The third component of human complement (C3) was digested with human leukocyte elastase (HLE). Digestion proceeds in two stages under controlled conditions of proteolysis. The initial event of HLE attack produces cleavage of the C3 a chain near the amino terminus forming an a' chain and releasing an 8500 molecular weight fragment (a4) which is immunologically and chemically similar to the C3a anaphylatoxin. The purified a 4 fragment is lacking in both anaphylatoxic or chemotactic activity. More extensive digestion of C3 with HLE results in a selective degradation of the a'chain to form three principle fragments of 36 000 (al), 28 000 (a2), and 24 000 (a3) molecular weight. Digestion of C3 with HLE either for extended periods of time (2 h ) or at high ratios of enzyme to C3 (1:20) does not affect the covalent integrity of the / 3 chain. Specificity of HLE cleavage sites on the protein substrate is consistent with that previously observed with model synthetic substrates. However, the ability of HLE to cleave the peptide linkage between aliphatic residues and a cationic residue was not predicted from model substrate studies. Fragments al to a 4 account for approximately 90% of the total a-chain structure. Fragments a2 and a 4 are not covalently bonded to the disulfide-linked complex of a l , 03, and @chain.

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We previously demonstrated in rabbits that various neutrophil chemotactic factors share an ability to induce recruitment of polymorphonuclear leukocytes (PMN) from bone marrow when administered intravenously (Jagels and Hugli, J Immunol 148:1119, 1992). In the study reported here, we investigated the effects of chemotactic factors on the expression of beta 2 integrins and L-selectin in vivo and the roles of these adhesionmolecules in the recruitment process. Leukocytosis was induced by infusion of either C5a (5 micrograms/kg), N-formyl-methionyl-leucyl-phenylalanine (f-MLP; 2.5 micrograms/kg), or tumor necrosis factor alpha (TNF-alpha; 100 ng/kg). C5a increased the expression of CD18 (the common subunit of beta 2 integrins) on PMN by nearly twofold and decreased levels of L-selectin by 50% within 15 minutes after administration. Levels of beta 2 integrins returned to baseline 2 to 3 hours after induction of leukocytosis. L-selectin remained depressed for more than 5 hours, demonstrating that shedding was induced in the recruited bone marrow leukocytes as well as in circulating PMN. In contrast to the response to C5a, TNF-alpha did not cause upregulation of CD18 or shedding of L-selectin. Levels of L-selectin were consistently increased 60 minutes after administration of TNF-alpha, coinciding with a rapid rise in the number of band-form PMN in the circulation. Intact IgG and F(ab)2 forms of the anti-CD18 monoclonal antibody IB4 or the anti-L-selectin antibody DREG-200 were administered intravenously 15 minutes before induction of leukocytosis by the chemotactic factors. Neither IB4 nor its F(ab)2 fragments blocked leukocytosis induced by C5a, f-MLP, or TNF-alpha. DREG-200 also did not block leukocytosis induced by f-MLP, C5a, or TNF-alpha. These results suggest that leukocyte emigration from the bone marrow into the circulation proceeds through interactions distinct from those involved in neutrophil chemotaxis and diapedesis. Shedding of L-selectin from C5a-recruited bone marrow leukocytes demonstrates activation of these cells in the recruitment process and may reflect a potential mechanism for their release. The dissimilar effects of C5a and TNF-alpha on expression of adhesion molecules may result from distinct stimulatory pathways and suggests differential activation states for cellular recruitment by these inflammatory factors.

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