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The implantation of foreign material carries a risk of infection which frequently is resistant to all treatment short of removing the implant. We have previously shown that these materials activate neutrophils by contact, leading to production of oxygen free radicals accompanied by release of granule products. Such activation further results in depletion of local host defenses, including the capacity of biomaterial-activated neutrophils to kill bacteria. Among the granule products released from neutrophils are small cationic antibacterial peptides (human neutrophil peptides [HNP]) known as defensins. Here we tested the hypothesis that defensins, released from activated neutrophils onto the surface of biomaterials, might play a role in the deactivation of subsequent neutrophil populations. Incubation of neutrophils with purified HNP resulted in a dose-related impairment of stimulus-induced oxygen radical production and of phagocytic killing. Furthermore, fresh neutrophils added to biomaterial-associated neutrophils exhibited impaired phagocytic killing. This impairment could be abrogated by antibody to HNP but not by an irrelevant antibody. Taken together, these observations support the idea that neutrophils activated at a material surface can create, by means of HNP release, an environment hostile to their microbicidal function and that of their infiltrating brethren.

Identification of mesenteric lymph node (MLN) bacteria showed that indigenous streptomycin-sensitive Escherichia coli could be recovered from MLN at least 48 h after this organism had been essentially eliminated from the cecal flora by antibiotics and replaced with exogenous streptomycin-sensitive E. coli JK. Additional experiments with antibiotic-treated rats also showed that indigenous streptomycin-sensitive E. coli could be recovered from the MLN 4 days after elimination of this organism from the cecal flora. These findings suggest that the time of bacterial translocation to MLN may be kinetically different from the time of recovery of bacteria from MLN and that the MLN may be a focus of infection with intestinal bacteria.

Neutrophils preexposed to high concentrations of activated complement or synthetic N-formyl methionyl peptides are inhibited in their subsequent spontaneous and chemotactic migratory responses. We have considered the possibility that a part of this nonspecific loss of migratory function may be attributable to the interaction of the leukocytes with reactive forms of oxygen deriving from the cytotaxin-induced burst of oxidative metabolic activity. For these studies we have assessed the effect of preexposure of neutrophils from patients with chronic granulomatous disease to cytotaxins on their subsequent migratory responses. We find that these responses are not altered by preexposure to either cytotaxin. Thus, there appears to be a functional relationship between deactivation and the ability of the normal neutrophil to undergo a cytotaxin-induced respiratory burst.

Human polymorphonuclear neutrophils have been preexposed to activated complement as zymosan-activated serum (ZAS) or to the chemotactic oligopeptide N-formyl methionylphenylalanine (F-Met-Phe). Spontaneous migration and chemotactic responses toward the deactivating and other cytotaxins were monitored after washing and resuspension of cells in cytotaxin-free medium. Two patterns of deactivation were observed. Preexposure of the leukocytes to high doses of ZAS or F-Met-Phe decreased all subsequent migratory responses. Preexposure of the leukocytes to lower doses of ZAS or F-Met-Phe decreased only a subsequent chemotactic response to the deactivating cytotaxin. These results suggest two mechanisms, or components, of chemotactic deactivation.

Anaerobes, in particular Bacteroides spp., are the predominant bacteria present in mixed intra-abdominal infections, yet their critical importance in the pathogenicity of these infections is not clearly defined. Succinic acid, a major fatty acid by-product of Bacteroides metabolism, was tested for its effect on neutrophil function to determine whether it might play a role in enhancing the virulence of Bacteroides-containing infections. At pH 5.5 but not pH 7.0, succinic acid at concentrations commonly found in clinical abscesses profoundly inhibits in vitro neutrophil function. It virtually obliterates phagocytic killing of Escherichia coli and reduces neutrophil random migration and chemotactic response to formyl-methionyl-leucyl-phenylalanine and C5a. These effects occur in conjunction with a reduced chemiluminescent peak and delayed time to the peak. The effect on neutrophils is only partially reversible by multiple washings. These findings suggest that succinic acid may be an important Bacteroides virulence factor when present in the microenvironment of a mixed intra-abdominal infection in which concentrations are high and the pH of the medium is reduced.

Light-producing reactions have been reported to occur after phagocytosis of opsonized particles by human polymorphonuclear neutrophils, eosinophils, and monocytes. Such chemiluminescence appears to be related to the generation of singlet oxygen, superoxide, and hydroxyl radicals, which have also been implicated as microbicidal agents. In examining the influences of various medium components on leukocyte chemiluminescence, we have observed that the amount of light measured is increased by addition of soluble protein, the amino acids tyrosine and tryptophane, or excess zymosan to the reaction medium. These agents appear to produce their effect not by increasing the rate of phagocytosis, but by providing substrate for secondary light-producing reactions. Polystyrene particles do not provide a suitable substrate for such secondary light-producing reactions. This is evidenced by the failure of ingested latex to stimulate high levels of chemiluminescence in the cellular system and their failure to augment light production in two noncellular chemiluminescent reactions. Some of the light generated in the cellular chemiluminescence response may derive from secondary reactions, which occur outside of the phagocyte. Support for this phenomenon is provided by two experiments. In one, addition of supplementary, nonopsonized zymosan to the reaction, after phagocytosis of opsonized zymosan is complete, resulted in an increased level of chemiluminescence. In another, addition of nonopsonized zymosan, together with latex particles, resulted in a significant increase in chemiluminescence. The results of the latter experiment also support our hypothesis that latex does not provide an appropriate substrate for secondary light-producing reactions. These observations suggest that leukocytes activated by phagocytosis generate electronically activated radicals which act intra- and extracellularly and that the amino acids tyrosine and trytophane may provide one substrate through which these agents act.

It is thought that the normal enteric microflora acts not only to prevent intestinal colonization but also to prevent subsequent systemic dissemination of ingested, potentially pathogenic bacteria. To determine the relative roles of specific components of the intestinal bacterial flora in bacterial translocation out of the gut, mice were given various antimicrobial agents to selectively eliminate specific groups of intestinal bacteria. The cecal flora and the translocating bacteria in mesenteric lymph nodes were monitored both before and after oral inoculation with antibiotic-resistant Escherichia coli C25. Orally administered streptomycin selectively eliminated cecal facultative gram-negative bacilli, orally administered bacitracin-streptomycin eliminated all cecal bacterial species except low numbers of aerobic sporeformers, and parenterally administered metronidazole selectively eliminated cecal anaerobic bacteria. Compared with control mice, only metronidazole-treated mice had significantly increased rates of dissemination of intestinal bacteria into mesenteric lymph nodes, indicating that the exclusive absence of anaerobic bacteria facilitated the translocation of the intestinal facultative bacteria. In a parallel experiment with streptomycin-resistant E. coli C25 as a marker, parallel results were obtained. Metronidazole increased the translocation of the marker strain and the indigenous strains of intestinal bacteria. Thus, anaerobes appeared to play a key role in confining indigenous bacteria to the gut. However, intestinal colonization and translocation of E. coli C25 occurred most readily after bacitracin-streptomycin treatment, suggesting that in addition to anaerobic bacteria, other bacterial groups may play a role in limiting the intestinal colonization and extraintestinal dissemination of E. coli C25.

Hepatic dysfunction is a poorly understood and highly lethal component of multiple-system organ failure. Both in vivo and in vitro studies of "liver" function have generally neglected hepatocyte-Kupffer cell interactions. In the following experiments, isolated hepatocytes were cocultivated with unstimulated peritoneal cells, predominately macrophages, which served as a readily available Kupffer cell analog. Coculture of hepatocytes with peritoneal cells resulted in little or no change in [3H]leucine incorporation into hepatocyte protein. When gentamicin-killed Escherichia coli cells (GKEC) were added to coculture, there was a marked decrease in hepatocyte [3H]leucine incorporation. In contrast, GKEC added to hepatocytes alone had no effect. Kinetic data revealed an 8-h delay before any significant decrease in leucine incorporation into hepatocyte protein after the addition of GKEC to the coculture. The maximal decrease in hepatocyte [3H]leucine incorporation occurred 24 h after GKEC were added. The decrease observed 24 h after GKEC were added disappeared almost completely after 48 h of coculture. Similar alterations in cocultured hepatocyte protein synthesis were observed after the addition of phorbol myristate acetate, lipopolysaccharide, or muramyl dipeptide, a component of bacterial peptidoglycan. Hepatocyte viability by trypan blue exclusion was unchanged, and gross morphology by light or electron microscopy was unaffected. We propose that during sepsis, macrophages (Kupffer cells) respond to circulating microbial products and mediate alterations in hepatocyte function. These experiments underscore the important role of Kupffer cell function in attempts to understand hepatic malfunction in multiple-system organ failure.

This new assay of viral cell-mediated immunity is sensitive, reproducible, and in many ways resembles the in vivo state. The spread of herpes simplex virus between adjacent monolayer cells was inhibited in the presence of spleen cells from guinea pigs sensitized to that virus. This in vitro control of viral growth was quantified by determining plaque size in monolayers to which were added sensitized spleen cells as opposed to nonsensitized or no spleen cells. The simple measurement of plaque size reduction as an in vitro test of viral cell-mediated immunity is described. In addition to correlating highly with skin testing and macrophage migration inhibition as a test of viral cell-mediated immunity, the ability of sensitized spleen cells to reduce plaque size developed by day 7, paralleling the onset of delayed cutaneous hypersensitivity. The specificity of this lymphocyte-mediated interaction was demonstrated by the inability of herpes simplex virus-sensitized spleen cells to alter the growth of vaccinia virus in cell culture. A ratio of sensitized spleen cells to monolayer cells of 6:1 resulted in significant plaque size reduction on both HEp-2 and conjunctiva monolayers. The data presented demonstrate the potential usefulness of plaque size reduction as a technically simple, specific, and more direct measure of cellular antiviral activity.