Cellular Inflammatory Responses of Man to Bacterial Endotoxin: A Comparison with PPD and Other Bacterial Antigens (1972)

Abstract Healthy male volunteers were inoculated intradermally with serial dilutions (10-5 to 10-1 µg/0.1 ml) of purified bacterial endotoxins derived from Salmonella typhosa, Escherichia coli, Pseudomonas species, and from wild type, rough B, and glucose deficient mutant strains of Salmonella typhimurium. Other volunteers were inoculated intradermally with serial dilutions (105 to 109/0.1 ml) of formalin or heat-killed, freshly washed whole Gram-negative bacteria—S. typhosa, E. coli, Shigella shiga, Pasteurella tularensis, Pseudomonas aeruginosa and Proteus morganii, or with similarly prepared Gram-positive bacteria—Staphylococcus aureus, Diplococcus pneumoniae, Streptococcus salivarius and Lactobacillus delbrueckii. Two dilutions (0.2 and 0.02 µg/0.1 ml) of preservativefree purified protein derivative (PPD) were also tested in tuberculin sensitive volunteers. Non-bacterial inflammants included ethanol and bromsulphalein. In each study, diluent alone (sterile pyrogen-free saline) was inoculated simultaneously. Biopsies were obtained at various time intervals under local anesthesia, and the cellular infiltrates analyzed in multiple sections prepared with hematoxylin and eosin and Giemsa stains. Each of the above preparations, in the dose ranges employed, evoked cellular inflammatory responses at 24 hr that were stereotyped, consisting of predominantly mononuclear perivascular infiltrations. In contrast, early (3- to 6-hr) cellular inflammatory responses were not stereotyped. The Gram-negative bacteria, as well as their purified endotoxins, usually evoked predominantly perivascular mononuclear infiltrates at 3 to 6 hr when inoculated in doses that elicited minimal inflammatory responses, i.e., responses slightly but unequivocally greater than those elicited by saline alone. As the dose of endotoxin or Gram-negative bacteria was increased, the intensity of the early cellular inflammatory response increased and became progressively more polymorphonuclear leucocytic in nature; polymorphonuclear predominance generally developed at concentrations 100-fold above the minimal inflammatory dose. Similar to endotoxin, PPD in sensitive volunteers usually evoked initial predominantly mononuclear responses. The time sequence, however, differed sharply from endotoxin in that the onset of the mononuclear infiltration was delayed for 6 to 10 hr. As with endotoxin, higher concentrations of PPD in some of the more reactive volunteers elicited earlier and predominantly polymorphonuclear leucocytic infiltration. In contrast to the Gram-negative bacteria or their endotoxins, and to PPD, the Gram-positive bacteria as well as the non-bacterial inflammants always elicited predominantly polymorphonuclear leucocytic responses at 3 to 6 hr, even with minimal inflammatory doses. Differences between the cellular composition of the early inflammatory response evoked by the Gram-positive and by the Gramnegative bacteria bore no relationship to serum-agglutinating antibody titers. The hypothesis is advanced that the early predominant mononuclear inflammatory infiltration evoked by the lower concentration of Gram-negative bacteria in man is mediated by their endotoxin content, and that the responsible chemotactic factors may be similar to those activated during the tuberculin response. The relationship of this hypothesis to the existing controversy regarding the early cellular response during delayed hypersensitivity reactions and to the inflammatory reactions seen during Gram-negative bacterial infection in man is considered.