Despite substantial investigative efforts, sepsis remains a major cause of morbidity and mortality in intensive care units (1, 2). Acute lung injury is recognized as a key component in the pathophysiology of sepsis. Numerous data have demonstrated that excessive infiltration of neutrophils is a rate-limiting step in septic lung damage (3, 4). For example, inhibition of LFA-1 (CD11a/CD18) and membrane-activated complex 1 (Mac-1) (CD11b/CD18) not only attenuates neutrophil infiltration but also protects against septic lung injury (5). Tissue navigation of leukocytes is coordinated by secreted chemokines, including two main groups (CC and CXC), based on structural properties (6). The CXC chemokines, such as macrophage inflammatory protein 2 (MIP-2), are considered to predominately attract neutrophils (7, 8). Besides their well-known role in wound healing and vascular hemostasis (9), accumulating data suggest that platelets also exert proinflammatory actions, including supporting pulmonary recruitment of neutrophils in sepsis (10). Indeed, a recent study in abdominal sepsis showed that platelet-derived CD40 ligand (CD40L, CD154) activates neutrophils and supports Mac-1-mediated neutrophil infiltration in septic lung injury (11). Thus, complex interactions between neutrophils and platelets may be operative in the pathophysiology of sepsis.
The clinical course of streptococcal infections ranges from uncomplicated cases to severe and fatal conditions, such as streptococcal toxic shock syndrome (STSS), in which mortality rates can exceed 50% (12). M protein expressed on the surface of Streptococcus pyogenes is an important virulence factor. There are more than 80 different serotypes, but the M1 serotype is most frequently associated with STSS (13). M1 protein exerts numerous proinflammatory actions, such as increased production of cytokines (14), chemokines (15), and tissue factor (16), which all may contribute to M1 protein-induced edema formation and tissue damage in the lung. Interestingly, previous reports have shown that M1 protein may directly bind and activate several cells of the innate immune system, including neutrophils (14, 17) and platelets (18). However, the individual roles of these cells in M1 protein-induced lung injury remain elusive.
Based on the considerations above, the aim of the present study was to define the role of neutrophils and platelets in M1 protein-induced inflammation and tissue injury in the lung.
MATERIALS AND METHODS
Male C57Bl/6 mice weighing 20 to 25 g were used. All animals were housed on a 12-h/12-h light-dark cycle and fed a laboratory diet and water ad libitum. All experimental procedures were approved by the Regional Ethical Committee for Animal Experimentation at Lund University, Sweden. Mice were anesthetized i.p. with 75 mg of ketamine hydrochloride (Hoffman-La Roche, Basel, Switzerland) and 25 mg of xylazine (Janssen Pharmaceutica, Beerse, Belgium) per kilogram of body weight.
A total of 47 C57Bl/6 mice were used in this study. Polymicrobial sepsis in mice (n = 4) was induced by cecal ligation and puncture (CLP) as previously described in detail (5). In brief, animals were anesthetized, and the abdomen was opened to exteriorize the cecum that was filled with feces by milking stool backward from the ascending colon. A ligature was placed below the ileocecal valve and then the cecum was punctured twice with a 21-gauge needle. The cecum was then returned into the peritoneal cavity, the abdominal wall was closed with a suture, and all CLP animals were killed 6 h later. M1 protein (provided as a gift from Prof Heiko Herwald, Department of Clinical Sciences, Section of Clinical and Experimental Infection Medicine, Lund University) was purified from a mutated S. pyogenes strain, making the likelihood that the preparation contains endotoxin close to zero. We also measured the endotoxin content of the M1 protein samples and confirmed that endotoxin levels were below the detection limit. Separate animals were challenged i.v. with 15 μg of M1 protein solved in 200 μL phosphate-buffered saline (PBS) (n = 5). Control animals received 200 μL PBS (i.v.) alone (n = 5) and killed 4 h later. Platelet depletion was induced by administration of an antibody directed against murine CD42b (GP1bα, rat IgG, 1.0 mg/kg; Emfret Analytics GmbH & Co KG, Wurzburg, Germany) i.p. 2 h before M1 protein injection (n = 5). In separate experiments, a monoclonal antibody directed against murine Gr-1 (RB6- 8C5, 75 μg/mouse; eBioscience Inc, San Diego, Calif) was given i.v. 24 h before M1 protein treatment to deplete mice of neutrophils (n = 5). Isotype-matched nonfunctional antibodies (nonimmune rat IgG for platelet depletion; Emfret Analytics GmbH & Co. KG; rat IgG2b, κ, for neutrophil depletion; eBioscience Inc) were used as controls (n = 5). Animals were killed 4 h after M1 protein administration. One lobe of the left lung was ligated and excised for edema measurement. The right lung was used for collecting bronchoalveolar lavage fluid (BALF) to quantify neutrophils. The remaining lungs were perfused with 20 mL of PBS and excised. One lobe was fixed in formaldehyde for histology, and the remaining lung tissue was weighed, snap-frozen in liquid nitrogen, and stored at −80°C for later myeloperoxidase (MPO) analysis and enzyme-linked immunosorbent assay.
Systemic platelet and leukocyte counts
Blood was collected from tail vein and mixed with Stromatol solution (Mascia Brunelli spa, Viale Monza, Milan, Italy; 1:50 dilution) for platelet counts or Türk's solution (0.2 mg gentian violet in 1 mL glacial acetic acid, 6.25% vol/vol; 1:20 dilution) for leukocyte differential counts. Cells were counted in a Burker chamber. Leukocytes were defined as monomorphonuclear and polymorphonuclear cells.
One lobe of the left lung was excised, washed in PBS, weighed and dried at 60°C for 72 h, and reweighed. The change in the ratio of wet weight to dry weight was used as indicator of lung edema formation.
Briefly, lung tissue was homogenized in 1 mL of 0.5% hexadecyltrimethylammonium bromide, then the samples were freeze-thawed once, after which the MPO activity in the supernatant was determined spectrophotometrically as the MPO-catalyzed change in absorbance in the redox reaction of H2O2 (450 nm, 25°C) as previously described in detail (19). Values were expressed as MPO units per gram of tissue.
Enzyme-linked immunosorbent assay
Macrophage inflammatory protein 2 levels in lung tissue were analyzed by enzyme-linked immunosorbent assay (R&D Systems Europe, Abingdon, Oxon, UK) using recombinant murine MIP-2 as standards. The minimal detectable MIP-2 concentrations are less than 0.5 pg/mL.
Blood was collected (1:10 acid citrate dextrose) 4 h after M1 protein injection and incubated (10 min, room temperature) with an anti-CD16/CD32 antibody to reduce nonspecific labeling and then incubated with PE-conjugated anti-Gr-1 (RB6-8C5; eBioscience Inc) and fluorescein isothiocyanate (FITC)-conjugated anti-Mac-1 (M1/70, integrin αM chain) antibody. Another set of samples was stained with FITC-conjugated anti-CD41 (MWReg30, integrin αIIb chain) and PE-conjugated anti-CD40L (MR1) antibodies (all antibodies except where indicated were purchased from BD Biosciences Pharmingen, San Jose, Calif). Cells were fixed, erythrocytes were lysed, and neutrophils and platelets were recovered after centrifugation and analyzed with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, Calif). A viable gate was used to exclude dead and fragmented cells.
Lung samples were fixed in 4% formaldehyde phosphate buffer overnight and then dehydrated and paraffin-embedded. Six-micrometer sections were stained with hematoxylin and eosin (H&E).
In vitro binding assays
Blood was collected from healthy animals and washed once in FACS buffer (10 mM HEPES, 0.1% bovine serum albumin in PBS) and incubated with unlabeled M1 protein (50 μg/mL, final concentration) and/or FITC-conjugated M1 protein (1 μg/mL, final concentration) together with unlabeled M1 protein (50 μg/mL, final concentration) for 1 h at 37°C. Cells were then stained with PE-conjugated anti-CD41 (MWReg30, integrin αIIb chain) and allophycocyanin-conjugated anti-Gr-1 (RB6-8C5; BD Biosciences Pharmingen) for flow cytometric analysis. Cells incubated with unlabeled M1 protein alone were used as negative controls.
Data are presented as mean values ± SEM. Statistical evaluations were performed using Kruskal-Wallis one-way analysis of variance on ranks followed by multiple comparisons versus control group (Dunnett method). P < 0.05 was considered significant, and n represents the number of animals.
M1 protein-induced neutrophil recruitment
Myeloperoxidase was used as a global indicator of neutrophil infiltration into the lung. M1 protein challenge increased pulmonary activity of MPO by more than 16-fold, i.e., from 0.6 ± 0.2 to 9.7 ± 0.4 U/g (Fig. 1A, P < 0.05 vs. control, n = 5). Administration of the anti-GP1bα antibody significantly reduced systemic platelet counts in the blood by more than 81%. However, platelet depletion had no effect on M1 protein-induced MPO levels in the lung (Fig. 1A, P > 0.05 vs. control antibody [Ctrl ab] + M1, n = 5). Moreover, cellular analysis of lavage fluid from the bronchoalveolar space showed that the number of neutrophils increased by more than 3-fold after M1 protein challenge (Fig. 1B, P < 0.05 vs. control, n = 5). Administration of anti-GP1bα antibody did not decrease M1 protein-induced neutrophil recruitment into the bronchoalveolar space (Fig. 1B, P > 0.05 vs. Ctrl ab + M1, n = 5). In contrast, we observed that the CLP-provoked increase in MPO activity and BALF neutrophils in the lung were significantly decreased in mice depleted of platelets (Fig. 1, A and B, P < 0.05 vs. Ctrl ab + CLP, n = 4). In addition, we found that M1 protein challenge increased pulmonary levels of MIP-2 from 3.3 ± 0.8 to 57.3 ± 5.8 ng/g in the lung (P < 0.05 vs. control, n = 5). Depletion of platelets had no effect on M1 protein-induced MIP-2 production in the lung (P > 0.05 vs. Ctrl ab + M1, n = 5).
M1 protein-induced lung injury
M1 protein injection caused clear-cut edema formation in the lung; i.e., wet-dry ratio increased from 4.5 ± 0.1 to 5.1 ± 0.1 (P < 0.05 vs. control, n = 5). Moreover, morphologic analysis revealed normal tissue architecture and lack of neutrophil infiltration in lungs from control animals, whereas M1 protein challenge caused destruction of the pulmonary structure and infiltration of neutrophils (Fig. 1C). Administration of the anti-GP1bα antibody had no effect on M1 protein-provoked lung edema (i.e., wet-dry ratio was 5.1 ± 0.1) or microarchitecture (Fig. 1C). In contrast, we observed that platelet depletion both reduced edema formation by 68% (P < 0.05 vs. Ctrl ab + CLP, n = 4) and maintained intact tissue architecture in CLP mice (Fig. 1C).
Platelet and plasma levels of CD40L
To further clarify the role of platelets in M1 protein-triggered lung injury, we examined surface expression of CD40L on platelets and plasma levels of soluble CD40L. In line with our previous findings (11), CLP caused a concomitant reduction in CD40L expression on platelets and increase in soluble CD40L levels in plasma (Fig. 2, P < 0.05 vs. control, n = 4). However, we observed that M1 protein challenge had no impact on platelet or plasma levels of CD40L, suggesting that platelets are not activated in response to M1 protein exposure (Fig. 2, P > 0.05 vs. control, n = 5).
Mac-1 expression on neutrophils
Considering that CD40L seems to regulate neutrophil expression of Mac-1 in abdominal sepsis (10), we next asked whether streptococcal M1 protein may directly activate neutrophils in the circulation despite no changes in soluble CD40L levels. Indeed, we found that M1 protein greatly increased Mac-1 expression on circulating neutrophils; i.e., mean fluorescence intensity values increased from 324 ± 15 in controls to 507 ± 45 in M1 protein-treated mice (Fig. 3A, P < 0.05 vs. control, n = 5). While platelet depletion markedly reduced CLP-induced Mac-1 expression (86% reduction; Fig. 3B, P < 0.05 vs. Ctrl ab + CLP, n = 4), we observed that depletion of platelets had no influence on neutrophil expression of Mac-1 provoked by M1 protein (Fig. 3A, P > 0.05 vs. Ctrl ab + M1, n = 5).
M1 protein binds preferentially to neutrophils but not to platelets
To study the interaction of M1 protein with neutrophils and platelets, we labeled M1 protein with FITC and studied the binding with flow cytometry. Interestingly, we found that the MFI values of platelets were similar in samples incubated with unlabeled and FITC-labeled M1 protein or with unlabeled M1 protein alone (Fig. 4). In contrast, a significant proportion of neutrophils bound FITC-labeled M1 protein (Fig. 4, P < 0.05 vs. platelets, n = 5).
M1 protein-induced lung injury is dependent of neutrophils
Considering that M1 protein seems to bind to neutrophils and not to platelets, we wanted to determine the role of neutrophils in M1 protein-induced lung injury. Administration of the anti-Gr-1 antibody reduced circulating neutrophils by more than 80%. It was found that neutrophil depletion decreased MPO activity by 66% (Fig. 5A, P < 0.05 vs. Ctrl ab + M1, n = 5) and the number of BALF neutrophils by 86% (Fig. 5B, P < 0.05 vs. Ctrl ab + M1, n = 5) as well as edema formation by 50% (Fig. 5C, P < 0.05 vs. Ctrl ab + M1, n = 5) in mice injected with M1 protein. In addition, we observed that depletion of neutrophils protected against M1 protein-induced damage to the lung microarchitecture (Fig. 5D).
Streptococcus pyogenes of the M1 serotype is frequently associated with severe streptococcal infections and the development of STSS. One of the most sensitive organs in STSS is the lung (20, 21). Our data show that i.v. administration of M1 protein caused a severe lung damage characterized by edema formation and massive accumulation of leukocytes in the bronchoalveolar space. Moreover, we provide evidence showing that depletion of platelets had no effect on infiltration of neutrophils or edema formation in M1 protein-mediated lung injury. In contrast, neutrophil depletion effectively attenuated pulmonary edema and tissue damage in M1 protein-induced acute lung injury. Taken together, these novel findings suggest that neutrophils but not platelets constitute a key target in the pathogenesis of streptococcal M1 protein-induced lung injury.
Accumulating data suggest that platelets may regulate tissue injury in sepsis by, at least in part, activating circulating neutrophils (10). Moreover, a recent in vitro study reported that M1 protein has the capacity to activate platelets and cause platelet aggregation (18). It was therefore of great interest to evaluate the functional role of platelets in M1 protein-provoked neutrophil activation and lung injury in vivo. Our results show that depletion of platelets has no effect on M1 protein-induced neutrophil expression of Mac-1, neutrophil infiltration, or edema formation in the lung, indicating that platelets do not play a significant role in septic lung damage caused by M1 protein. In contrast, we could confirm that platelets indeed play a key role in neutrophil activation and lung injury in abdominal sepsis as shown previously (10, 11). This discrepant role of platelets is in line with the notion that the cellular and molecular mechanisms regulating immune cell activation and tissue injury in response to different bacteria and toxins are distinctly unique (22-24). Instead of platelets, we found that neutrophils play a dominant role in M1 protein-induced edema and tissue injury in the lung. This observation is in line with a previous study showing that neutrophil-derived secretion contributes to pulmonary damage in response to M1 protein challenge (25). Notably, although our present findings point to a dominating role of neutrophils in mediating M1 protein-induced lung damage, these results do not necessarily exclude that also other immune cells, such as monocytes and T cells, may contribute to acute lung injury in streptococcal infections, knowing that M1 protein has been reported to bind to these immune cells as well (14, 26). CD40L is expressed on the surface of resting platelets and upon activation platelets secrete soluble CD40L into the plasma (27). A recent study showed that platelet-derived CD40L mediates neutrophil activation and Mac-1 expression in abdominal sepsis (11). In the present study, we confirmed that surface expression of CD40L on platelets decreased concomitantly with an increase in the levels of soluble CD40L in a model of CLP. In contrast, we observed herein that surface expression on platelets and soluble levels in plasma of CD40L were unchanged in response to M1 protein challenge, indicating that M1 protein is not an effective stimulator of platelets in vivo. These findings may seem to contrast with a recent study reporting that M1 protein may bind to platelets and promote platelet aggregation in vitro (18). Nonetheless, we found also that M1 protein does not bind effectively to platelets, whereas M1 protein bound in a clear-cut fashion to neutrophils. In addition, M1 protein fails to activate mouse platelets in whole blood ex vivo (unpublished observation). This discrepancy may be related to the fact that Shannon et al. (18) observed that M1 protein binding to platelets was critically dependent on the presence of IgG antibodies directed against M1 protein and that these antibodies might be lacking in C57Bl/6 mice. Indeed, they observed that M1 protein, activated platelets, and IgG are colocalized at the site of infection in biopsies from septic patients, therefore platelet activation (18). Moreover, our data show that M1 protein upregulates Mac-1 on neutrophils in a platelet-independent manner. Considered to together, these findings suggest that M1 protein binds directly to and activates neutrophils leading to increased Mac-1 expression and accumulation of neutrophils in the lungs in response to systemic challenge with M1 protein. In this context, it should be noted that our findings do not exclude the possibility that platelets may be activated and start to aggregate secondarily to M1 protein-induced activation of neutrophils. Indeed, neutrophil-mediated activation of platelets has been observed in several conditions, such as glomerular injury (28), hypercholesterolemia (29), and abdominal sepsis (30). Moreover, a study showed that M1 protein can bind and activate monocytes via binding to TLR2 causing tissue factor release (14, 16), which, in turn, could activate platelets.
In conclusion, our data suggest that streptococcal M1 protein induces lung damage via direct activation of neutrophils independently of platelets. Thus, these findings indicate that platelets do not play an important role in acute lung injury caused by M1 protein. Instead, targeting neutrophil functions seems more relevant than inhibiting platelet activation in severe infections triggered by streptococcal M1 protein.
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