Jun-ichi Tanabe

Induction of neutrophil chemotactic factor production by staurosporine in rat peritoneal neutrophils

1 Incubation of rat peritoneal neutrophils in medium containing various concentrations of staurosporine (6.4–64 nM) increased the neutrophil chemotactic activity in the conditioned medium in a time‐ and concentration‐dependent manner. 2 Separation of the neutrophil chemotactic activity in the conditioned medium by isoelectric focusing revealed that staurosporine (64 nM) stimulated the production of basic (pH>8) neutrophil chemotactic factors, while TPA (12‐O‐tetradecanoylphorbol 13‐acetate, 49 nM) stimulated the production of both basic (pH>8) and acidic (pH 5) neutrophil chemotactic factors. 3 Determination by immunoassay of cytokine‐induced neutrophil chemoattractant (CINC)‐1, ‐2α, ‐2β and ‐3 in the conditioned medium at 4 h revealed that staurosporine (64 nM) and TPA (49 nM) strongly stimulated the production of CINC‐3 (staurosporine, 133.0±3.8; TPA, 26.7±1.0; control, 0.32±0.01 ng ml−1, means±s.e.mean from four samples) compared to CINC‐1 (staurosporine, 55.0±1.2; TPA, 12.2±0.3; control, 0.56±0.01 ng ml−1), and CINC‐2α (staurosporine, 1.09±0.03; TPA, 0.90±0.02; control, <0.10 ng ml−1). CINC‐2β was below the detectable amount (<0.078 ng ml−1). 4 The level of CINC‐3 mRNA in the peritoneal neutrophils was determined by reverse transcription‐polymerase chain reaction. Staurosporine (64 nM) and TPA (49 nM) enhanced the level of CINC‐3 mRNA time‐dependently, but had no effect on GAPDH mRNA levels. 5 Production of staurosporine‐induced neutrophil chemotactic factor was inhibited by the protein kinase C inhibitors, H‐7 (IC50, 12.3 μM), calphostin C (IC50, 0.77 μM) and Ro 31‐8425 (24.3% inhibition at 10 μM), and by the tyrosine kinase inhibitor, genistein (IC50, 68.5 μM). Production of TPA‐induced neutrophil chemotactic factor was also inhibited by both inhibitors. 6 Both the staurosporine‐ and the TPA‐induced increase in CINC‐3 mRNA levels were suppressed by H‐7 and genistein.

Pharmacological analysis of neutrophil chemotactic factor production by leucocytes and roles of PAF in allergic inflammation in rats

1 The role of platelet activating factor (PAF) in neutrophil infiltration in air pouch type allergic inflammation in rats was investigated. 2 Neutrophil infiltration into the pouch fluid 8 h after injection of the antigen (azobenzenearsonate‐conjugated acetyl bovine serum albumin) solution into the air pouch of immunized rats was inhibited dose‐dependently by treatment with PAF antagonists (CV‐3988, L‐652,731 and Y‐24,180) in parallel with the decrease in neutrophil chemotactic activity in the pouch fluid. 3 Four hours after injection of the antigen solution into the air pouch of immunized and non‐immunized rats, there was no significant difference between the two groups in the number of total leucocytes, neutrophils, mononuclear cells and eosinophils in the pouch fluid. However, when the infiltrated leucocytes were incubated in medium, chemotactic factor production by leucocytes from immunized rats was greater than that from non‐immunized rats. 4 When leucocytes from non‐immunized rats were preincubated for various periods in the medium containing 10 or 50 nM of PAF, washed, and further incubated in the medium containing no PAF, chemotactic factor production was not stimulated. 5 The increase in the chemotactic activity in the conditioned medium was not suppressed by the 5‐lipoxygenase inhibitor, AA861. In addition, the chemotactic activity in the conditioned medium was not inhibited by the PAF antagonists. 6 Incubation of the infiltrated leucocytes in the medium containing the protein synthesis inhibitor, cycloheximide, inhibited chemotactic factor production in a concentration‐dependent manner in parallel with the decrease in uptake of [3H]‐leucine into the acid‐insoluble fraction of leucocytes. 7 Separation of the chemotactic activity in the conditioned medium by isoelectric focusing revealed that the leucocyte infiltrated into the pouch fluid produce two kinds of factors, viz. leucocyte‐derived neutrophil chemotactic factor‐1 (LDNCF‐1) and LDNCF‐2 of which pI values are 4–5 and above 8, respectively. 8 The results indicate that PAF has no significant role in leucocyte activation to produce chemotactic factors, and that neutrophil chemotactic factors produced by infiltrated leucocytes are not PAF or leukotriene B4 but are produced through a protein synthesis mechanism. 9 The mechanism of action of PAF antagonists on neutrophil infiltration into the inflammatory locus is discussed.

Possible roles of protein kinases in neutrophil chemotactic factor production by leucocytes in allergic inflammation in rats

1 In an air pouch‐type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium. 2 To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined. 3 When the infiltrated leucocytes were incubated for 4h in medium containing the non‐selective protein kinase inhibitor K‐252a (1–100 ng ml−1, 2.14–214 nm), the tyrosine kinase inhibitor genistein (1–50 μg ml−1, 3.7–185 μm), and the more selective protein kinase C inhibitor H‐7 (5–100 μg ml−1, 13.7–274 μm); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration‐dependent manner, but the adenosine 3′:5′‐cyclic monophosphate (cAMP)‐dependent protein kinase inhibitor H‐89 (1–1000 ng ml−1, 2.24–2240 nm) showed no effect. 4 Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte‐derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF‐2. Treatment of the leucocytes with K‐252a, genistein, and H‐7, but not H‐89, inhibited production of both LDNCF‐1 and LDNCF‐2. 5 These results suggest that activation of tyrosine kinase and protein kinase C., but not cAMP‐dependent protein kinase, is responsible for the production of LDNCF‐1 and LDNCF‐2. 6 The steroidal anti‐inflammatory drug dexamethasone and the protein synthesis inhibitor cyclohex‐imide inhibited neutrophil chemotactic factor production in a concentration‐dependent manner. Time‐course experiments showed that the inhibitory effect by dexamethasone was apparent even 30min after the incubation. 7 Mechanism for inhibiting the production of LDNCF‐1 and LDNCF‐2 by dexamethasone is also discussed.

Characterization of Leukocyte-Derived Neutrophil Chemotactic Factor-2 and Its Possible Roles in Neutrophil Infiltration in Allergic Inflammation in Rats

This study sought to clarify the responsible chemotactic factor for neutrophils in allergic inflammation in rats. When the leukocytes collected from the pouch fluid 4 h after injection of the antigen solution into the air pouch were incubated, the neutrophil chemotactic activity in the conditioned medium increased time-dependently with higher levels for the leukocytes from the immunized rats than the nonimmunized ones. The chemotactic activity did not result from cytokine-induced neutrophil chemoattractant (CINC) because CINC concentrations in the conditioned medium were low. Neutrophil chemotactic factors in the conditioned medium were separated by isoelectric focusing into two factors, leukocyte-derived neutrophil chemotactic factor (LDNCF)-1 and LDNCF-2. The activity of LDNCF-2 was more than 75% of the total chemotactic activity in the conditioned medium. LDNCF-2 was purified by gel chromatography and reverse-phase HPLC. The N-terminal amino acid sequence (1-20) of the purified LDNCF-2 was identical to the 32-51 amino acid sequence of the pro-form of rat macrophage inflammatory protein (MIP)-2. Higher levels of MIP-2 mRNA in the leukocytes from the immunized rats than that from the non-immunized rats were proved by the reverse transcription-polymerase chain reaction. In vivo, concentrations of CINC in the pouch fluid were low, and did not represent the chemotactic activity in the pouch fluid. These results suggest that LDNCF-2 (MIP-2) is an important chemotactic factor for neutrophils in the allergic inflammation in rats.

Possible participation of macrophage inflammatory protein 2 in neutrophil infiltration in allergic inflammation in rats

Recombinant rat macrophage inflammatory protein 2 (MIP-2) was prepared from E. coli transfected with a glutathione-S-transferase (GST)-MIP-2 fusion protein expression vector. A polyclonal antibody to rat MIP-2 was then obtained from rabbits by immunization with recombinant rat MIP-2. Using the polyclonal antibody which selectively suppressed neutrophil chemotactic activity of MIP-2, the role of MIP-2 in neutrophil infiltration in allergic inflammation in rats was studied. In an air pouch-type allergic inflammation model in rats, neutrophil infiltration into the pouch fluid increased with time after antigen challenge. Neutrophil chemotactic activity in the pouch fluid collected 8 h after antigen challenge was diminished by anti-MIP-2 antibody. In addition, when leukocytes that had infiltrated into the pouch fluid collected 4 h after antigen challenge were incubated, neutrophil chemotactic activity in the conditioned medium increased time-dependently, and the activity was neutralized by anti-MIP-2 antibody. Furthermore, when anti-MIP-2 antibody was injected into the pouch 6 h after antigen challenge, neutrophil infiltration into the pouch fluid during the next 2 h was suppressed. These findings indicate that MIP-2 plays an important role in neutrophil infiltration in rat allergic inflammation.

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.

Dexamethasone inhibits the production of macrophage inflammatory protein 2 in the leukocytes in rat allergic inflammation

In the air pouch-type allergic inflammation model in rats, when the infiltrated leukocytes in the pouch fluid collected 4 h after antigen challenge were incubated, neutrophil chemotactic activity in the conditioned medium increased time-dependently. They produced neutrophil chemotactic factors, viz. leukocyte-derived neutrophil chemotactic factor (LDNCF)-2, a major component, and LDNCF-1, a minor component. When the infiltrated leukocytes were incubated in the presence of dexamethasone, neutrophil chemotactic activity in the conditioned medium decreased in a concentration-dependent manner, and production of LDNCF-2 and LDNCF-1 was inhibited. Because purified LDNCF-2 had been found to be identical with rat macrophage inflammatory protein 2 (MIP-2), effects of dexamethasone on the level of MIP-2 mRNA in the leukocytes were investigated. Using the reverse transcription-polymerase chain reaction technique, it was demonstrated that dexamethasone suppressed the level of MIP-2 mRNA in the leukocytes. These results indicate that dexamethasone inhibits MIP-2 production at the transcription level.