Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis

Abstract

Objective: Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis.

Design: Prospective randomized animal investigation and in vitro studies.

Setting: Preclinical academic research laboratory.

Subjects: Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies.

Interventions: Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis.

Measurements and main results: Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis.

Conclusions: Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses, making this a promising new treatment strategy for sepsis.

Figure 1. Pnx1-dependent and independent mechanisms contribute to PMN activation in sepsis

(A) Blood samples of wild-type (WT) and homozygous pnx1 KO mice (pnx1^−/−) were stimulated in vitro with W-peptide (100 μM, 15 min at 37°C) and PMN activation was assessed. (B-C) Heterozygous (pnx1^+/−) or homozygous (pnx1^−/−) pnx1 KO and WT mice were subjected to CLP and PMN activation was determined after 4 h. Panel B shows representative flow cytometer results. Panel C shows cumulative data from mice subjected to CLP or sham surgery. (D) Plasma AST levels of pnx1 KO and WT mice subjected to sham surgery or CLP were assessed 4 h after CLP. Data are expressed as mean ± SEM of 5 animals in each group (ANOVA; *p<0.05, **p<0.01, ***p<0.001).

Figure 2. Pnx1-dependent mechanisms contribute to host immune defense in sepsis

(A) WT mice were subjected to sham surgery (t=0) or CLP for the indicated times. Then bacterial counts (colony forming units; CFU/ml) were determined in peritoneal lavage, blood, and bronchoalveolar lavage (BALF) samples. (B) Heterozygous (pnx1^+/−) and homozygous (pnx1^−/−) pnx1 KO and WT mice were subjected to CLP for 4 h, sacrificed, and bacterial counts determined in blood, BALF, spleen, liver, lungs, and peritoneal lavage. Data shown represent the mean and SEM of 3–5 mice in each group. Statistical comparisons between groups were made with ANOVA, *p<0.05, **p<0.01, ***p<0.001. (C) WT mice and homozygous (pnx1^−/−) pnx1 KO mice were subjected to CLP and survival rates were recorded for a period of 72 h following CLP. Each group consisted of 11 animals. Survival statistics were assessed using the Kaplan-Meier test, *p<0.05.

Figure 3. Inhibition of P2 receptor signaling reduces host organ damage but also host immune defense in sepsis

(A) Three different strategies with the aim to reduce PMN activation and preserving host defense were employed, administering suramin subcutaneously at a dose of 4 μl/g BW of a 4-mM solution either 10 min before or 1 h or 3 h after CLP. (B–C) Animals were sacrificed 18 h after CLP and PMN activation was assessed. Panel B shows the percentage of activated (CD11b⁺) PMNs and panel C shows the abundance of CD11b on the surface of PMNs. (D–E) AST levels or bacteria counts in the circulation of mice treated as described above were determined as readouts of host organ damage or host immune defense, respectively. Data shown are expressed as mean ± SEM of 8–12 animals per group (ANOVA; *p<0.05, **p<0.01, ***p<0.001).

Figure 4. Removing systemic ATP improves host immune defense and survival in sepsis

(A) WT mice were randomly divided into three groups. Control mice received normal saline vehicle prior to CLP; suramin was administered subcutaneously as a bolus (16 nmol/g BW) 10 min prior to CLP; and apyrase was administered intraperitoneally as a bolus (0.6 IU/g BW) 30 min prior to CLP. Animals were sacrificed after 18 h and PMN activation and plasma AST levels were determined. Data are expressed as percent change compared to untreated controls and represent the means ± SEM of 8–12 animals per group (ANOVA; *p<0.05, **p<0.01, ***p<0.001). (B) Body temperature was recorded with a rectal temperature probe before surgery (t=−1 h), at the end of surgery (t=0 h), and throughout the 72-h observation period following CLP. Each data point represents the mean ± SEM of 6 animals in each group. Differences among the three groups were analyzed with ANOVA (*p<0.05 control vs. apyrase; ^#p<0.05; ^##p<0.01 apyrase vs. suramin). (C) Survival rates in the three treatment groups were recorded for a period of 72 h after CLP. Each group consisted of 11 animals. Survival statistics were assessed using the Kaplan-Meier test (*p<0.02 control vs. apyrase; ^$p<0.05 control vs. suramin; ^##p<0.01 apyrase vs. suramin). (D) Numbers of bacteria in the blood and the peritoneum of mice with or without apyrase treatment were assessed 18 h after CLP. Data are expressed as means ± SEM of 17–21 animals per group (blood) and 6–10 animals per group (peritoneum). Comparisons between groups were made with ANOVA (**p<0.005, ***p<0.0001 control vs. apyrase). (E) ATP concentrations and total PMN counts in the peritoneal lavage of CLP mice without (control) and with apyrase treatment were determined (n=3 per group). Comparisons between groups were made with ANOVA (**p<0.005, ***p<0.0001 control vs. apyrase).

Figure 5. Removing systemic ATP improves PMN chemotaxis

Freshly isolated primary human PMNs were exposed to a chemotactic gradient using a micropipette loaded with fMLP (100 nM). Individual cell traces were recorded with time lapse imaging and analyzed with image processing software. (A) Representative images of cell traces showing chemotaxis of PMNs to the chemotactic source (micropipette tip at right edge of each image); 20x objective. (B) Increasing concentrations of exogenous ATP were added in the presence or absence of apyrase and PMN chemotaxis was assessed (see also video 1). Individual cell traces were aligned with their starting points at coordinate x=y=0 μm and the micropipette tip at coordinate x=0, y=200 μm. (C) The velocity of migration was calculated from the total distance each cell traveled divided by the observation time. Gradient sensing ability of cells was estimated by determining the percentage of PMNs that migrated in the correct direction, i.e., on a migration path that did not deviate by more than 60° from a straight line between their starting points and the micropipette tip. The experiments shown were performed with cells isolated from at least three different healthy individuals. Data are expressed as means ± S.D. of n=20–40 cells from each donor.