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Blood, 1 June 2006, Vol. 107, No. 11, pp. 4399-4406. Prepublished online as a Blood First Edition Paper on February 7, 2006; DOI 10.1182/blood-2005-09-3776.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Platelet depletion in mice increases mortality after thermal injuryFrom the Julian and Eunice Cohen Laboratory for Surgical Research, Department of Surgery (Immunology), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Department of Surgery, Massachusetts General Hospital, Boston; and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX.
Platelets play a fundamental role in maintaining hemostasis and have been shown to participate in innate and adaptive immunity. However, the role of platelets in the immune response to injury remains undefined. We tested the importance of platelets in the host response to serious injury in a newly developed platelet-deficient mouse model. Wild-type and platelet-depleted C57BL/6J mice underwent a 25% full-thickness total body surface area thermal or sham injury. Platelet-deficient mice showed survival of 51% at 48 hours after injury compared with 94% to 100% survival in experimental control mice (P < .001). Necropsy and histology ruled out hemorrhage and hypovolemia as causes of death. Percentages of peripheral blood monocytes (P < .01) and neutrophils (P < .05) were increased between 36 and 48 hours after thermal injury in platelet-deficient mice compared with control mice. Plasma levels of TNF  (P < .001), IL-6 (P < .001), and MCP-1 (P < .05) were also elevated by 24 hours whereas levels of TGF 1 were reduced between 24 and 36 hours following injury in platelet-depleted mice (P < .001) compared with control mice. Our findings demonstrate for the first time that platelets play a critical protective role during the host response to injury. Moreover, our findings suggest that platelets and, more importantly, platelet-derived TGF1 modulate the systemic inflammatory response occurring after injury.
Platelets are the smallest but most abundant cell type found in the circulation and range in numbers from 150 x 109/L to 400 x 109/L in humans.1 They are enucleated cells derived from the fragmentation of megakaryocytes and contain preformed compartmentalized proteins as well as messenger RNA.1,2 While platelets primarily function to maintain hemostasis, they also participate in tissue repair and wound remodeling and in antimicrobial host defense.1,3-5 Thus, platelets can be perceived as nomadic "sentinels" capable of responding instantly to chemical changes in their environment and acting as a first line of defense after injury or bacterial invasion.
The activation of platelets in response to injury or bacterial infection initiates the formation of platelet aggregates and the expression of cell adhesion molecule receptors and costimulatory molecules such as P-selectin (CD62P), CD40, and CD154.5-12 Activated platelets are also capable of releasing proinflammatory cytokines such as interleukin-1 beta (IL-1
We undertook this study because we believe that platelets may participate in the intercellular communication network between injured tissue and the immune system. This may occur as part of the innate immune response, through the expression of platelet TLRs, or through other specific ligand-receptor interactions. Thus, an injury-induced change in the number of circulating platelets or platelet-derived products or alterations in platelet function may be detrimental. This has been suggested by the clinical observation that platelet deficiency correlates with a higher mortality after severe trauma and after sepsis.21,22 However, the underlying mechanisms associated with these observations have not been closely examined. Thus, the purpose of this study was to determine the effect of injury on platelets and to evaluate the role of platelets in the host response to injury. Using polyclonal antibodies to deplete platelets, we developed a platelet-deficient mouse model to test the influence of platelets on the host response to injury. We demonstrate that platelets play a significant protective role in survival following injury. We also show that injury leads to measurable changes in the numbers of circulating platelets. Finally, we demonstrate that platelets appear to control innate immune system reactivity after thermal injury, since platelet-deficient mice display significantly higher levels of circulating proinflammatory cytokines (TNF
Animals Six- to 8-week-old pathogen-free C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and acclimated for at least 1 week prior to use. All mice were maintained in an accredited virus antibody–free facility in accordance with guidelines of the National Institutes of Health and the Harvard Medical Area Standing Committee on Animals. Mouse injury model Groups of C57BL/6J mice were either sham or thermal injured in accordance with an injury protocol that has been approved by the National Institutes of Health and Harvard Medical Area Standing Committee on Animals.23 Briefly, 8- to 10-week-old C57BL/6J mice were anesthetized with ketamine (125 mg/kg) + xylazine (6 mg/kg), and the dorsal fur was shaved. Mice were placed in a template exposing a 25% total body surface area. The exposed skin was immersed in 90°C water for 9 seconds. This has been shown to produce an anesthetic full-thickness burn. Control mice were anesthetized, shaved, and exposed to 24°C water. After sham or thermal injury, mice were resuscitated by intraperitoneal injection of 1 mL sterile normal saline solution. The resulting survival rate from this form of injury is at least 95%.24 Time course study Sham- and thermal-injured mice were killed at sequential intervals after thermal injury immediately after carbon dioxide asphyxiation. Whole blood was collected by cardiac puncture into 1-mL syringes containing 50 µL 169 mM EDTA (ethanolamine diamine tetra-acetic acid; Sigma, St Louis, MO). The blood was transferred to sterile 2.0-mL polypropylene microcentrifuge tubes containing an additional 50 µL EDTA. Duplicate 30-µL aliquots were removed for analysis of platelet numbers, leukocyte numbers, and subsets, using the Hemavet 850 (Drew Scientific, Farmington, CT). Plasma was prepared from the remaining anticoagulated blood samples by centrifugation at 16 000g for 20 minutes at 4°C. Plasma samples were stored for cytokine determination. Affinity purification of rabbit anti–mouse platelet antibody
Rabbit anti–mouse platelet antiserum was purchased from Inter-Cell Technologies (Jupiter, FL) and was affinity purified on a protein G column (Pharmacia, Uppsala, Sweden). Briefly, the affinity column was prepared by washing with 20–column bed volumes of Tris (tris(hydroxymethyl)aminomethane)–buffered saline containing sodium azide (50 mM Tris-HCl, pH = 7.4; 150 mM NaCl; 0.05% NaN3). The rabbit Ig–containing antiserum was loaded on to the column, which was then washed with 20 volumes of PBS (pH = 7.4). The Ig was eluted with 50 mM glycine-HCl (pH = 3.0; Sigma) into tubes containing neutralization buffer (1 M Tris-HCl, pH = 8.0; 1.5 mM NaCl; 1 mM EDTA; Sigma). Fractions containing the antiplatelet Ig were pooled and sterilized by 0.22-µm filtration (Millipore, Burlington, MA). The sterile Ig–containing fractions were mixed with END-X B52 beads (Associates of Cape Cod Incorporated, Falmouth, MA) to ensure that the resulting antiplatelet Ig ( Platelet depletion in vivo
Mice were treated with either an affinity-purified normal rabbit immunoglobulin (CTL-Ig; Inter-Cell Technologies) or the affinity-purified rabbit anti–mouse platelet polyclonal immunoglobulin ( Development of a platelet-depleted thermal-injury model
Groups of mice were pretreated with either CTL-Ig or Cytokine determinations
Plasma levels of TNF Data analysis The GraphPad Prism 4.0 for Windows software program (GraphPad Software, San Diego, CA) was used for all statistical calculations. Data from survival studies were analyzed using the Kaplan-Meier and log-rank tests. Hematologic and plasma cytokine values were analyzed using the nonparametric Mann-Whitney test. Differences were considered significant when the P value was less than or equal to .05.
The effect of thermal injury on circulating platelet numbers Experiments were performed to determine the effect of thermal injury on circulating platelets at different times after injury (Figure 1A). Platelet numbers in thermal-injured wild-type (WT) mice were significantly reduced by 1.5 hours after injury, resulting in a transient but mild thrombocytopenia when compared with WT sham-injured controls (P < .05). This reduction in platelet numbers persisted for at least 12 hours (P < .05) and then returned to normal or elevated levels over time from 24 to 168 hours after injury (data not shown). Hematocrit levels were also measured in the same blood samples from thermal-injured and sham-injured mice to determine whether the observed reduction in platelet numbers in thermal-injured mice occurred as a consequence of hemorrhage or hemodilution. Hematocrit levels from thermal-injured mice were significantly higher at the earliest time point (0.5 hours) when compared with sham controls (P < .05; Figure 1B). Hematocrit levels, however, were within normal limits at all other time points examined (1.0-18.0 hours). This suggests that the observed reduction in circulating platelets was not simply due to hemorrhage or hemodilution. To further determine whether platelet-deficient thermal-injured mice displayed hemorrhage, we performed necropsy of sham versus burn mice at 0.5 hours after injury. We did not observe any signs of hemorrhage in thermal-injured platelet-deficient mice.
Development of a mouse model of platelet depletion following thermal injury
Since thermal injury significantly alters circulating platelet numbers, we wanted to develop a model to test whether platelets might modulate the host response to injury. We reasoned that developing a platelet-deficient mouse would allow us to investigate this question. Using a unique rabbit anti–mouse platelet polyclonal antibody (
Survival of mice to thermal injury after platelet depletion
Thrombocytopenia has been shown to correlate with a poor clinical prognostic outcome in human trauma patients.21,22 Therefore, we were interested in testing whether platelet-deficient mice demonstrated differences in survival following thermal injury. Mice were treated 3 days prior to burn or sham injury with either CTL-Ig (n = 38) or Hematologic parameters in mice following thermal injury To determine the mechanisms through which platelets might protect mice following thermal injury, we assessed hematologic parameters including sequential determinations of platelet numbers, hematocrit, absolute leukocyte numbers, and percentages of leukocyte subsets after injury in groups of sham- and thermal-injured control and platelet-deficient mice.
Platelets. Since the majority of platelet-depleted mice died between 24 and 48 hours after injury, we evaluated platelet numbers during these time points. Platelet numbers remained significantly lower in
Hematocrit. We then evaluated hematocrit levels in sham- and thermal-injured mice that were treated with either CTL-Ig or
Leukocytes. Whole blood leukocyte numbers and leukocyte subsets were evaluated in sham- and thermal-injured mice. As shown in Table 1, absolute white blood cell counts and lymphocyte counts were markedly greater at 24 hours in thermal-injured mice that were treated with
Because absolute numbers of both leukocytes and leukocyte subsets were unremarkable at later time points (36 and 48 hours), we evaluated the percentages of each leukocyte subset in control and experimental mice (Table 2). The percentages of circulating lymphocytes were similar in thermal-injured mice treated with either CTL-Ig or PLT-Ig at 24 and 48 hours, whereas the percentages of lymphocytes were significantly lower in the PLT-Ig group at 36 hours when compared with the CTL-Ig group (P < .05). While the percentages of monocytes were similar in the 2 groups 24 hours after thermal injury, a marked increase in the percentages of monocytes in the PLT-Ig–treated group was observed when compared with the CTL-Ig–treated group at 36 (P < .01) and 48 (P < .005) hours. Although the percentage of neutrophils was lower at 24 hours after injury in the PLT-Ig–treated group, this percentage was significantly increased at 36 hours (P < .05) followed by significant decrease at 48 hours (P < .01).
Plasma cytokine levels in mice following thermal injury
As shown in Table 2, we found significant changes in circulating leukocyte subsets in
Platelets are known to express ligands and receptors for costimulatory molecules. It is also known that platelets, once activated, release cytokines including transforming growth factor beta-1 (TGF
We next wanted to determine more exactly when circulating TGF
Since TGF 1 levels peaked at 1.5 hours following injury and correlated with a significant decrease in the number of circulating platelets, we wanted to determine the levels of plasma TGF1 at this time point in thermal-injured mice that were platelet deficient. As shown in Figure 7, plasma levels of TGF1 were significantly lower in thermal-injured platelet-deficient mice (P < .001) when compared with thermal-injured CTL-Ig–treated mice.
While platelets are traditionally thought to be regulators of hemostasis and coagulation, there is now accumulating evidence that platelets may also be important in the development and progression of inflammatory processes.12,16,17,19,20 Recent studies have demonstrated that platelets participate in the development of both innate and adaptive immune responses.14,15 Thus, it can be perceived that platelets constantly sense and respond to changes in their microenvironment including danger signals such as cytokines or bacterial products released in response to injury or invasion by microorganisms.
The purpose of this study was 2-fold. First, we wanted to determine whether major thermal injury alters circulating platelet abundance. Second, we wanted to determine whether platelets contribute to the host response after injury. Our results show that there is a significant decrease in circulating platelets that leads to a transient thrombocytopenia after thermal injury in the mouse, similar to findings in rats that have undergone sublethal trauma29 and clinical observations in thermal-injured patients.22 Although thermal injury did affect platelet numbers, it was still unclear whether platelets contributed to the host immune response after injury. In addition, it was found that severe injury results in platelet activation and alterations in platelet function. Here, mortality was found to be associated with a decreased platelet function.6 We report here for the first time the use of a novel mouse model of platelet deficiency to more clearly define the role of platelets in the injury response. As demonstrated in our survival studies, thermal injury led to a significantly decreased survival in mice that were made platelet deficient. Our observations of normal hematocrit, no abnormal necropsy findings, and normal histology strongly suggest that mortality in most of the platelet-depleted mice did not occur as a consequence of hemorrhage or hypovolemic shock and that a reduction in circulating platelet numbers significantly influenced survival following thermal injury by other mechanisms. Hence, our findings are consistent with the clinical observations that a deficiency in platelets combined with severe trauma correlates with increased mortality and with the observation that burn patients who undergo episodes of thrombocytopenia have a poor outcome.21,22 Our findings, together with the published clinical observations, suggest that platelets are important in maintaining immune homeostasis after a major injury. Furthermore, the observed thrombocytopenia early after injury may occur as a direct consequence of platelet activation and generation of platelet-leukocyte aggregates and of platelet-derived microparticles. Both of these phenomena have been described in patients after severe trauma and sepsis.21,22,30,31 Our observation of thrombocytopenia in thermal-injured mice may be an important feature of the host response to injury. Our findings, together with published clinical observations, suggest that platelet transfusion and/or immunotherapy with cytokines such as TGF
Little information is available on the hematologic and immune alterations that accompany injury-induced thrombocytopenia. In order to investigate these questions, both leukocyte numbers and cytokine levels were evaluated in our platelet-depleted experimental mice. We found that the overall leukocyte and lymphocyte numbers were increased at 24 hours after thermal injury in these mice. Upon closer analysis of the leukocyte subsets, we found that the percentage of circulating monocytes increased significantly at the 36- and 48-hour time points, with only a transient increase in neutrophils in thermal-injured platelet-deficient mice. It is possible that these increased percentages of circulating monocytes and, to a lesser extent, neutrophils may occur as a result of reduced tissue extravasation as a consequence of significantly reduced levels of circulating activated platelets, platelet-derived microparticles, platelet-derived TGF In our studies, we found that thermal injury of platelet-deficient mice led to increased mortality. Although the mechanisms responsible for this outcome were unknown initially, hemorrhage was excluded as cause of death, since necropsy and histology findings were unremarkable and hematocrit values were normal in these mice. Indeed, there have been no reports of platelet depletion in mice with antibodies resulting in mortality due to low platelet counts. Depletion of platelets in mice using polyclonal antibodies or monoclonal antibody cocktails has been used to study the role of platelets in wound healing and the role of platelet caspases in the development of cerebral malaria.3,35,36 However, one study clearly demonstrated that low platelet counts after treatment with low doses of polyclonal antibodies did not cause hemorrhage.37
Thermal injury of platelet-deficient mice led to an exaggerated systemic inflammatory response culminating with increased mortality and was characterized by excessive and prolonged release of IL-6 and MCP-1 and by a marked increase in TNF . These increases in plasma cytokines may have occurred as a result of increased production by both immune and nonimmune tissues possibly as a consequence of reduced levels of platelet-derived TGF1. This idea is supported by the reported evidence that proinflammatory cytokines including IL-1, TNF, IL-6, and MCP-1 production are regulated by TGF1. Moreover, IL-6 has been demonstrated to upregulate MCP-1 production.38-42 Indeed, we clearly show that TGF1 levels are significantly decreased for up to 36 hours after thermal injury of platelet-deficient compared with control mice. Our present findings correlate well with those of Yeh et al,43 who demonstrated that there was an increase in mortality due to sepsis in burn patients that had deficient levels of serum TGF1 and enhanced levels of IL-6. In contrasting our finding of drastically decreased TGF1 after thermal injury in platelet-deficient mice, we also showed that TGF1 levels were significantly increased early, from 1.5 to 12 hours after injury in WT mice. This sustained increase in TGF1 levels during this early time period may be critical in defining the extent of the systemic response to injury. This is further supported by clinical findings in burn patients.43 Curiously, we also found that this early increase in TGF1 parallels the observed transient injury-induced thrombocytopenia. Thus, release of platelet-derived proteins including TGF1 and the development of a transient thrombocytopenia may be reflective of platelet activation. Together, these findings further support published results indicating that platelets are a significant source of this cytokine. Indeed, Grainger et al28 have shown that while human platelets possess only about 4000 TGF1 molecules/cell, they account for the majority of the circulating TGF1 (45 ng/mL). This contrasts with mononuclear cells and granulocytes, which contain 12 000 and 6000 molecules/cell, respectively, and yet only account for 2.7 ng/mL of the circulating TGF1.28 Thus, our observed injury-induced thrombocytopenia may be a reflection of platelet activation and TGF1 release. Our group and others have reported that injury induces an early state of immune suppression that remits over time.24,44-52 Our findings suggest that this may relate to the early systemic increase in TGF1. It is also plausible that increased TGF1 may trigger the activation or function of regulatory T cells.44 Thus, in the absence of adequate levels of circulating TGF1, injury-induced immune activation may become exacerbated, resulting in increased mortality. Future studies will be necessary to further understand the role of platelets and platelet-derived products in modulating the host response to injury.
A role for platelets in the host response to injury may occur initially through platelet activation, which is followed by release of TGF
In summary, we describe a novel mouse model of platelet deficiency to study the host immune response to injury. We also demonstrate that platelets may be a critical component of this response. Finally, we clearly demonstrate that TGF
The authors thank Adam Delisle for his excellent technical support.
Submitted September 20, 2005; accepted January 26, 2006.
Prepublished online as Blood First Edition Paper, February 7, 2006; DOI 10.1182/blood-2005-09-3776.
Supported by grants from the National Institutes of Health (GM062119-03, J.A.M.; and GM05766406, J.A.L.), the Julian and Eunice Cohen Laboratory for Surgical Research, and the Surgical Infection Society Junior Faculty Research Fellowship (P.H.L.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Peter H. Lapchak, Julian and Eunice Cohen Laboratory for Surgical Research, Department of Surgery (Immunology), Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115; e-mail: plapchak{at}partners.org or plapchak{at}rics.bwh.harvard.edu.
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Top
Abstract
Introduction
were determined by flow cytometry using the BD Cytometric Bead Array (CBA; Becton Dickinson, Waltham, MA) mouse inflammation kit following the manufacturer's recommendations. Plasma levels of TGF
) or thermal injured (n = 8, 
). At each time point tested, mice were killed, whole blood was removed by cardiac puncture, and platelet numbers were determined (A). Normal range for platelet numbers is 592 x 109/L (592 x 103/µL) to 2972 x 109/L (2972 x 103/µL), as outlined by the data analysis printout for the Hemavet 850 from Drew Scientific. Values below the dotted line represent thrombocytopenia. Hematocrits were determined from the same blood sample at each time point (B). The gray vertical bar represents the normal limits for hematocrit (.35-.45 [35.1%-45.4%]), as outlined by the data analysis printout for the Hemavet 850 from Drew Scientific. Data are representative of 2 independent experiments (mean ± SEM). Data were analyzed using the Mann-Whitney test. Differences were considered significant when *P < .05.
) or 
) and killed 72 hours later. Anticoagulated whole blood was collected by cardiac puncture, and platelet numbers (A) and hematocrit (B) were determined (mean ± SEM). Data were analyzed using the Mann-Whitney test. Differences were considered significant when *P < .05.
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