-sheet and C-terminal alpha-helix in the monomer structure, as observed in human IL-8, but non-trivial differences are also observed.

A stress-sensitive chemokinergic neuronal pathway in the hypothalamo-pituitary system.

Recently we found that cytokine-induced neutrophil chemoattractant influenced anterior pituitary hormone release in vitro. These observations prompted us to investigate the possibility of the existence of cytokine-induced neutrophil chemoattractant in the hypothalamus. Immunohistochemistry showed that cytokine-induced neutrophil chemoattractant-like immunoreactivity existed in the paraventricular hypothalamic nucleus, the supraoptic nucleus, both the internal and the external layers of the median eminence and the posterior pituitary. Since the paraventricular hypothalamic nucleus plays a pivotal role in response to stressful stimuli, we examined the effect of a single episode of immobilization stress on cytokine-induced neutrophil chemoattractant messenger RNA expression in the paraventricular hypothalamic nucleus. Immobilization stress induced strong hybridization signals of cytokine-induced neutrophil chemoattractant messenger RNA in the parvocellular and magnocellular subdivision of the paraventricular hypothalamic nucleus within 15 min, and cytokine-induced neutrophil chemoattractant-like immunostaining intensity in the posterior pituitary started to increase around the periphery of the posterior lobe at 30 min after stress and extended to the whole lobe at 1 h after stress. The increase in the serum cytokine-induced neutrophil chemoattractant in response to stress showed a kinetically biphasic pattern. A first phase occurred within 15 min which may be due to an immediate release of stored cytokine-induced neutrophil chemoattractant in the neurohypophysis, since hypophysectomy completely blocked this phase. A second phase may reflect the release of newly synthesized cytokine-induced neutrophil chemoattractant in the paraventricular hypothalamic nucleus and/or peripheral cytokine-induced neutrophil chemoattractant, since hypophysectomy could not reduce this phase. These data suggest that cytokine-induced neutrophil chemoattractant in the paraventricular hypothalamic nucleus was immediately synthesized in response to stress, and then released into the peripheral blood via the hypothalamo-neurohypophysial system, revealing the presence of a stress-sensitive chemokinergic neuronal pathway in the hypothalamo-pituitary system.

Induction of an eosinophil chemotactic factor production from T lymphocytes by a B cell lymphoma line.

Production of an eosinophil chemotactic factor (ECF) from human mononuclear leukocytes (MNL) was induced by coculture with an irradiated B cell lymphoma line, BALL-1. BALL-1 induced ECF production from OKT4-positive T lymphocytes without evident IL-2 production. Treatment of MNL with anti-IL-2 antibody failed to suppress the BALL-1-induced ECF production, whereas the treatment strongly inhibited IL-2-induced ECF production. Control supernatants of BALL-1 cells alone did not induce ECF production. BALL-1 fixed with periodate-lysine-paraformaldehyde, but not acetone or ethanol, induced evident ECF production. The isoelectric point of BALL-1-induced ECF (m.w. 10-30 kD) was around pI 7, whereas that of the IL-2-induced ECF was around pI 5. A combination of monoclonal antibodies against IL-3, IL-5, and GM-CSF suppressed the activity of the IL-2-induced ECF but not that of the BALL-1-induced ECF. BALL-1-induced ECF suppressed a respiratory burst from an eosinophilic cell line (YY-1) induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine, whereas the IL-2-induced ECF did not, suggesting that the biological function of these two ECF is different, at least in the effect on respiratory burst of eosinophils. From the present results we propose that one reason for infiltration of eosinophils into the stroma of tumors is that some tumor cells can stimulate OKT4-positive T lymphocytes to produce an ECF, and that eosinophils attracted by this ECF exhibit biological functions which are different from those of eosinophils attracted by other ECF.

Induction of zymosan-air-pouch inflammation in rats and its characterization with reference to the effects of anticomplementary and anti-inflammatory agents.

Induction of an experimental inflammation of the air-pouch type with the aid of zymosan (known to activate the alternative pathway of the complement system) was carried out in an attempt to induce a reproducible inflammatory model suitable for quantitative studies. Rats were injected subcutaneously with 8 ml of air on the dorsal surface to make an air-pouch, followed 24h later by 4 ml of 1.6% (w/v) zymosan suspension. This induced inflammatory responses. Treatment with zymosan suspension provoked exudation of fluid, accumulation of polymorphnuclear leukocytes (PMN) in the pouch and the development of granulation tissue as a wall of the pouch. Approximately half of the PMN in the pouch formed a characteristic layer of aggregated cells sticking onto the inner surface of the pouch wall. They were counted after being completely disaggregated by means of incubation with trypsin. In preliminary experiments with potential anti-inflammatory drugs, local application of a novel anti-complementary agent, K-76COONa, inhibited leukocyte accumulation in the pouch, whereas the potent anti-inflammatory agent, dexamethasone, was ineffective. By contrast, exudation of the pouch fluid was effectively inhibited by dexamethasone but not by K-76COONa.

Mechanism of anti-inflammatory action of glucocorticoids: re-evaluation of vascular constriction hypothesis.

1 The question whether constriction of local vessels is essential for the anti-inflammatory action of glucocorticoids in carrageenin-induced granulomatous inflammation was studied. 2 The vasodilator prostaglandin E1 injection into the granuloma pouch fluid increased the exudation of plasma protein into the granuloma tissue. 3 Noradrenaline significantly reduced plasma exudation, possibly through alpha-adrenoceptor stimulation. 4 Cortisol and dexamethasone in doses sufficient to inhibit vascular permeability were without effect on the blood content in the granuloma tissue. 5 The results suggest that constriction of local vessels does not play an essential role in the anti-exudative effect of glucocorticoids in chronic proliferation inflammation.

Role of bradykinin in the vascular permeability response induced by carrageenin in rats.

1 Bradykinin in carrageenin-induced inflammatory pouch fluid was measured by an enzyme immunoassay method. 2 The bradykinin showed a single peak in the 30-60 min period after the challenge and then decreased quickly, and there was a correlation between the bradykinin level and exudation of fluorescein-labelled bovine serum albumin in the first 60 min period. 3 Captopril (an inhibitor of kininase II) elevated both the bradykinin level in the inflammatory pouch fluid and vascular permeability, while DL-2-mercaptomethyl-3- guanidinoethylthiopropanoic acid (an inhibitor of kininase I) had no effect. 4 Soybean trypsin inhibitor (SBTI) inhibited the vascular permeability response in parallel with the decrease in the bradykinin level. 5 A bradykinin-degrading activity appeared in the pouch fluid within 1 h after the challenge and increased with time. 6 In the period of 3.5-4 h, bradykinin levels were suppressed below the sensitivity limit of the assay, i.e. 0.07 nm ml-1, in spite of active generation. This was because degradation of bradykinin was very rapid in this late stage. Nevertheless, bradykinin still played a definite role in sustaining a high level of vascular permeability response in the late stage in conjunction with prostaglandins.

Stimulation of neutrophil adherence to vascular endothelial cells by histamine and thrombin and its inhibition by PAF antagonists and dexamethasone.

1. In order to clarify the roles of platelet-activating factor (PAF) in histamine- and thrombin-induced neutrophil adhesion to vascular endothelial cells, the effects of several PAF antagonists were examined. The effects of the glucocorticoid dexamethasone were also examined in order to gain further insight into the anti-inflammatory actions of glucocorticoids. 2. In culture, histamine and thrombin stimulated the adherence of rat peritoneal neutrophils to human endothelial cells from the umbilical vein. They did not stimulate neutrophil adherence in the absence of endothelial cells, suggesting that the target cells for the histamine- and thrombin-induced adherence of neutrophils were endothelial cells, not neutrophils. 3. Several PAF antagonists, such as CV-3988, L-652,731 and Y-24,180 inhibited the histamine- and thrombin-induced neutrophil adherence in a concentration-dependent manner. Indomethacin failed to inhibit it. 4. Dexamethasone, a steroidal anti-inflammatory drug, did not inhibit the histamine- and thrombin-induced adherence of neutrophils to endothelial cells when the drug was present only during the 20 min incubation period for the adherence assay. When the endothelial cells were preincubated for 3 h with dexamethasone, the adherence of neutrophils to endothelial cells induced by histamine or thrombin was not inhibited. 5. When the neutrophils were preincubated for 3 h with dexamethasone, the histamine- and thrombin-induced adherence of neutrophils to endothelial cells was inhibited. 6. Our studies indicate that: (a) adherence of neutrophils to endothelial cells induced by histamine and thrombin is mediated by PAF production since PAF antagonists inhibited the adherence of neutrophils; and (b) neutrophils, not endothelial cells, are the target cells through which dexamethasone acts to inhibit adherence.

Vascular permeability responses and the role of prostaglandin E2 in an experimental allergic inflammation of air pouch type in rats.

Rats were sensitized with azobenzene arsonate-conjugated acetyl bovine serum albumin. An allergic inflammation was induced in the preformed air pouch in the dorsum of the sensitized rats by injecting the antigen dissolved in a 2% sodium carboxymethyl cellulose solution into the air pouch. Time course changes of vascular permeability, accumulated pouch fluid volume and prostaglandin E2 (PGE2) levels in the pouch fluid were compared in sensitized and non-sensitized rats to characterize the allergic inflammatory reaction. Effects of three cyclo-oxygenase inhibitors (indomethacin, diclofenac sodium and tiaprofenic acid) on vascular permeability and accumulated pouch fluid volume 4 and 24 h after the immunological challenge injection were examined to elucidate a possible role of PGE2 in the inflammatory response. Four h after initiating the allergic reaction, although the level of PGE2 in the pouch fluid reached a high level, the vascular permeability response, measured over the period 3.5-4 h, was not suppressed by treatment with the three cyclo-oxygenase inhibitors and neither was the pouch fluid volume measured over the period 0-4 h. However, vascular permeability and accumulated pouch fluid volume at 24 h were suppressed by the cyclo-oxygenase inhibitors in a dose-dependent manner. These observations suggest that in this model, endogenous PGE2 does not affect oedema formation measured at 4 h. However, oedema formation measured at 24 h may be dependent on PGE2 generation.

Infiltration of neutrophils by intrapleural injection of tumour necrosis factor, interleukin-1, and interleukin-8 in rats, and its modification by actinomycin D.

1. To assess in vivo chemotactic activity of tumour necrosis factor (TNF), interleukin-1 (IL-1), IL-8, and cytokine-induced neutrophil chemoattractant (CINC), we injected these cytokines into the pleural cavity of rats. 2. CINC (0.1-1 microgram) and recombinant human IL-8 (rhIL-8, 0.2-5 micrograms) caused neutrophil infiltration into the rat pleural cavity in a dose-dependent fashion, peaking at 3 h. The number of leukocytes in the peripheral blood did not change significantly. 3. RhTNF alpha and rhIL-1 alpha also induced neutrophil accumulation. The dose response curves of rhTNF alpha (0.67 ng-6.7 micrograms) and rhIL-1 alpha (0.45 ng-4.5 micrograms) at 3 h were bell shaped. On the other hand, unlike CINC and rhIL-8, rhTNF alpha and rhIL-1 alpha caused transient marked leukopenia at 3 h in a simple dose-dependent fashion. 4. Concomitant injection of actinomycin D dose-dependently and completely at 10 micrograms inhibited neutrophil infiltration induced by rhTNF alpha (0.67 microgram) and rhIL-1 alpha (0.45 microgram) at 3 h. However, that induced by CINC or rhIL-8 was not affected by actinomycin D. 5. Peaking at 1 h, CINC production in the pleural cavity was found after intrapleural injection of rhTNF alpha (0.67 microgram) or rhIL-1 alpha (0.45 microgram), but not after that of rhIL-8 (5 micrograms). The CINC production induced by rhTNF alpha or rhIL-1 alpha and the neutrophil infiltration was suppressed by concomitant injection of actinomycin D (1 and 10 micrograms). 6. These results indicate that CINC and IL-8 themselves are direct chemoattractants for neutrophils, whereas TNF and IL-1 induce neutrophil infiltration indirectly via newly synthesized mRNA for chemotactic protein including CINC, which may be involved in neutrophil emigration at local inflammatory sites in rats.