Rat macrophage inflammatory protein-1alpha, a CC chemokine, acts as a neutrophil chemoattractant in vitro and in vivo.

Recombinant rat macrophage inflammatory protein-1alpha (rMIP-1alpha) at a concentration of 3x10(-8) M had strong neutrophil chemotactic activity, though the potency of rMIP-1alpha was less than that of cytokine-induced neutrophil chemoattractant (CINC)-1 at lower concentrations. In addition, rMIP-1alpha induced neutrophil chemotaxis in vivo when rMIP-1alpha was injected into the preformed air-pouch on the back of rats. The adhesion of rMIP-1alpha-treated neutrophils to fibrinogen significantly increased, reaching a maximum adhesion at 10(-8) M. Stimulation of neutrophils with rMIP-1alpha induced a transient increase in intracellular free [Ca2+] dose-dependently. rMIP-1alpha 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-1alpha. Supporting these findings, an additive increase in chemotactic potency was found when both rMIP-1alpha and CINC-were added to the lower wells of Boyden chamber in vitro. In addition, high levels of rMIP-1alpha were detected in the inflammatory site of air-pouch/carrageenan-induced inflammation in rats. Our results suggest that rMIP-1alpha acts as a neutrophil chemoattractant and, together with CINCs, plays an important role in infiltration of neutrophils into inflammatory sites in rats.

Differential production of chemokines by phagocytosing rat neutrophils and macrophages.

In this study, rat neutrophils and macrophages produced cytokine-induced neutrophil chemoattractants (CINCs) and rat macrophage inflammatory protein-1 alpha (MIP-1 alpha) in different patterns during phagocytosis of heat-killed yeast cells in vitro. The cultured supernatants of the phagocytosing rat neutrophils and macrophages had chemotactic activities toward neutrophils, and the chemotactic potencies were markedly inhibited by anti-CINCs IgGs or/and anti-MIP-1 alpha IgG, suggesting that CINCs and MIP-1 alpha are major neutrophil chemoattractants produced by the phagocytosing neutrophils and macrophages. Dexamethasone suppressed the production of CINCs and MIP-1 alpha by the phagocytosing cells in a dose-dependent manner. Our results demonstrate significant differences in the production of CINCs and MIP-1 alpha by neutrophils and macrophages during phagocytosis of yeast cells and thus may suggest the different contribution of each chemokine to neutrophil recruitment in the processes of inflammation in rats.

Effects of cytokine-induced neutrophil chemoattractants (CINCs) on shape change, adhesiveness and phagocytosis of rat neutrophils.

Rat cytokine-induced neutrophil chemoattractants (CINC)-1, -2 alpha, -2 beta and -3 belong to alpha-chemokines and are known to be potent chemoattractants for rat neutrophils in vivo and in vitro. In this study, the abilities of CINCs to affect several functions of rat neutrophils are estimated. All CINCs at a concentration of 10(-8) M induced the polarization of rat neutrophils. The adhesion of each CINC-treated neutrophil to fibrinogen significantly increased, reaching a maximal adhesion at 10(-8) M of each CINC. The CINCs enhanced the phagocytosis of heat-killed yeast cells by neutrophils in a dose-dependent manner. In contrast, CINCs did not induce the production of reactive nitrogen intermediates. The results suggest that CINCs play a significant role in neutrophil infiltration and phagocytosis at inflammatory sites in rats.

Identification and characterization of receptor for cytokine-induced neutrophil chemoattractant-3 on rat neutrophils.

Cytokine-induced neutrophil chemoattractants (CINCs), members of alpha-chemokines, are considered to be major neutrophil chemotactic factors in rats. Recombinant CINC-3/rat macrophage inflammatory protein-2 (MIP-2) having an additional tyrosine residue at the carboxyl terminus (CINC-3-Tyr) was constructed, purified, radiolabeled with 125I, and used for binding studies. The specific binding of 125I-labeled CINC-3-Tyr (125I-CINC-3-Tyr) to rat neutrophils reached a plateau after approximately 60 min at 4 degrees C. This binding of 125I-CINC-3-Tyr could be reversed by adding an excess amount of unlabeled CINC-3-Tyr. Scatchard analysis revealed approximately 12,000 binding sites per cell on rat neutrophils with an apparent Kd value of 120 pM. Chemical cross-linking experiments suggested that the rat neutrophil CINC-3 receptor is a mass of approximately 69 kDa. Taken together, our results strongly suggest that rat neutrophils express a high affinity receptor for CINC-3.

Evidence for two putative receptors mediating chemotactic activity of cytokine-induced neutrophil chemoattractants (CINCs) for rat neutrophils.

Recently we have found that only cytokine-induced neutrophil chemoattractant (CINC)-3 induces an increase in intracellular [Ca2+] in rat neutrophils stimulated first with CINC-1, CINC-2 alpha or CINC-2 beta, and hypothesized that rat neutrophils have another receptor specific for CINC-3 in addition to a common receptor for all CINCs [Shibata F., et al., Eur. J. Biochem., 231, 306 (1995)]. To evaluate this hypothesis, chemotactic activity of these CINCs in vitro was determined by using rat neutrophils together with each CINC. In the presence of CINC-1, migration of neutrophils toward CINC-1, CINC-2 alpha and CINC-2 beta was inhibited, but that toward CINC-3 was not. Similar results were obtained when CINC-2 alpha or CINC-2 beta was added to neutrophils. However, addition of CINC-3 to neutrophil suspension in the upper chamber resulted in the complete inhibition of the neutrophil migration toward each CINC. The results support our hypothesis and indicate both the putative receptors can mediate chemotaxis of rat neutrophils.

Differential changes in the concentrations of cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in exudate during rat lipopolysaccharide-induced inflammation.

Recently we have purified four neutrophil chemotactic factors from conditioned medium of rat granulation tissue. Two chemokines besides cytokine-induced neutrophil chemoattractant-1 (CINC-1, formerly called CINC) and CNC-3/macrophage inflammatory protein-2 (MIP-2) were novel chemoattractants, designated as CINC-2 alpha and CINC-2 beta. In the present report, we developed an enzyme-linked immunosorbent assay (ELISA) specific for CINC-2. The biotin-streptavidin sandwich ELISA for CINC-2 beta detected CINC-2 alpha and CINC- 2 beta equally well between 1 ug/mI and 300 ng/ml, but did not show cross-reactivity with CINC-1 and CINC-3. The concentrations of CINC-1 and CINC-2 in the exudate during rat lipopolysaccharide (LPS)-induced inflammation were determined. The CINC-1 concentration in the exudate reached a maximum at 4 h after LPS injection, whereas the CINC-2 level steadily increased up to 8 h. The number of infiltrated cells in the exudate increased linearly until 6 h and gradually up to 8 h. Increase in the cell number was correlated with total concentrations of CINC-1 and CINC-2. The results suggest that CINC-2 as well as CINC-1 plays an important role in accumulation of neutrophils into the inflammatory lesion of LPS- induced inflammation in rats.

Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8.

We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run-on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required.

Recombinant production and biological properties of rat cytokine-induced neutrophil chemoattractants, GRO/CINC-2 alpha, CINC-2 beta and CINC-3.

Recently we found four cytokine-induced neutrophil chemoattractants, CINC-1, CINC-2 alpha, CINC-2 beta and CINC-3/macrophage inflammatory protein 2 (MIP-2), in conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in rats [Nakagawa, H., Komorita, N., Shibata, F., Ikesue, A., Konishi, K., Fujioka, M. & Kato, H. (1994) Biochem. J. 301, 545-550]. In the present report, we describe recombinant production of CINC-2 alpha, CINC-2 beta and CINC-3 in Escherichia coli, and biological properties of these chemokines. Neutrophil chemotactic activities of CINC-2 alpha and 2 beta in vitro were the same as the activity of CINC-1. CINC-3 had an activity comparable to other CINCs, but showed a decrease at high concentrations. Stimulation of neutrophils with CINCs induced an increase in intracellular [Ca2+] dose-dependently. CINC-3 was more potent than the other CINCs and still induced an increase in intracellular [Ca2+] in rat neutrophils stimulated first with other CINCs. CINC-2 alpha, CINC-2 beta and CINC-3 induced a comparable response to CINC-1 in the release of cathepsin G from rat neutrophils. Injection of CINC-2 alpha, 2 beta and 3 into preformed air-pouch on the back of rat induced infiltration of neutrophils to an extent similar to that caused by the injection of CINC-1. These data indicate CINC-2 alpha, 2 beta and 3 as well as CINC-1 are chemoattractants specific for neutrophil in vivo.