Efficacy of locally delivered polyclonal immunoglobulin against Pseudomonas aeruginosa peritonitis in a murine model

Abstract

Infectious peritonitis results from bacterial contamination of the abdominal cavity. Conventional antibiotic treatment is complicated both by the emergence of antibiotic-resistant bacteria and by increased patient populations intrinsically at risk for nosocomial infections. To complement antibiotic therapies, the efficacy of direct, locally applied pooled human immunoglobulin G (IgG) was assessed in a murine model (strains CF-1, CD-1, and CFW) of peritonitis caused by intraperitoneal inoculations of 10(6) or 10(7) CFU of Pseudomonas aeruginosa (strains IFO-3455, M-2, and MSRI-7072). Various doses of IgG (0.005 to 10 mg/mouse) administered intraperitoneally simultaneously with local bacterial challenge significantly increased survival in a dose-dependent manner. Local intraperitoneal application of 10 mg of IgG increased animal survival independent of either the P. aeruginosa or the murine strains used. A local dose of 10 mg of IgG administered up to 6 h prophylactically or at the time of bacterial challenge resulted in 100% survival. Therapeutic 10-mg IgG treatment given up to 12 h postinfection also significantly increased survival. Human IgG administered to the mouse peritoneal cavity was rapidly detected systemically in serum. Additionally, administered IgG in peritoneal lavage fluid samples actively opsonized and decreased the bacterial burden via phagocytosis at 2 and 4 h post-bacterial challenge. Tissue microbial quantification studies showed that 1.0 mg of locally applied IgG significantly reduced the bacterial burden in the liver, peritoneal cavity, and blood and correlated with reduced levels of interleukin-6 in serum.

FIG. 1

Dose-response curve for locally applied intra-abdominal IgG against P. aeruginosa IFO-3455. The CF-1 mouse survival (n = 10 to 25 animals/group) at day 10 postchallenge with 10⁷ CFU injected intraperitoneally simultaneous with a separate single injected dose of IgG (0.005, 0.05, 0.2, 0.5, 1.0, 5.0, or 10 mg per animal) is shown. IgG therapy increased the percent survival in a dose-dependent manner. The data represent the mean survival of IgG-treated mice from four different experiments. The difference in survival between the 0.5- and 10-mg/animal IgG dose groups is statistically significant (ANOVA with Tukey’s test; P < 0.008).

FIG. 2

CF-1, CD-1, and CFW mouse strain survival assessed for 10 days in the peritonitis model with a single local intraperitoneal injection of 10 mg of IgG or placebo treatment (5% dextrose) against a lethal dose of 10⁷ CFU of P. aeruginosa IFO-3455 injected intraperitoneally (n = 10 to 35 animals/group). IgG treatment resulted in significantly increasing survival compared to placebo (5% dextrose) treatment in all three mouse strains (ANOVA with Tukey’s test; P < 0.001).

FIG. 3

Pathogen strain influence on mouse survival (day 10) in a peritonitis model with local IgG administration. Three strains of P. aeruginosa were each separately injected intraperitoneally (dose = 10⁷ CFU) simultaneously with separate, single injections of 10 mg of pooled human IgG or placebo (5% dextrose) (n = 10 animals/group). IgG treatment significantly increased the survival rates compared to the placebo treatment (z test; P < 0.05).

FIG. 4

Survival in the mouse peritonitis model (day 10) influenced by time of local IgG administration relative to bacterial challenge. Single IgG injections intraperitoneally (10 mg) were administered prior to (prophylaxis), simultaneously with (challenge), or after (therapy) lethal intraperitoneal injections of P. aeruginosa IFO-3455 (10⁷ CFU, n = 10 to 25 animals/group). All IgG-treated groups marked with an asterisk showed significantly increased survival rates compared to the placebo (5% dextrose) treatment group (z test and ANOVA with Tukey’s test; P < 0.05).

FIG. 5

ELISA detection of human IgG levels in murine serum (⧫) and peritoneal lavage fluid (▵) after bacterial challenge following intraperitoneal injection of IgG and intraperitoneal lethal injections of P. aeruginosa IFO-3455 (10⁷ CFU, n = 3 or 4 animals per time point in each group) for 7 days. Human IgG is detectable rapidly in mouse serum after intraperitoneal administration and remains detectable by ELISA methods in both serum and mouse peritoneal lavage fluid for up to 7 days. (Inset) Human IgG levels in mouse serum and peritoneal lavage fluid in the first 12 h postadministration.

FIG. 6

Bacterial burden at various tissue sites assessed 6 h after intraperitoneal injection of 1.0 mg of IgG against 10⁶ CFU of P. aeruginosa IFO-3455 given intraperitoneally. Mice (n = 10) peritoneal lavage fluid (PL), liver homogenate, and blood analysis yielded CFU values that show that IgG treatment significantly decreased the bacterial burden compared to the placebo treatment (0.2 M glycine) after 6 h in all samples (t test; P < 0.05). The PL and blood bar graphs represent the log₁₀ CFU/milliliter, and the liver bar graph shows the log₁₀ CFU/gram of liver.

FIG. 7

Serum IL-6 levels after intraperitoneal injection of 1.0 mg of IgG against a nonlethal intraperitoneal dose (10⁶ CFU) of P. aeruginosa IFO-3455. Serum IL-6 levels of CF-1 mice (n = 5 to 25) were determined by ELISA. IgG treatment decreased IL-6 levels significantly compared to the control (P = 0.04) by 6 h after the bacterial challenge. A saline-plus-glycine placebo treatment without a bacterial challenge was used to determine normal background IL-6 levels in CF-1 mice. Treatment with saline plus 10 mg of IgG without a bacterial challenge shows that IL-6 levels resulting from IgG treatment alone are not significantly different from the normal background IL-6 levels (t test; P < 0.05).

FIG. 8

In vitro opsonophagocytic assay with mouse peritoneal lavage shows enhanced bacterial clearance with IgG. Murine peritoneal lavage fluid was assayed in vitro 2 h after intraperitoneal dosing with human IgG and intraperitoneal challenge with 10⁷ CFU of P. aeruginosa IFO-3455. Peritoneal lavage fluid of mice treated with 10 mg of IgG significantly reduced the levels of bacteria compared to placebo (5% dextrose)-treated mice both immediately after peritoneal lavage and 2 h later (t test; P < 0.05). The presence of human IgG facilitated the clearance of bacteria from lavage fluid, whereas the control lavage fluid exhibited bacterial growth during the incubation.