Sepsis-induced changes in macrophage co-stimulatory molecule expression: CD86 as a regulator of anti-inflammatory IL-10 response

Abstract

Background: Sepsis remains a substantial risk after surgery or other trauma. Macrophage dysfunction, as a component of immune suppression seen during trauma and sepsis, appears to be one of the contributing factors to morbidity and mortality. However, whereas it is known that the ability of macrophages to present antigen and express major histocompatibility complex MHC class II molecules is decreased during sepsis, it is not known to what extent this is associated with the loss of co-stimulatory receptor expression. Our objectives in this study were, therefore, to determine if the expression of co-stimulatory molecules, such as CD40, CD80, or CD86, on peritoneal/splenic/liver macrophages were altered by sepsis (cecal ligation [CL] and puncture [CLP] or necrotic tissue injury (CL) alone; and to establish the contribution of such changes to the response to septic challenge using mice that are deficient in these receptors.

Methods: To address our first objective, male C3H/HeN mice were subjected to CLP, CL, or sham (n = four to six mice/group), and the adherent macrophages were isolated from the peritoneum, spleen, or liver at 24 h post-insult. The macrophages were then analyzed by flow cytometry for their ex vivo expression of CD40, CD80, CD86, and/or MHC II.

Results: The expression of CD86 and MHC II, but not CD40 or CD80, were significantly decreased on peritoneal macrophages after the onset of sepsis or CL alone. In addition, CD40 expression was significantly increased in Kupffer cells after sepsis. Alternatively, splenic macrophages from septic or CL mice did not show changes in the expression of CD80, CD86, or CD40. To the degree that the loss of CD86 expression might contribute to the changes reported in macrophage function in septic mice, we subsequently examined the effects of CLP on CD86 -/- mice. Interestingly, we found that, unlike the background controls, neither the serum IL-10 concentrations nor the IL-10 release capacity of peritoneal macrophages from septic CD86 -/- mice were increased.

Conclusion: Together, these data suggest a potential role for the co-stimulatory receptor CD86/B7-2 beyond that of simply promoting competent antigen presentation to T-cells, but also as a regulator of the anti-inflammatory IL-10 response. Such a role may implicate the latter response in the development of sepsis-induced immune dysfunction.

FIG. 1

Expression of MHC II and CD86 on peritoneal macrophages 24 h post-CLP or CL. Peritoneal macrophages were isolated from C3H/HeN mice 24 h after surgery (CL, CLP or Sham-CLP [Sham]). For co-stimulatory molecule expression, peritoneal macrophages were stained with MHC II (A) and CD86 (B) with F4/80 Abs. The results are presented as percentage positively stained with Abs. *Significant difference at p < 0.05 versus sham, ANOVA followed by Tukey’s multiple comparisons test, mean ± SEM; n = four to six animals/group.

FIG. 2

Expression of CD80, CD86 and CD40 on Kupffer cells 24 h post-CLP. Liver macrophage (Kupffer cells, F4/80⁺ cells) were isolated from C3H/HeN mice 24 h after surgery (CLP or Sham-CLP [Sham]). For co-stimulatory molecule expression, Kupffer cells were stained with CD80, CD86, or CD40 with F4/80 Abs. The results are presented as percentage positively stained with Abs. *Significant difference at p < 0.05 versus sham, ANOVA followed by Tukey’s multiple comparisons test, mean ± SEM; n = four to six animals/group.

FIG. 3

Expression of MHC II and CD86 on splenic macrophages 24 h post-CLP or CL. Splenic macrophages (F4/80⁺ cells) were isolated from C3H/HeN mice 24 h after surgery (CL, CLP or Sham-CLP [Sham]). For co-stimulatory molecule expression, splenocytes were stained with MHC II (A) and CD86 (B) with F4/80 Abs. The results are presented as percentage positively stained with Abs. *Significant difference at p < 0.05 versus sham, ANOVA followed by Tukey’s multiple comparisons test, mean ± SEM; n = four to six animals/group.

FIG. 4

IL-6 and IL-10 levels in plasma of CD86 +/+ (background control) or CD86 -/- mice 24 h post-CLP. Plasma was collected from each animal 24 h after surgery. IL-6 (A) or IL-10 (B) levels were measured by ELISA. n = 4 animals/group. Significance indicated by *p < 0.05 versus sham, ^#p < 0.05 versus CD86 +/+ CLP, ANOVA followed by Tukey’s multiple comparisons test.

FIG. 5

Levels of IL-2 in conditioned medium of cultured splenocyte isolated 24 h post CLP or Sham-CLP [Sham] from CD86 +/+ and CD86 -/- mice, stimulated for 24 h with plate-bound anti-CD3. Triplets of equal number (10⁶) of cells/mL were cultured for 24 h (37°C, 5% CO₂). The supernatants were collected and IL-2 measurement was made by ELISA. n = 4 independent repeat experiments. *Significant difference at p < 0.05 versus sham, ANOVA followed by Tukey’s multiple comparisons test.

FIG. 6

Levels of IL-6 (A) and IL-10 (B) in conditioned medium of cultured peritoneal macrophages, isolated 24 h post CLP or Sham-CLP [Sham] from CD86 +/+ and CD86 -/- mice, stimulated for 24 h with LPS. Triplets of equal number (10⁶) of cells/mL were cultured for 24 h (37°C, 5% CO₂). The supernatants were collected and IL-2 measurement was made by ELISA. n = 4 independent repeat experiments. Significance indicated by *p < 0.05 versus sham, ^#p < 0.05 versus CD86 +/+ CLP, ANOVA followed by Tukey’s multiple comparisons test.