Effects of anti-C5a antibodies on human polymorphonuclear leukocyte function: chemotaxis, chemiluminescence, and lysosomal enzyme release.

Previous investigation has demonstrated that in vivo complement activation can produce acute lung injury. Complement component C5a has been implicated as a key factor in this damage. In addition, C5a is thought to play a central role in mediating polymorphonuclear leukocyte (PMN) function. Studies suggest that administering antibodies to C5a might play a role in attenuating lung injury in animal models of sepsis. To evaluate further the effects of anti-C5a antibodies, we compared the effects of anti-human C5a des-Arg monoclonal (MAb) and polyclonal (PAb) antibodies on PMN functions including chemotaxis, chemiluminescence, and lysosomal release. PMN chemotaxis was assayed in Boyden chambers using 0.5% zymosan-activated serum (ZAS) as a source of C5a and 0.5% normal human serum (NHS) as a control. PMN chemiluminescence was measured by scintillation counting using ZAS as a stimulant and NHS as control. In addition, the lysosomal marker enzyme beta-D-glucuronidase was spectrophotometrically determined to assess lysosomal release. The PMN chemotactic response to ZAS was completely abolished with MAb and PAb anti-C5a antibodies (p less than 0.01). Control antibodies had no effect on ZAS-stimulated chemotaxis. The anti-C5a MAb markedly inhibited PMN chemotaxis at concentrations ranging from 20 to 0.2 microgram/ml, and was approximately 30 times more potent than the PAb. ZAS-stimulated PMN chemiluminescence was markedly decreased in response to monoclonal antibodies to C5a. In contrast, the control antibody did not inhibit ZAS-stimulated PMN chemiluminescence. Anti-C5a antibodies also significantly attenuated the release of the lysosomal enzyme beta-D-glucuronidase from ZAS-stimulated PMN. Anti-C5a antibody treatment did not cause a significant lytic effect when incubated with PMN, as demonstrated by the absence of the cytoplasmic marker lactate dehydrogenase in the supernatant. These studies suggest that in states of complement activation, MAbs and PAbs may decrease PMN functions including chemotaxis, chemiluminescence, and lysosomal enzyme release.

Generation of a protective human monoclonal for the treatment of gram-negative sepsis.

Using a combination of Epstein-Barr virus transformation and cell fusion to a mouse/human heteromyeloma cell line, we have generated a human monoclonal antibody, D234, that recognizes cross-reactive determinants on the lipopoly-saccharides (LPS) of Gram-negative bacteria. Direct binding to a series of rough mutant LPSs and smooth LPSs demonstrates the broad cross-reactivity of this monoclonal. D234 inhibits Re LPS-induced chemiluminescence. In a murine model of Gram-negative sepsis, D234 given after infection significantly increases survival. This antibody may have therapeutic potential for the treatment of life-threatening Gram-negative infections in humans.

Characterization of murine monoclonal antibodies that recognize neutralizing epitopes on human C5a.

We generated a panel of 10 murine monoclonal antibodies (MAbs) that recognize human complement fragment C5a. These MAbs were characterized for their ability to immunoprecipitate 125I-labeled C5a, bind C5a in solid-phase enzyme immunoassay, and block 125I-labeled C5a binding to polymorphonuclear leukocytes. Four of these MAbs had affinity constants for C5a in the 1 X 10(9) to 3 X 10(9) M-1 range. These MAbs blocked C5a-induced neutrophil polarization and chemiluminescence. They blocked the ability of passively administered C5a to cause neutropenia in rabbits. These anti-C5a neutralizing MAbs may have potential therapeutic use in states of complement activation.