Endotoxin-induced lung inflammation is independent of the complement membrane attack complex

Abstract

Several products of the activated complement system are known to modulate endothelial cell function in vitro. It has been shown that the membrane attack complex (MAC) (C5b-C9) can enhance tumor necrosis factor alpha (TNF-alpha)-induced expression of P- and E-selectin and intercellular adhesion molecule type 1 in cell cultures of human umbilical vein endothelial cells. In the present study the potential role of this synegism for lung injury during endotoxin-mediated septic shock in vivo was examined using a model of C6-deficient PVG (C-) (RT1(C)) rats and the congenic PVG (C+) (RT1(C)) strain. Following administration of a high (5 mg/kg) or low (0.5 mg/kg) dose of lipopolysaccharide (LPS) (Escherichia coli O55:B5), we determined the expression of cytokines, chemokines, and adhesion molecules as well as the recruitment of leukocytes in the lung. Challenge with intraperitoneal i.p. injections of LPS resulted in a strong induction of TNF-alpha, interleukin-1alpha/beta, cytokine-induced neutrophil chemoattractant, interferon-inducible protein 10, macrophage inflammatory proteins 1alpha and 2, macrophage chemotactic protein 1, and P-selectin. However, there were no significant differences between PVG (C-) and PVG (C+) rats. Immunoperoxidase staining showed a similar increase of lung infiltration by CD11b/c(+) leukocytes in both rat strains. We therefore conclude that the described synergism between TNF-alpha and the MAC of the complement system on the induction of endothelial adhesion molecules is dispensable for inflammatory processes during endotoxin-mediated septic shock in vivo.

FIG. 1

Hemolytic complement activity of PVG (C+) and PVG (C−) serum. Hemolytic complement activity of serum from PVG (C+) rats was expressed as the CH₅₀ titer, which ranged between 40 and 80 (50% of hemolysis of presensitized sheep RBC). Serum of PVG (C−) rats had no hemolytic complement activity.

FIG. 2

C6 production in PVG (C+) and PVG (C−) rats. Western blot analysis of serum samples from PVG (C+) and PVG (C−) rats was performed using a goat anti-human C6 polyclonal Ab. Polyclonal goat anti-human IgG Ab was used as a control. A strong band corresponding to C6 in the serum of PVG (C+) rats was detectable at 95 kDa. The serum of PVG (C−) rats and the control was negative for C6.

FIG. 3

Lung cytokine production. Cytokine release of TNF-α, IL-6, IL-1β, and IL-1α in PVG (C−) and PVG (C+) rats was determined by an RNase protection assay 4 h after i.p. challenge with LPS (5 or 0.5 mg/kg LPS). Data are expressed as percent RNA levels relative to the L32 standard. There was no significant differences in cytokine induction between PVG (C−) and PVG (C+) rats (n = 4).

FIG. 4

Induction of lung chemokine expression after LPS (5 mg/kg) challenge. Total RNA was isolated from the lungs of PVG (C−) and PVG (C+) rats. Expression of MCP-1, MIP-1α, KC, IP-10, and MIP-2 was determined before (open symbols) or 4 h after (solid symbols) i.p. challenge with LPS. Expression of chemokines was examined by RT-PCR analysis. To normalize mRNA levels, the cDNA titer of each chemokine, defined as the final dilution yielding detectable amplification products, was divided by the GAPDH titer derived from the same cDNA template. Each symbol represents the result from the lungs of a single rat (n = 4).

FIG. 5

RT-PCR analysis of lung adhesion molecule induction. Representative gels (n = 4) of the RT-PCR analysis of the adhesion molecules P-selectin, E-selectin, and VCAM-1, 4 h after challenge with 5 mg of LPS/kg, are shown. The GAPDH controls of the same samples are also depicted. Serial cDNA dilutions (1:3) were used as template for PCR analysis. The most intense induction was found for P-selectin.

FIG. 6

Lung adhesion molecule induction. Induction of lung adhesion molecules 4 h after LPS challenge with 5 mg of LPS/kg was determined by RT-PCR as described in the legend to Fig. 4. Total RNA was isolated from the lungs of PVG (C−) and PVG (C+) rats. There was a significant induction of P-selectin after LPS challenge, but there was no difference between PVG (C−) and PVG (C+) rats (n = 4). The adhesion molecules E-selectin, ICAM-1, and VCAM-1 were not significantly induced by LPS challenge with 5 mg of LPS/kg.

FIG. 7

Cellular infiltration of CD11b/c+ cells after LPS induction. LPS challenge is associated with an intense accumulation of CD11b/c⁺ leukocytes in the lungs. Lungs were removed 4 h following i.p. injection of 5 mg of LPS/kg. For immunohistochemical analysis of leukocyte infiltration, sections of snap-frozen tissue samples of PVG (C+) (B) or PVG (C−) rats (C) were incubated with MAb CD11b/c. Control sections were incubated with isotype-matched control Ab (A). MAb reactivity was detected using an immunoperoxidase technique. (Magnification, × 180).