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The Journal of Immunology, 2006, 177: 1264-1271.
Copyright © 2006 by The American Association of Immunologists

Signaling via Platelet-Activating Factor Receptors Accounts for the Impairment of Neutrophil Migration in Polymicrobial Sepsis1

Susana E. Moreno2,*, José C. Alves-Filho2,*, Fabrício Rios-Santos*, João S. Silva{dagger}, Sérgio H. Ferreira*, Fernando Q. Cunha* and Mauro M. Teixeira3,{ddagger}

* Departamento de Farmacologia and {dagger} Departamento de Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil; and {ddagger} Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Minas Gerais, Brazil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a systemic inflammatory response that results from the inability of the immune system to limit bacterial spread during an ongoing infection. Recently, we have documented an impaired neutrophil migration toward the infectious focus in severe sepsis. This impairment seems to be mediated by circulating cytokines, chemokines, and NO. Platelet-activating factor (PAF) plays an important role in the orchestration of different inflammatory reactions, including the release of cytokines, chemokines, and free radicals. Using a PAFR antagonist, PCA-4248, and PAFR-deficient mice, we investigated whether signaling via PAFR was relevant for the failure of neutrophils to migrate to the site of infection after lethal sepsis caused by cecum ligation and puncture in mice. In PAFR-deficient mice or mice pretreated with PCA-4248 (5 mg/kg) and subjected to lethal sepsis, neutrophil migration failure was prevented, and bacterial clearance was more efficient. There was also reduced systemic inflammation (low serum cytokine levels), lower nitrate levels in plasma, and higher survival rate. Altogether, the results firmly establish a role for PAFR in mediating the early impairment of neutrophil migration toward the infectious focus. Blockade of PAFR may prevent the establishment of severe sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Platelet-activating factor (PAF)4 is a potent phospholipid mediator synthesized by a large number of cells, including platelets, endothelial cells, macrophage, and neutrophils (1, 2). Its biological activity is mediated through a G protein-linked receptor (PAFR) that is expressed on the surface of a variety of cell types (1, 2, 3) and regulated through a rapid degradation by a subfamily of phospholipases A2, the PAF-acetylhydrolases (4, 5). In the inflammatory response, PAF has well-characterized actions, mediating the activation of leukocytes to produce cytokines, NO, and other inflammatory mediators and recruitment of these cells to inflammatory site (1, 2). Because of its widespread production and action, PAF production and action on PAFR have been implicated in the pathophysiology of various inflammatory diseases, including asthma, systemic lupus erythematosus, rheumatoid arthritis, and Crohn’s disease (6, 7).

Sepsis is a systemic inflammatory response that results from the inability of the immune system to control bacterial spread during an ongoing infection. There are evidences that PAF concentrations are increased and PAF acetylhydrolase activity decreased in plasma of septic patients or experimental endotoxemia, suggesting that PAF might be a mediator of sepsis and septic shock (8, 9, 10, 11, 12, 13). In fact, it is reported that PAF itself is capable of eliciting many symptoms associated with endotoxic shock (7). Moreover, the overexpression of the PAFR increases lethality in response to LPS administration in mice (14), and the administration of PAFR antagonists to animals and humans protect them from the deleterious effects of LPS (15, 16, 17, 18, 19, 20). However, clinical trials using recombinant human PAF acetylhydrolase or PAFR antagonists failed to reduce the mortality of severe septic patients, although a substantial reduction in organ dysfunction was achieved (21, 22, 23).

Recently, we have shown that severe sepsis induced by cecal ligation and puncture (CLP) or Staphylococcus aureus inoculation is associated with impaired neutrophil recruitment into sites of infection (24, 25, 26, 27). This impairment of neutrophil migration resulted in augmented number of bacteria in the peritoneal cavity and blood which was associated with high mortality. On the other hand, in sublethal sepsis, the bacterial infection was restricted to the peritoneal cavity, neutrophil migration was not suppressed and no significant mortality was observed. The mechanisms involved in the impairment of neutrophil migration are not completely understood, but it may be due to excessive release of proinflammatory chemokines/cytokines and a concomitant increase in NO derived from inducible NO synthase (25, 26, 28, 29).

In the present study, using a PAFR antagonist and PAFR-deficient (PAFR–/–) mice, we investigated the role of PAFR signaling for the failure of neutrophil migration into the infectious focus and in the outcome of polymicrobial sepsis induced by CLP. We found that PAFR signaling results in an impaired neutrophil migration toward the infectious focus followed by bacteremia, increase of systemic cytokines, and high mortality. Thus, PAFR signaling is detrimental in severe polymicrobial sepsis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

BALB/C (8- to 10-wk-old) mice obtained from the facility of the School of Medicine of Ribeirão Preto were housed in cages in temperature-controlled rooms and received food and water ad libitum. PAFR–/– mice were generated as previously described and backcrossed or at least 10 generations into a BALB/c background (30). All experiments were conduced in accordance with the ethical guidelines of the School of Medicine of Ribeirão Preto, University of São Paulo.

Sepsis model

Sepsis was induced through CLP as previously described with slight modification (31). Briefly, mice were anesthetized with tribromoethanol (250 mg · kg–1), a 1-cm midline incision was made on the anterior abdomen, and the cecum was exposed and ligated below the ileocecal junction without causing bowel obstruction. A single puncture was made through the cecum using a 21- or 16-gauge needle to induce nonlethal (NL-CLP) and lethal (L-CLP) sepsis, respectively. In another set of experiment, seven punctures were made through the cecum using a 21-gauge to induce moderate sepsis (M-CLP). The cecum was returned to the abdomen, and the peritoneal wall and skin incision were closed. All animals received 1 ml of saline s.c. immediately after the surgery.

Experimental protocol

The mice were pretreated s.c. with vehicle (saline; 0.2 ml) or with the PAFR antagonist 2-(phenylthio)ethyl-5-methoxycarbonyl-2,4,6-trimethyl-1,4-dihydropyridine-3-carboxylate (PCA-4248; 5 mg · kg–1). Thirty minutes later, mice were subjected to NL-CLP, M-CLP, or L-CLP. In another set of experiments, the animals subjected to L-CLP were treated after CLP procedure (5 mg/kg, s.c., 4 and 24 h after L-CLP). After sepsis induction, we determined neutrophil migration into peritoneal cavity, leukocyte rolling, and adhesion to mesenteric microcirculation, bacterial load in the peritoneal exudates, and blood and cytokines and nitrate concentration in serum. The survival rate of mice was determined daily for 21 days after sepsis induction. The experiments were repeated two to three times. This used dose of PCA-4248 did not interfere with basal cytokine levels and neutrophil migration observed in normal animals (data not shown).

Neutrophil migration to the peritoneal cavity after sepsis induction

Neutrophil migration was assessed 6 h after sepsis induction. The animals were killed, and the cells present in the peritoneal cavity were harvested by introducing 1.5 ml of PBS containing 1 mM EDTA. Total counts were performed with a cell counter (Coulter AC T series analyzer; Beckman Coulter), and differential cell counts were conducted on cytocentrifuge slides (Cytospin 3; Shandon Southern Products) stained by the May-Grünwald-Giemsa (Rosenfeld) method. The results are expressed as the number of neutrophils per cavity.

Intravital microscopy of leukocytes to assess rolling and adhesion to the mesentery after sepsis induction

The leukocyte parameters were examined as described previously (25). Briefly, mice were anesthetized with tribromoethanol (250 mg · kg–1, diluted in saline), and the mesenteric tissue was withdrawn for microscopic examination. The animals were maintained on a special board thermostatically controlled at 37°C, with a transparent platform on which the tissue to be transilluminated was placed. The preparation was kept moist and warm by irrigating the tissue with warmed (37°C) Ringer Locke’s solution (pH 7.2–7.4), containing 1% gelatin.

A 500-line television camera was incorporated onto a triocular Zeiss microscope to facilitate observation of the enlarged image (x3400) on the video screen. Images were recorded on a video recorder with a long-distance objective (x40) with a 0.65-numerical aperture. Vessels selected for study were third-order venules, defined according to their branch-order location within the microvascular network. These vessels corresponded to postcapillary venules, with a diameter of 12–18 µm. Rolling leukocytes (rollers) were defined as those white blood cells that moved at a lower velocity than erythrocytes in the same stream. The number of rolling leukocytes was determined at 10-min intervals. These leukocytes moved at a sufficiently slow pace as to be individually visible and were counted as they rolled past a 100-µm length of venule. Rolling was assessed 2 h after CLP sepsis had commenced. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for >30 s (32). The number of adherent cells (stickers) was expressed as the number per 100-µm length of venule investigated 4 h after CLP sepsis commencement.

Bacterial counts in the peritoneal exudates and blood

Bacterial count was assessed as described previously (33). Briefly, mice were killed 6 h after sepsis induction. For peritoneal lavage fluid, the peritoneal cavity was washed with 1.5 ml of sterile PBS, and aliquots of serial dilutions of these peritoneal fluids were plated on Muller-Hinton agar dishes (Difco) and incubated at 37°C; CFU were analyzed after 24 h. The results were expressed as log of CFU per peritoneal cavity. For bacteremia, blood was collected at the same time under sterile conditions, and 10 µl of blood was plated on Muller-Hinton agar dishes and incubated at 37°C; CFU were analyzed after 24 h, and the results were expressed as log of CFU per milliliter of blood.

Determination of cytokine levels

The animals were killed 6 h after sepsis induction, the blood was harvested, and the concentrations of TNF-{alpha}, IL-6, and IL-10 were determined by using a double-ligand ELISA. The results are expressed as picograms per milliliter. The peritoneal exudate was also harvested, and the concentration of KC was determined by ELISA. The results are expressed as nanograms per milliliter.

Determination of serum nitrite concentration

The nitrate concentration in serum samples was determined by enzymatic reduction of nitrate with nitrate reductase, as described previously (34).

Statistical analysis

The data (except for the survival curves) are reported as the means ± SEM of values obtained from two different experiments. The means of different treatments were compared by ANOVA. If significance was determined, individual comparisons were subsequently tested with Bonferroni’s t test for unpaired values. Bacterial counts were analyzed by the Mann-Whitney U test. The survival rate was expressed as the percentage of live animals, and a log-rank test ({chi}2 test) was used to determine differences between survival curves. A value of p ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The blockade of PAFR improves the resistance against polymicrobial sepsis

Initial experiments were designed to examine the involvement of PAFR signaling in the outcome of polymicrobial sepsis. To this end, mice were pretreated with a PAFR antagonist (PCA-4248; 5 mg/kg, s.c.) or vehicle (control) and subjected to NL-CLP, M-CLP, or L-CLP by CLP model. As shown in Fig. 1A, all vehicle- and PCA-4248-pretreated animals subjected to NL-CLP survived throughout the observation period (21 days). Moreover, both vehicle- and PCA-4248-pretreated animals subjected to M-CLP presented survival rates of ~50% (Fig. 1A). On the other hand, 100% of vehicle-treated mice subjected to L-CLP died within 3 days, and the pretreatment with PCA-4248 significantly protected the animals against L-CLP-induced lethality. There was 50% of survival at day 6 in PCA-4248-treated mice, and protection was sustained throughout the observation period (Fig. 1B). Since the clinical intervention is not routinely performed as preventive, we also performed a set of experiment using a posttreatment protocol. PCA-4248 was given in two doses with a 24-h interval after CLP, beginning 4 h after the lethal CLP procedure. Although the posttreatment was less effective than the pretreatment, it also conferred significant protection against lethality (25%) until day 9 after sepsis induction. After this point, the survival rates of the posttreated group was not significantly different to the untreated animals (Fig. 1B). Altogether, these findings suggest that PAFR signaling is detrimental in the early phase of lethal sepsis, whereas this receptor appeared to play no major role in nonlethal or moderate sepsis. Thus, the remaining of this investigation focused on identifying the detrimental role of PAF on lethal sepsis using the pretreatment protocol.


Figure 1
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  		FIGURE 1. PAFR antagonist, PCA-4248, improves resistance against polymicrobial sepsis. A, Survival rates of animals pretreated with vehicle (control) or PCA-4248 (PCA; 5 mg · kg–1) and submitted to NL-CLP and M-CLP were determined daily up to 21 days after surgery. B, Survival rates of animals that receive vehicle (control), pretreated (5 mg · kg–1, 30 min before CLP), or posttreated with PCA-4248 (5 mg · kg–1, s.c., 4 and 24 h after CLP) and submitted to L-CLP were determined daily up to 21 days after surgery. The experiment was repeated three times. Results are expressed as percent survival. The survival rate of the PCA-4248-pretreated animals was significantly different from animals that received only vehicle after L-CLP with p < 0.05, Mantel-Cox log-rank test (n = 12–20).

 
Effect of PAFR blockade on rolling, adhesion, and migration of neutrophil and on chemokine production in mice subjected to polymicrobial sepsis

We have demonstrated that a marked impairment of neutrophil migration into the infectious focus is observed in lethal sepsis, which is associated with a high mortality rate (24, 26). In an attempt to investigate whether PAF is involved in this process, the recruitment of neutrophils into the peritoneal cavity was determined in vehicle- and PCA-4248-pretreated mice subjected to L-CLP. In agreement with our previous data (24, 26), the results in Fig. 2A show that animals subjected to NL-CLP presented a marked neutrophil migration into the peritoneal cavity, increasing gradually from 6 to 12 h after surgery. However, mice subjected to L-CLP displayed an impaired neutrophil migration. Indeed, in the latter group, the migration of neutrophils was 5-fold smaller than that observed in mice subjected to NL-CLP (Fig. 2A), despite the fact that the number of holes and the number of bacteria that fall into the peritoneal cavity of L-CLP mice are higher than those of NL-CLP mice. Moreover, using intravital microscopy to visualize leukocyte-endothelial cell interactions, we found that the impairment of neutrophil migration was correlated with a significant reduction in the number of rolling and adherent leukocytes to postcapillary venules of the mesentery when compared with NL-CLP mice (Fig. 2B). When mice subjected to L-CLP were pretreated with PCA-4248, the reduction of rolling and adhesion of leukocytes to postcapillary venules (Fig. 2B) and the impairment of migration of neutrophils into peritoneal cavity (Fig. 2A) were reduced.


Figure 2
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  		FIGURE 2. Effects of PAFR blockade on rolling, adhesion, and migration of neutrophil in mice subjected to polymicrobial sepsis. A, Neutrophil migration into the peritoneal cavity was performed 6 and 12 h after NL-CLP or lethal L-CLP surgery. The animals were pretreated with PCA-4248 (5 mg · kg–1) or received only vehicle as control. Results are expressed as means ± SEM (n = 10). *, p < 0.05 compared with NL-CLP group; #, p < 0.05 compared with animals treated with vehicle subjected to L-CLP (ANOVA, followed by Bonferroni’s test). B, Leukocyte rolling (left set of bars) and adhesion (right set of bars) to venular mesentery in animals pretreated with PCA-4248 (5 mg · kg–1) or vehicle, using in vivo intravital microscopy assay. The rolling and adherence parameters were evaluated 2 and 4 h after NL-CLP or L-CLP surgery, respectively. The experiments were repeated twice. The results are expressed as mean ± SEM (n = 6). *, p < 0.05 compared with NL-CLP group; #, p < 0.05 compared with animals treated with vehicle subjected to L-CLP (ANOVA, followed by Bonferroni’s test).

 
Because neutrophil migration is a complex process that involves several chemotactic factors that include CXC chemokines, we also evaluated the production of the CXC chemokine KC after blockade of PAFR. We observed that production of KC at the site of infection was not affected by blockade of PAFR as compared with untreated L-CLP group (NL-CLP, 3.34 ± 0.43; L-CLP, 4.70 ± 0.35; and L-CLP+PCA-4248, 4.14 ± 0.27 ng/ml; n = 5). Thus, the beneficial effect of PAFR inhibition did not correlate with an enhancement of CXC chemokines at the site of infection. Taken together, these results suggest that PAFR signaling underlies the failure of neutrophils to migrate into the infection focus during lethal sepsis.

PAFR blockade improves bacterial clearance in mice subjected to polymicrobial sepsis

The next series of experiments was designed to investigate the effects of PAFR blockade on the bacterial load in the infectious focus (peritoneal exudate) and bacteremia (blood) 6 h after CLP. As shown in Fig. 3, A and B, vehicle-treated mice subjected to NL-CLP did not present detectable bacterial counts in exudate or blood. In contrast, vehicle-treated mice subjected to L-CLP failed to control infection, as demonstrated by the increased number of bacteria in the peritoneal cavity and blood. Pretreatment with PCA-4248 induced a marked reduction of bacterial counts in the peritoneal cavity and blood in mice subjected to L-CLP (Fig. 3), clearly demonstrating that the PAFR blockade results in the improvement of bacterial clearance. It is important to reinforce that the number of bacteria that transmigrate to peritoneal cavities of L-CLP mice is higher than that of NL-CLP mice. Thus, although the number of neutrophil that migrated to peritoneal cavity of L-CLP mice treated with PAFR antagonist was similar to that observed in NL-CLP mice (Fig. 2A), the ratio of neutrophil:bacteria present in peritoneal cavity is different. It appears that the blockade of PAFR re-established the impaired neutrophil migration only partially, possibly explaining why the control of infection was not totally efficient in PAFR antagonist-treated mice.


Figure 3
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  		FIGURE 3. PAFR blockade improves bacterial clearance in mice subjected to polymicrobial sepsis. Bacterial counts in the exudate (A) and blood (B) from animals subjected to NL-CLP and L-CLP surgery, which were pretreated with PCA-4248 (5 mg · kg–1) or vehicle. Quantification of the bacteria was performed 6 h after sepsis induction, and the results are expressed as log of CFU/milliliter (n = 9). The experiment was repeated twice. *, p < 0.05 compared with NL-CLP group; #, p < 0.05 compared with animals treated with vehicle subjected to L-CLP (ANOVA, followed by Bonferroni’s test).

 
The systemic inflammatory response is attenuated after PAF blockade in mice during polymicrobial septic peritonitis

High levels of systemic inflammatory cytokines might contribute to organ injury and shock during sepsis. To elucidate the potential role of PAFR signaling in controlling the cytokine storm that accompanies a lethal septic response, we next investigated serum levels of cytokines 6 h after CLP. The results in Fig. 4 show that serum concentrations of TNF-{alpha}, IL-6, and IL-10 were significantly increased in vehicle-treated mice subjected to L-CLP as compared with the NL-CLP group. Pretreatment with PCA-4248 markedly reduced the systemic cytokine levels in mice subjected to lethal sepsis (Fig. 4), indicating that the systemic inflammatory response is attenuated in these animals.


Figure 4
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  		FIGURE 4. Systemic concentration of TNF-{alpha}, IL-10, and IL-6 are attenuated after PAFR blockade in mice during polymicrobial sepsis. TNF-{alpha} (A), IL-10 (B), and IL-6 (C) levels in animals subjected to NL- and L-sepsis induced by CLP, which were pretreated with PCA-4248 (5 mg · kg–1) or vehicle. The cytokine levels in serum were determined at 6 h after sepsis induction. The experiment was repeated twice. Results are expressed as means ± SEM (n = 6). *, p < 0.05 compared with NL-CLP group; #, p < 0.05 compared with animals treated with vehicle subjected to L-CLP (ANOVA, followed by Bonferroni’s test).

 
PAFR–/– mice are also protected from the lethal effects of polymicrobial sepsis

To confirm the detrimental role of PAFR signaling in lethal sepsis, we also conducted experiments using PAFR–/– mice. The results in Fig. 5 show that PAFR-deficient mice presented a significant enhancement of the survival rate after L-CLP as compared with wild-type (WT) mice (Fig. 5A). In animals subjected to NL-CLP, there was no detectable lethality in PAFR–/– and WT mice (Fig. 5A). In PAFR–/– mice subjected to L-CLP, there was a reduction of the impairment of migration of neutrophils to the infectious focus, and the bacterial counts in blood were significantly reduced as compared with WT mice (Fig. 5, B and C). Although the bacterial counts in Figs. 5C and 3B differ in intensity, probably reflecting interassay variation, it is clear that either pharmacological or genetic inhibition of PAFR promoted similar reduction of infection indices. Peritoneal exudate levels of KC were similar in WT and PAFR–/– mice subjected to L-CLP (L-CLP WT, 4.70 ± 0.35; and L-CLP PAF–/–, 4.26 ± 0.23 ng/ml; n = 5). These results are consistent with data obtained with the PAFR antagonist.


Figure 5
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  		FIGURE 5. Likely to PAFR antagonism, the PAFR–/– mice are protected from the lethal effects of polymicrobial sepsis. Survival rate (A), neutrophil migration into peritoneal cavity (B), bacterial count in blood (C), and TNF-{alpha}, IL-10, and IL-6 levels in serum (D) were evaluated in PAFR–/– and WT mice subjected to NL- and L-polymicrobial sepsis induced by CLP. A, The survival rate of PAFR–/– was significantly different from WT mice after L-CLP; p < 0.05, Mantel-Cox log-rank test (n = 12). B–D, *, p < 0.05 compared with NL-CLP group; #, p < 0.05 compared with WT mice subjected to L-CLP (n = 5; ANOVA, followed by Bonferroni’s test). The experiments were repeated twice.

 
We have previously demonstrated that the systemic production of NO induced by circulating inflammatory cytokines contributes to impairment of neutrophil migration into the infectious focus in lethal sepsis (24, 26, 28). Thus, we assessed serum levels of cytokines and nitrate in WT and PAFR-deficient mice submitted to L-CLP. The results in Fig. 5D and Table I show that serum concentrations of TNF-{alpha}, IL-6, IL-10, and nitrate were significantly increased in WT mice subjected to L-CLP as compared with NL-CLP group. In contrast, the circulating levels of TNF, IL-6, and nitrate reduced significantly in PAFR–/– mice subjected to L-CLP.


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  		Table I. Serum nitrate concentration 6 h after polymicrobial sepsis induced by CLPa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Studies from our and other laboratories demonstrate that neutrophil migration to the infectious focus is extremely important for the local control of bacterial growth, preventing bacterial dissemination, and, consequently, evolution of sepsis. It was observed that neutrophil migration to the infectious focus is impaired in severe sepsis induced by CLP or S. aureus infection, and this phenomenon is associated with an increase of bacterial count at the site of infection, bacteremia, and high mortality (24, 25, 26). It seems that an early and inappropriate systemic inflammatory response, characterized by elevated levels of plasma cytokines, chemokines, and NO mediates the impairment in the migration of neutrophils (25, 26, 28). There are evidences indicating that PAF, an inflammatory phospholipid mediator, induces several pathophysiological dysfunctions, which are observed in both animal models and in human sepsis (6, 35). In this study, we investigated whether PAFR signaling is involved on the failure of neutrophil migration to the infectious focus observed in lethal sepsis.

Our data clearly demonstrate that the absence of PAFR signaling, as assessed by using a PAFR antagonist and PAFR-deficient mice, provided significant protection from CLP-induced mortality in lethal sepsis. The survival advantage provided by blockade of PAFR signaling was associated with an enhancement of neutrophil migration into the infectious site, i.e., there was a reduction of failure of the migration of neutrophils in PAFR antagonist-treated or PAFR–/– mice (Figs. 2A and 5B). Moreover, the use of intravital microscopy revealed that the blockade of PAFR avoided the reduction of leukocyte-endothelial cell interactions (rolling and adhesion) observed in mice subjected to lethal CLP (Fig. 2B), supporting the concept that the impairment of an adequate neutrophil-endothelial interaction in lethal sepsis involves PAFR signaling. Reinforcing this hypothesis, it was observed that the peritoneal levels of KC (a neutrophil chemotactic chemokine) in septic mice were not affected by blockade of PAFR or in PAFR–/– mice. The survival rate of animals subjected to moderate septic insult (M-CLP) was not affected by treatment with the PAFR antagonist (Fig. 1A). In this context, in a recent study, we demonstrated that animals submitted to M-CLP did not present failure of neutrophil migration to the site of infection (36). Altogether, these findings suggest that the main harmful effect of PAF in lethal sepsis is the mediation of the failure of the migration of neutrophils.

Although the PAFR blockade increased the rolling and adhesion of the neutrophils on endothelial cells, as discussed above, there is also the possibility that a reduction of apoptosis could account for at least part of the increased number of neutrophils accumulated in the infection focus of L-CLP mice treated with PAFR antagonist. Indeed, there is evidence to suggest that apoptosis is increased during sepsis (37). As the available evidence in the literature suggests that PAF delays neutrophil apoptosis (38, 39), it seems unlikely that blockade of PAFR would lead to an increased accumulation of neutrophils after L-CLP.

PAFR antagonists may improve resistance against the systemic inflammatory response and lethal shock induced by administration of a high dose of LPS (16, 17, 18, 19, 20). The CLP sepsis model applied in our study differs from the endotoxic shock model because it also has an infectious process. As a consequence, beneficial effects for the outcome of sepsis caused by CLP may not only require attenuation of the systemic inflammatory response but also depend on activation or re-establishment of an efficient local host defense. We observed that the animals pretreated with PAFR antagonist had a marked reduction of bacterial counts in peritoneal cavity and blood, which was confirmed in PAFR–/– mice (Figs. 3 and 5C). Altogether, the results show that the enhanced neutrophil migration observed in PAFR antagonist-treated or PAFR–/– mice was accompanied by an improvement of bacterial clearance and reduction of the systemic spread of bacteria. Improved bacterial clearance and reduction of bacteremia may account for the increased survival rate after PAFR blockade. In this context, we have previously demonstrated that there was a positive association between bacteremia and mortality (25, 26).

The systemic inflammatory response is considered a central pathogenic event in severe sepsis. High levels of systemic inflammatory cytokines are implicated on the development of multiple organ failure and shock (40, 41). Among the cytokines, several reports have demonstrated that IL-6 levels are a major prognostic indicator of sepsis severity (42, 43, 44). Moreover, as mentioned previously, elevated levels of plasma cytokines, chemokines, and NO play a crucial role in mediating the impairment of the migration of neutrophils into the site of infection (24, 26, 28). Our results show that, during lethal CLP, there was a dysregulated elevation of systemic TNF-{alpha} and IL-6 levels and that PAFR blockade significantly reduced the levels of these cytokines (Figs. 4 and 5D). Furthermore, nitrite production, an index of inducible NO synthase activity, in response to lethal CLP was diminished significantly in PAFR–/– mice as compared with WT mice, suggesting that PAFR signaling may enhance NO production during severe sepsis (Table I). The latter results are consistent with studies demonstrating a close temporal relationship between the appearance of PAF and NO synthesis during sepsis (45, 46, 47, 48, 49) and results showing that PAF contributes to the induction of TNF-{alpha} after LPS injection in mice (50) and LPS activation of macrophages in vitro (51). Altogether, these data provide evidence that PAFR signaling is an important determinant of the systemic inflammatory response evolution during lethal sepsis.

Several studies have shown that systemic IL-10 is expressed in elevated concentrations during sepsis (24, 52, 53). Although some studies have shown that IL-10 produced concomitantly with TNF-{alpha} and IL-1 may counteract the proinflammatory effects of these cytokines (54, 55), anti-inflammatory predominance in sepsis seems to be associated with increased severity of infection and as a consequence increased end-organ damage and increased mortality (56, 57). In fact, van der Poll et al. (52) reported that plasma concentrations of IL-10 remained invariably high only in nonsurviving patients, while it significantly decreased in survivors. Thus, the imbalance between pro- and anti-inflammatory mediators is related to the severity and mortality of sepsis (57, 58). Our results show that the production of IL-10 increased in the serum of control L-CLP mice and reduced by blockade of PAFR (Figs. 4B and 5D). Interestingly, the reduction of IL-10 occurred concomitantly with the decrease of TNF-{alpha} and IL-6, reinforcing that the blockade of PAFR protect animals from established severe sepsis.

The evidence described above argues that the severity of sepsis correlates with levels of cytokines, nitrate, and neutrophil migration to the site of infection. However, the mortality was higher in L-CLP mice treated with PAFR antagonist (~40%) than that observed in NL-CLP (0%) (see Fig. 1). In the two groups, there were similar levels of cytokines, nitrate, and neutrophil migration (see Figs. 2 and 4). Interestingly, a similar percentage of the animals of the L-CLP group treated with the PAFR antagonist also had higher CFU in the exudates and blood than those observed in the NL-CLP group (see Fig. 3), which might explain the increased mortality in the former experimental group. A possible explanation to the greater lethality in PAFR antagonist-treated L-CLP mice is that the number of bacteria that transmigrates to peritoneal cavities of L-CLP is higher than that in NL-CLP, a reflection of the larger bore of the needle used to induce damage in the L-CLP group. Thus, although the number of neutrophils that migrated to peritoneal cavity of L-CLP mice treated with the PAFR antagonist was similar to that observed in NL-CLP mice (Fig. 2A), the ratio of neutrophil:bacteria present in peritoneal cavity was different, i.e., a similar number of neutrophils was involved in the control of different number of bacteria. This may be a possible explanation as to why the control of infection in PAFR antagonist-treated mice submitted to L-CLP was not totally efficient, why the bacteria reached the circulation, and survival was reduced. Taken together, the results demonstrate that the blockade of PAFR re-established only partially the neutrophil migration impairment, suggesting that other mediators, such as cytokines/chemokines (59) and leukotrienes (60), are also mediating the failure of neutrophil migration and other harmful events of L-CLP.

Our results demonstrating that the blockade of PAFR signaling reduced the impairment of neutrophil migration toward the infectious focus and reduced lethality may suggest that the inhibition of PAFR signaling might be an adequate strategy for the treatment of sepsis. This tenet is further reinforced by the studies showing that serum levels of PAF acetylhydrolase, which inactivates PAF, are decreased in severe sepsis (61, 62) and that PAFR antagonists improve resistance against the lethal effects of experimental endotoxemia (16, 17, 18, 19, 20). However, clinical trials with recombinant human PAF acetylhydrolase or PAFR antagonists have not been shown to reduce the mortality of patients with severe sepsis (21, 22, 23). Similarly, we observed that the protective effect of PAFR antagonist reduced when it was used as posttreatment protocol (Fig. 1B). Our studies may provide an interesting possibility to explain this apparent contradiction between good effects of PAFR antagonists experimentally but not in clinical trials. In the trials, the treatment of patients was started when the shock syndrome was advanced, and, hence, when the impairment of neutrophil migration was already established. In this context, we have demonstrated that neutrophils obtained from patients with severe sepsis present reduced chemotactic activity (34).

In summary, the present study identifies, for the first time to our knowledge, a fundamental role for PAFR in mediating the failure of neutrophils to migrate to infection focus during severe sepsis. The results suggest that PAFR antagonists used in the early stage of shock are potential drugs for immunotherapy of sepsis since they might reduce the impairment of neutrophil migration and, as consequence, avoid the spread of bacteria and the systemic inflammatory response syndrome.


   Acknowledgments
 
We are grateful to Giuliana Bertozi, Fabíola Leslie Mestriner, and Ana Kátia dos Santos for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico, and Programa de Núcleos de Excelência. Back

2 S. E. M. and J. C. A.-F. contributed equally to the work. Back

3 Address correspondence and reprint requests to Professor Mauro M. Teixeira, Department of Bioquimica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, Minas Gerais 31270-901, Brazil. E-mail address: mmtex{at}icb.ufmg.br Back

4 Abbreviations used in this paper: PAF, platelet-activating factor; CLP, cecum legation and puncture; PAFR–/–, PAF deficient; NL-CLP, nonlethal sepsis CIP, L-CLP; lethal sepsis CIP; M-CLP, moderate CLP; PCA-4248, 2-(phenylthio)ethyl-5-methoxycarbonyl-2,4,6-trimethyl-1,4-dihydropyridine-3-carboxylate; WT, wild type. Back

Received for publication May 29, 2005. Accepted for publication April 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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