A and B: LPS and peptidoglycan associated lipoprotein (PAL) induce much more TNF and IL-6 in 20% human whole blood than in 20% mouse whole blood. Data are reflective of 5 experiments. C and D: Heat killed E. coli bacteria induce much more TNF and IL-6 in 20% human whole blood than in 20% mouse whole blood. Data are reflective of 2 experiments. In all panels, human blood is depicted by solid bars, and mouse blood is depicted by open bars. Data are shown for mouse whole blood in all panels but cytokine responses are so low that they are barely visible in panels A–C despite high stimuli concentrations.

A. 10% mouse serum (open triangles) suppresses LPS-induced TNF production from human monocytes relative to 10% human serum (closed squares); reflective of 4 experiments. B. Dose dependent suppression of mouse serum (open triangle) on TNF induced from human monocytes by 1 ng/ml LPS in comparison with human serum (closed squares); reflective of 3 experiments. C. Mouse serum (10%) (open triangles) suppresses TNF from murine elicited peritoneal macrophages more than human serum (10%)(closed squares); reflective of 5 experiments. D. Dose effect of suppression of mouse (open triangles) and human (closed squares) serum on TNF induced from murine elicited peritoneal macrophages by 200 ng/ml LPS; reflective of 5 experiments.

A. TNF production by human monocytes in response to increasing amounts of LPS in the presence of 5% human serum (open squares) or 5% sera from different mouse strains: Balb/c (close squares), C57Bl/6 (crosses), LPS binding protein (LBP) deficient mice (diamonds), and CD14 deficient mice (closed triangles). B. LPS-induced TNF by human monocytes in the presence of 5% native mouse serum after exposure of mouse serum to trypsin-conjugated beads or sham beads (n=2 for monocytes; similar results were obtained using mouse macrophages, n=2). C. The inhibitory effect of mouse serum on human monocytes does not require their simultaneous presence with the TLR agonists. The sera (5%) were either added for 3h and then cells were extensively washed before activation for 18h with LPS or Pam3CysSK4 (P3C), (labeled Serum pretreatment); or cells were left without serum for 3h and then cultured for 18h in the presence of human or mouse serum (5%) and LPS or Pam3CysSK4 (P3C), (labeled Simultaneous); representative of 2 experiments. Similar results were obtained with BMDM, n = 2.

A. Electrophoretic mobility shift assay (EMSA) to determine the presence of NF-κB in nuclei of human monocytes activated for 45 minutes by LPS (10 ng/ml) in the presence of 5% mouse or human serum. B. Quantification of NF-κB complexes identified by EMSA, n=2. C. Activation of p38 MAPK and erk MAPK in human monocytes cultured in the presence of 5% human or mouse serum and in the absence or presence of LPS (1 ng/ml), and quantification of phosphorylated forms as compare to native form. D. Assessment of TNF mRNA by qPCR in human monocytes cultured for 3 h in the presence of human or mouse serum (10%) and either LPS (10 ng/ml) or Pam3CysSK4 (P3C),(100 ng/ml), n = 3.

Data are shown for concentration of TNF in culture supernatants from elicited mouse peritoneal macrophages incubated with 20ng/ml LPS for 18h in the presence of 20% serum (A) or 5% serum (B) from each species. LD50 or LD100 or shock for each species is listed in Table 2, and was derived from prior publications, as described in Methods. For rhesus monkey, chicken and turtles, symbols represent published doses of LPS at which there were no effect. In each plot, turtle serum was tested but information regarding lethality was obtained for lizards, as stated in Methods. For the data shown, the correlation remained significant when this point was removed from analysis. Open symbols were chosen to represent species with LD50 or shock < 1 mg LPS/kg and closed symbols were chosen to represent species with LD50 or shock > 5 mg LPS/kg.