CD44 deficiency is associated with enhanced Escherichia coli-induced proinflammatory cytokine and chemokine release by peritoneal macrophages

Abstract

CD44 is involved in several immune responses, such as cellular adhesion, migration, proliferation, and activation. Peritonitis is an important cause of sepsis, and Escherichia coli is one of the major pathogens involved therein. We sought to determine the role of CD44 in the host response to E. coli-induced abdominal sepsis and to assess the function of CD44 in the activation of primary peritoneal macrophages by E. coli or lipopolysaccharide (LPS) purified from this bacterium by using wild-type (WT) and CD44 knockout (KO) mice. CD44 KO mice already demonstrated enhanced CXC chemokine levels in peritoneal lavage fluid at 6 h after infection, whereas tumor necrosis factor alpha (TNF-alpha) and interleukin-6 levels were elevated at 20 h postinfection. In line with this, CD44 KO mouse peritoneal macrophages released more TNF-alpha and macrophage inflammatory protein 2 (MIP-2) than did WT cells upon stimulation with E. coli or LPS in the presence of autologous serum. In contrast, plasma TNF-alpha levels were lower in CD44 KO mice and CD44 KO blood leukocytes secreted similar amounts of TNF-alpha and MIP-2 upon ex vivo incubation with E. coli or LPS. The proinflammatory phenotype of CD44 KO macrophages was not associated with an altered expression of inhibitors of Toll-like receptor signaling, whereas it could be partially reversed by addition of WT serum. CD44 deficiency did not impact on leukocyte recruitment into the peritoneal cavity or organ failure. These data suggest that CD44 differentially influences cytokine and chemokine release by different leukocyte subsets.

FIG. 1.

CD44 KO mice demonstrate enhanced TNF-α, IL-6, and chemokine (MIP-2, KC, and LIX) production in peritoneal lavage fluid. Cytokine (TNF-α, IL-1β, IL-6, and IL-10) and chemokine (MIP-2, KC, LIX, and MCP-1) levels in peritoneal lavage fluid at 6 and 20 h after i.p. injection of 1 × 10⁴ CFU of E. coli into WT (black bars) and CD44 KO (white bars) mice are shown. Data are expressed as means ± SEM; n = 8 mice/group/time point. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus WT mice at the same time point. B.D. means below detection.

FIG. 2.

CD44 KO mice demonstrate decreased TNF-α and MCP-1 levels in plasma. Cytokine (TNF-α, IL-1β, IL-6, and IL-10) and chemokine (MIP-2 and MCP-1) levels in plasma at 6 and 20 h after i.p. injection of 1 × 10⁴ CFU of E. coli into WT (black bars) and CD44 KO (white bars) mice are shown. Data are expressed as means ± SEM; n = 8 mice/group/time point. *, P < 0.05 versus WT mice at the same time point.

FIG. 3.

CD44 does not impact on distant organ injury. Representative hematoxylin-and-eosin staining of lung (A and B) and liver (C and D) tissue at 20 h after i.p. injection of 1 × 10⁴ CFU of E. coli into WT (A and C) and CD44 KO (B and D) mice is shown. Original magnifications: liver, ×10; lung, ×20. Plasma concentrations of ASAT (E), ALAT (F), creatinine (G), and urea (H) in WT (black bars) and CD44 KO (white bars) mice are also shown. Data are expressed as means ± SEM; n = 8 mice/group.

FIG. 4.

CD44 KO peritoneal macrophages demonstrate increased TNF-α and MIP-2 release. TNF-α (A and B) and MIP-2 (C and D) release by WT (▪) and CD44 KO (□) peritoneal macrophages after 2, 6, and 18 h of stimulation with 200 ng/ml LPS (A and C) or an equivalent of 1 × 10⁷ CFU/ml heat-killed E. coli (B and D) is shown. Data are expressed as means ± SEM; n = 5 mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus WT mice at the same time point.

FIG. 5.

CD44 deficiency does not influence TNF-α and MIP-2 release by whole blood. TNF-α (A and B) and MIP-2 (C and D) release by WT (black bars) and CD44 KO (white bars) whole blood after 6 and 24 h of stimulation with 80 ng/ml LPS (A and C) or an equivalent of 4 × 10⁶ CFU/ml heat-killed E. coli (B and D) is shown. Data are expressed as means ± SEM; n = 5 mice/group.

FIG. 6.

CD44 deficiency does not influence IRAK-M and A20 mRNA expression in peritoneal macrophages. IRAK-M (A and B) and A20 (C and D) mRNA expression levels in WT (▪) and CD44 KO (□) peritoneal macrophages corrected for B2M at 2 and 6 h after LPS (A and C) or heat-killed E. coli (B and D) stimulation are shown. Data are expressed as means ± SEM; n = 5 mice/group.

FIG. 7.

WT serum abolishes increased TNF-α and MIP-2 release by CD44 KO peritoneal macrophages. TNF-α (A and B) and MIP-2 (C and D) release by WT (black bars, WT serum) and CD44 KO (white bars, CD44 KO serum; gray bars, WT serum) peritoneal macrophages after 18 h of stimulation with LPS (A and C) or heat-killed E. coli (B and D) is shown. Data are expressed as means ± SEM; n = 5 mice/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus the indicated group.