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Comparative Study
. 2010 Dec 1;185(11):6930-8.
doi: 10.4049/jimmunol.1002300. Epub 2010 Nov 1.

Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival

Florin L Craciun  1 , Elizabeth R Schuller, Daniel G Remick
Affiliations
Comparative Study

Early enhanced local neutrophil recruitment in peritonitis-induced sepsis improves bacterial clearance and survival

Florin L Craciun et al. J Immunol. .

Abstract

Neutrophils are critical for the rapid eradication of bacterial pathogens, but they also contribute to the development of multiple organ failure in sepsis. We hypothesized that increasing early recruitment of neutrophils to the focus of infection will increase bacterial clearance and improve survival. Sepsis was induced in mice, using cecal ligation and puncture (CLP); blood samples were collected at 6 and 24 h; and survival was followed for 28 d. In separate experiments, peritoneal bacteria and inflammatory cells were measured. Septic mice predicted to die based on IL-6 levels (Die-P) had higher concentrations of CXCL1 and CXCL2 in the peritoneum and plasma compared with those predicted to live (Live-P). At 6 h, Live-P and Die-P had equivalent numbers of peritoneal neutrophils and bacteria. In Die-P mice the number of peritoneal bacteria increased between 6 and 24 h post-CLP, whereas in Live-P it decreased. The i.p. injection of CXCL1 and CXCL2 in naive mice resulted in local neutrophil recruitment. When given immediately after CLP, CXC chemokines increased peritoneal neutrophil recruitment at 6 h after CLP. This early increase in neutrophils induced by exogenous chemokines resulted in significantly fewer peritoneal bacteria by 24 h [CFU (log) = 6.04 versus 4.99 for vehicle versus chemokine treatment; p < 0.05]. Chemokine treatment significantly improved survival at both 5 d (40 versus 72%) and 28 d (27 versus 52%; p < 0.02 vehicle versus chemokines). These data demonstrate that early, local treatment with CXC chemokines enhances neutrophil recruitment and clearance of bacteria as well as improves survival in the CLP model of sepsis.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Mice predicted to die in the acute phase of CLP sepsis had higher peritoneal and plasma levels of CXCL1 and CXCL2 than did those predicted to survive. By 24 h post-CLP, mice predicted to die had significantly lower peritoneal to plasma concentration gradients of CXCL1 and CXCL2. CXCL1 was measured by ELISA, and CXCL2 was measured by microarray immunoassay. The peritoneal concentration was calculated as described in the Materials and Methods. The concentration gradient was expressed as the ratio between the concentration of the chemokine in the peritoneum and the concentration in plasma. For A, C, D, and F, at 6 h n = 9 for Live-P and 11 for Die-P, and at 24 h n = 24 for Live-P and 13 for Die-P. For B and E, at 6 h n = 9 for Live-P and 11 for Die-P, and at 24 h n = 29 for Live-P and 14 for Die-P. Results are shown as mean ± SEM. p < 0.05 when compared with Live-P at 6 h (a); Die-P at 6 h (b); Live-P at 24 h (c).
FIGURE 2
FIGURE 2
Die-P mice had higher levels of pro- and anti-inflammatory mediators both locally (in the peritoneal cavity) and systemically (in plasma). Average z-scores for all the pro- and anti-inflammatory mediators measured for each mouse were calculated as described in the statistical analysis section of Materials and Methods. For A and B, at 6 h n = 9 for Live-P and 11 for Die-P, and at 24 h n = 29 for Live-P and 14 for Die-P. For C and D, at 6 h n = 9 for Live-P and 11 for Die-P, and at 24 h n = 24 for Live-P and 13 for Die-P. Results are shown as mean ± SEM. p < 0.05 when compared with Live-P at 6 h (a); Die-P at 6 h (b); Live-P at 24 h (c).
FIGURE 3
FIGURE 3
Relation between survival prediction and peritoneal or circulatory numbers of neutrophils. Total numbers of neutrophils (A) and macrophages (C) present in the peritoneum were calculated based on total and differential cell counts. Circulating counts of neutrophils (B) were obtained on a Hemavet instrument. For A, C, at 6 h n = 9 for Live-P and 11 for Die-P, and at 24 h n = 20 for Live-P and 10 for Die-P. For B, at 6 h n = 9 for Live-P and 11 for Die-P, and at 24 h n = 19 for Live-P and 9 for Die-P. Results are shown as mean ± SEM. p < 0.05 when compared with Live-P at 6 h (a); Die-P at 6 h (b); Live-P at 24 h (c).
FIGURE 4
FIGURE 4
Peritoneal bacterial load in relation to survival prediction. At 6 h post-CLP equivalent numbers of bacteria were present in both groups. The Live-P group controlled the infection and had fewer bacteria at 24 h post-CLP. The counts obtained after incubating in aerobe and anaerobe conditions were added in a single value that was log transformed. At 6 h n = 9 for Live-P and 11 for Die-P, and at 24 h n = 12 for Live-P and 11 for Die-P. Results are shown as mean ± SEM. p < 0.05 when compared with Live-P at 24 h (c).
FIGURE 5
FIGURE 5
Peritoneal neutrophil recruitment 4 h after local injection of CXCL1 and CXCL2 chemokines. Mice were injected i.p. with saline (200 μl), low-dose chemokines (100 ng CXCL2 + 10 ng CXCL1), or high-dose chemokines (500 ng CXCL2 + 50 ng CXCL1). A shows representative peritoneal lavage cytospin slides from the saline and high-dose chemokine-injected mice (Diff-Quick stain; original magnification ×1000). For B, C, and D, n = 4 for all the groups. Results are shown as mean ± SEM. p < 0.05 when compared with saline (a); 100 ng CXCL2 + 10 ng CXCL1 (b).
FIGURE 6
FIGURE 6
Peritoneal and circulating numbers of neutrophils after injection of chemokines immediately following CLP. Mice were injected i.p. with saline (200 μl) or chemokines (500 ng CXCL2 + 50 ng CXCL1). The i.p. injection of chemokines increased the early, 6 h, recruitment of neutrophils into the peritoneum. For A, C, at 6 h n = 10 for saline and 10 for chemokines, and at 24 h n = 20 for saline and 19 for chemokines. For B, at 6 h n = 9 for saline and 7 for chemokines, and at 24 h n = 27 for saline and 26 for chemokines. Results are shown as mean ± SEM. p < 0.05 when compared with saline at 6 h (a); chemokines at 6 h (b); saline at 24 h (c).
FIGURE 7
FIGURE 7
Peritoneal bacterial counts after injection of chemokines immediately following CLP. Mice were injected i.p. with saline (200 μl) or chemokines (500 ng CXCL2 + 50 ng CXCL1). The counts obtained after incubating in aerobe and anaerobe conditions were added in a single value that was log transformed. In mice injected i.p. with exogenous recombinant chemokines, compared with the saline-injected mice, there was a significant reduction in the growth of bacteria. At 6 h n = 10 for saline and 10 for chemokines, and at 24 h n = 20 for saline and 20 for chemokines. Results are shown as mean ± SEM. p < 0.05 when compared with saline at 6 h (a); saline at 24 h (c).
FIGURE 8
FIGURE 8
Improved 28-d survival after injection of chemokines immediately following CLP. Mice were injected i.p. with saline (200 μl) or chemokines (500 ng CXCL2 + 50 ng CXCL1). For saline n = 30, and for chemokines n = 29. The log rank test was performed to compare the two survival curves.
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