Martin G. Schwacha

Macrophages and post-burn immune dysfunction

The activation of a pro-inflammatory cascade after burn injury appears to be important in the development of subsequent immune dysfunction, susceptibility to sepsis and multiple organ failure. Macrophages are major producers of pro-inflammatory mediators and their productive capacity for these mediators is markedly enhanced following thermal injury. Thus, macrophage hyperactivity (as defined by increased productive capacity for pro-inflammatory mediators) appears to be of critical importance in the development of post-burn immune dysfunction. This review will focus on the current state of knowledge with regards to the role of macrophages in the development of post-burn immune dysfunction. Particular areas of discussion include: nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems, macrophages and the T-helper (Th)-1/Th-2 cytokine responses, alterations in macrophages signal transduction and a potential role for gamma/delta T-cells in the development of macrophage hyperactivity following thermal injury. A more comprehensive understanding of the relationship between macrophage activity and post-burn immune dysfunction will hopefully provide the basis for improved therapeutic regimes in the treatment of burn patients.

High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this “divide-and-conquer” strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 different proteins with 1494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 “classic” cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways.

Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Rα). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

Androgen and estrogen receptors in splenic T lymphocytes: effects of flutamide and trauma-hemorrhage.

The endogenous sex steroids, testosterone and beta-estradiol, play a major role in inflammatory processes. They regulate several cytokine genes by interaction with their intracellular receptors that are, essentially, transcription factors. Because T-lymphocyte functions are altered following trauma-hemorrhage in male mice, we investigated whether (i) receptors for androgen (AR) and estrogen (ER) are present in splenic T lymphocytes, (ii) receptor expressions are altered following trauma-hemorrhage, and (iii) pretreatment of male mice with the AR antagonist, flutamide, alters receptor expressions and IL-6 release. Analysis of nuclear extracts indicated the presence of AR and ER in splenic T lymphocytes. No difference in receptor expressions between males and females or following trauma-hemorrhage was observed. Pretreatment of males with flutamide, however, led to increased ER expression in T lymphocytes of sham and trauma-hemorrhaged animals. This suggested that flutamide is capable of inducing the expression of another receptor belonging to a different gonadal steroid. Because response elements for AR and ER are present in the promoter region of the IL-6 gene, release of IL-6 and expression of signal transducer and activator of transcription 3 (STAT3) were analyzed as functional parameters in splenic T lymphocytes. Trauma-hemorrhage decreased IL-6 release by T lymphocytes and the release was restored to sham levels with flutamide pre-treatment. Similarly, STAT3 expression was decreased in T lymphocytes following trauma-hemorrhage and the expression was restored by flutamide pre-treatment. These data collectively demonstrate the importance of gonadal steroids in the regulation of splenic T-lymphocyte functions.

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll‐like receptors (TLR). Although mitogen‐activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP ∼35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF‐α, interleukin‐6 (IL‐6), monocyte chemoattractant protein‐1 (MCP‐1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF‐α, MCP‐1, and KC production by Kupffer cells were significantly increased following trauma‐hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG‐stimulated TLR9 signaling increased TNF‐α and IL‐6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, ‐4, and ‐9 signaling. In contrast, TNF‐α and IL‐6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma‐hemorrhage. J. Cell. Physiol. 210: 667–675, 2007.

High-frequency percussive ventilation and low tidal volume ventilation in burns: a randomized controlled trial.

Objectives:In select burn intensive care units, high-frequency percussive ventilation is preferentially used to provide mechanical ventilation in support of patients with acute lung injury, acute respiratory distress syndrome, and inhalation injury. However, we found an absence of prospective studies comparing high-frequency percussive ventilation with contemporary low-tidal volume ventilation strategies. The purpose of this study was to prospectively compare the two ventilator modalities in a burn intensive care unit setting. Design:Single-center, prospective, randomized, controlled clinical trial, comparing high-frequency percussive ventilation with low-tidal volume ventilation in patients admitted to our burn intensive care unit with respiratory failure. Setting:A 16-bed burn intensive care unit at a tertiary military teaching hospital. Patients:Adult patients ≥18 yrs of age requiring prolonged (>24 hrs) mechanical ventilation were admitted to the burn intensive care unit. The study was conducted over a 3-yr period between April 2006 and May 2009. This trial was registered with ClinicalTrials.gov as NCT00351741. Interventions:Subjects were randomly assigned to receive mechanical ventilation through a high-frequency percussive ventilation-based strategy (n = 31) or a low-tidal volume ventilation-based strategy (n = 31). Measurements and Main Results:At baseline, both the high-frequency percussive ventilation group and the low-tidal volume ventilation group had similar demographics to include median age (interquartile range) (28 yrs [23–45] vs. 33 yrs [24–46], p = nonsignificant), percentage of total body surface area burn (34 [20–52] vs. 34 [23–50], p = nonsignificant), and clinical diagnosis of inhalation injury (39% vs. 35%, p = nonsignificant). The primary outcome was ventilator-free days in the first 28 days after randomization. Intent-to-treat analysis revealed no significant difference between the high-frequency percussive ventilation and the low-tidal volume ventilation groups in mean (± sd) ventilator-free days (12 ± 9 vs. 11 ± 9, p = nonsignificant). No significant difference was detected between groups for any of the secondary outcome measures to include mortality except the need for “rescue” mode application (p = .02). Nine (29%) in the low-tidal volume ventilation arm did not meet predetermined oxygenation or ventilation goals and required transition to a rescue mode. By contrast, two in the high-frequency percussive ventilation arm (6%) required rescue. Conclusions:A high-frequency percussive ventilation-based strategy resulted in similar clinical outcomes when compared with a low-tidal volume ventilation-based strategy in burn patients with respiratory failure. However, the low-tidal volume ventilation strategy failed to achieve ventilation and oxygenation goals in a higher percentage necessitating rescue ventilation.

The role of γδ T cells in the regulation of neutrophil-mediated tissue damage after thermal injury

Thermal injury induces an inflammatory response that contributes to the development of secondary tissuedamage. Neutrophil recruitment and activation are in part responsible for this tissue damage. Although γδ T cells have been shown to regulate the inflammatory responses in tissues that are prone to neutrophil‐mediated injury post‐burn, their role in the induction of secondary tissue injury post‐burn remains unknown. To study this, γδ T cell‐deficient (γδ TCR−/−) and wild‐type (WT) mice were subjected to thermal injury or sham procedure, and tissue samples were isolated 1–24 h thereafter. Burn injury induced neutrophil accumulation in the lung and small intestines of WT mice at 1–3 h post‐injury. No such increase in neutrophil tissue content was observed in γδ TCR−/− mice. An increase in tissue wet/dry weight ratios was also observed in these organs at 3 h post‐burn in WT but not in γδ TCR−/− mice. A parallel increase in plasma and small intestine levels of the chemokines macrophage‐inflammatory protein‐1β (chemokine ligand 4) and keratinocyte‐derived chemokine (CXC chemokine ligand 1) were observed in injured WT mice but not in injured γδ TCR−/− mice. Increased activation (CD120b expression) of the circulating γδ T cell population was also observed at 3 h post‐burn in WT mice. These results indicate the γδ T cells, through the production of chemokines, play a central role in the initiation of neutrophil‐mediated tissue damage post‐burn.

The PI3K/Akt Pathway Mediates the Nongenomic Cardioprotective Effects of Estrogen Following Trauma-hemorrhage

Objective:To determine whether the nongenomic actions of E2 have any beneficial effect on cardiac function following trauma-hemorrhage and whether those effects are mediated via the PI3K/Akt pathway. Summary Background Data:Since studies suggest that both genomic and nongenomic pathways are involved in mediating the salutary effects of 17β-estradiol (estradiol) following trauma-hemorrhage, we examined if the nongenomic effects of estradiol on cardiac function after trauma-hemorrhage involve the PI3K/Akt pathway. Methods:Male Sprague-Dawley rats (∼300 g) underwent trauma-hemorrhage (mean blood pressure, 40 mm Hg for 90 min, then resuscitation). Estradiol conjugated to bovine serum albumin (BSA) (estradiol-BSA; 1 mg/kg estradiol) with or without estrogen receptor antagonist (ICI 182,780), PI3K inhibitor (Wortmannin), or vehicle was injected intravenously during resuscitation. At 2 hours after trauma-hemorrhage or sham operation, cardiac output, stroke volume, heart rate, mean arterial pressure, and ±dP/dt were measured. Cardiomyocyte PI3K, p-Akt, Akt protein expressions and apoptosis were also determined. One-way ANOVA and Tukeys test were used for statistical analysis. Results:Cardiac output, stroke volume, and ±dP/dt decreased significantly after trauma-hemorrhage. Administration of estradiol or estradiol-BSA significantly improved these parameters of cardiac function. Although trauma-hemorrhage decreased cardiomyocyte PI3K protein expression and Akt phosphorylation (p-Akt), estradiol or estradiol-BSA treatment following trauma-hemorrhage prevented such decreases in cardiomyocyte PI3K protein expressions and p-Akt. The increase in cardiomyocyte apoptosis was also prevented in rats receiving estradiol-BSA. Co-administration of ICI 182,780 or Wortmannin abolished beneficial effects of estradiol-BSA on cardiac functions following trauma-hemorrhage. Conclusion:The PI3K/Akt pathway plays a critical role in mediating the nongenomic salutary effects of estradiol on cardiac function following trauma-hemorrhage.

The Role of MIP-1α in the Development of Systemic Inflammatory Response and Organ Injury following Trauma Hemorrhage

Although MIP-1α is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1α plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1α-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 ± 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum α-glutathione transferase, TNF-α, IL-6, IL-10, MCP-1, and MIP-1α and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1α KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1α plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1α, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1α levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.

Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Akt-dependent HO-1 up-regulation

Protein kinase B (Akt) is known to be involved in proinflammatory and chemotactic events in response to injury. Akt activation also leads to the induction of heme oxygenase (HO)‐1. Up‐regulation of HO‐1 mediates potent, anti‐inflammatory effects and attenuates organ injury. Although studies have shown that 17β‐estradiol (E2) prevents organ damage following trauma‐hemorrhage, it remains unknown whether Akt/HO‐1 plays any role in E2‐mediated attenuation of hepatic injury following trauma‐hemorrhage. To study this, male rats underwent trauma‐hemorrhage (mean blood pressure, ∼40 mmHg for 90 min), followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E2 (1 mg/kg body weight), E2 plus the PI‐3K inhibitor (Wortmannin), or the estrogen receptor (ER) antagonist (ICI 182,780). At 2 h after sham operation or trauma‐hemorrhage, plasma α‐GST and hepatic tissue myeloperoxidase (MPO) activity, IL‐6, TNF‐α, ICAM‐1, cytokine‐induced neutrophil chemoattractant‐1, and MIP‐2 levels were measured. Hepatic Akt and HO‐1 protein levels were also determined. Trauma‐hemorrhage increased hepatic injury markers (α‐GST and MPO activity), cytokines, ICAM‐1, and chemokine levels. These parameters were markedly improved in the E2‐treated rats following trauma‐hemorrhage. E2 treatment also increased hepatic Akt activation and HO‐1 expression compared with vehicle‐treated, trauma‐hemorrhage rats, which were abolished by coadministration of Wortmannin or ICI 182,780. These results suggest that the salutary effects of E2 on hepatic injury following trauma‐hemorrhage are in part mediated via an ER‐related, Akt‐dependent up‐regulation of HO‐1.