Characterization of leukocyte-derived neutrophil chemotactic factor-2 and its possible roles in neutrophil infiltration in allergic inflammation in rats.

Abstract

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.