No abstract

Severe injury causes a dramatic host response that disrupts immune homeostasis and predisposes the injured host to opportunistic infections. Because Toll-like receptors (TLRs) recognize conserved microbial Ags and endogenous danger signals that may be triggered by injury, we wanted to determine how injury influences TLR responses. Using an in vivo injury model, we demonstrate that injury significantly increased TLR2-and TLR4-induced IL-1␤, IL-6, and TNF-␣ production by spleen cells. This influence of injury on TLR reactivity was observed as early as 1 day after injury and persisted for at least 7 days. The outcome of similar studies performed using TLR4-mutant C57BL/10ScN/Cr mice revealed that TLR2 responses remained primed, thus suggesting that injury-induced priming can occur independently of endogenous TLR4 signaling. Increased TLR4 reactivity was also observed in vivo, because LPS-challenged injured mice demonstrated significantly higher cytokine expression levels in the lung, liver, spleen, and plasma. Macrophages and dendritic cells were the major source of these cytokines as judged by intracellular cytokine staining. Moreover, ex vivo studies using enriched macrophage and dendritic cell populations confirmed that T cells did not contribute to the enhanced TLR2 and TLR4 responses. The results of flow cytometry studies using TLR2-and TLR4-MD-2-specific Abs indicated that injury did not markedly alter cell surface TLR2 or TLR4-MD-2 expression. Taken together, these findings establish that injury primes the innate immune system for enhanced TLR2-and TLR4-mediated responses and provides evidence to suggest that augmented TLR reactivity might contribute to the development of heightened systemic inflammation following severe injury.

ObjectivePatients with serious traumatic injury and major burns and an animal model of burn injury were studied to determine the effect of injury on the production of cytokines typical of the T helper-2 lymphocyte phenotype as opposed to the T helper-1 phenotype and on the production of interleukin-1 2. Summary Background DataPerturbations of natural and adoptive immunity are related to the increased susceptibility to infection manifested by seriously injured and burn patients. Earlier work has shown that impaired adoptive immunity after injury is characterized by diminished production of interleukin-2 (IL-2), a product of Th lymphocytes. Exposure of naive Th cells to certain antigens and cytokines causes conversion to either the Th-1 or the Th-2 phenotype. Th-1 cells produce IL-2 and interferon-gamma (IFN-T) and initiate cellular immunity. Th-2 cells secrete interleukin-4 (IL-4) and interleukin-10 (IL-10) and stimulate production of certain antibodies. Conversion to the Th-1 phenotype is facilitated by , and conversion to the Th-2 phenotype is promoted by IL-4. The authors believed that serious injury might cause conversion of Th cells to the Th-2 as opposed to the Th-1 phenotype rather than generalized Th suppression. MethodsThe authors studied circulating peripheral blood mononuclear cells (PBMC) from 16 major burn and 8 trauma patients on 32 occasions early after injury and from 13 age-and sex-matched healthy individuals for cytokine production after phytohemagglutinin stimulation. Also studied was a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from burn mice, 10 to 12 per group, were studied after activation by concanavalin A or by the bacterial antigen Staphylococcus aureus Cowan strain for cytokine production and cytokine messenger RNA expression as determined by reverse transcriptase polymerase chain reaction. Burn mice were compared with sham-burn controls and attention was focused on day 10 after burn injury, a time when IL-2 production and resistance to infection are 482

No abstract

No abstract

Tissue injury and infection produce significant alterations in host metabolic and immune homeostasis. It is increasingly clear that many of these changes result from a complex cascade of mononuclear phagocyte-derived endogenous mediators. Among the more important is a group of host proteins called cytokines, which play an integral role in mediating the host response to tissue injury and infection. Of these proteins, tumour necrosis factor (TNF) and interleukin (IL) types 1 and 6 have received much attention for their pathophysiological roles in infection and trauma. Evidence is reviewed for the involvement of these cytokines in the characteristic alterations in the metabolic and immune responses to such injury. These endogenous mediators initiate an integrated fuel substrate and hormonal adjustment to trauma and sepsis, and help to provide optimal metabolic homeostasis for systemic host defences. Widespread tissue injury, especially when associated with fulminant sepsis, may, however, precipitate massive release of TNF, IL-1 and IL-6, triggering a series of reactions involving multiple organs, and culminating in the 'sepsis syndrome'. New therapies designed to downregulate this aberrant response, either by neutralizing endotoxin directly or by blocking the release or actions of these cytokines, are reviewed. Although these treatments hold much promise for the future management of severely traumatized and infected patients, careful evaluation of both the benefits and complications of therapy is needed before widespread clinical use can be recommended.

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1−/−) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-α, IL-1β, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1−/−, CD4−/−, or CD8−/− mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8−/− mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4−/− mice produced cytokines at significantly higher levels, equivalent to those in Rag1−/− mice. Moreover, reconstitution of Rag1−/− or CD4−/− mice with WT CD4+ T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4+ T cells into CD4+CD25+ and CD4+CD25− subpopulations before their adoptive transfer into Rag1−/− mice showed that CD4+CD25+ T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4+CD25− T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4+CD25+ T regulatory cells in controlling host inflammatory responses after injury.

The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope 18 O-labeled ″universal″ reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the 18 O-labeled ″universal″ reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on 16 O/ 18 O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label- NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dualquantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinity depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free 16 O-MS intensity data extracted from the same data sets. The results showed that the 18 O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the 18 O-reference for accurate quantitation makes this strategy especially attractive when a large number of biological samples are involved in a study where label-free quantitation may be problematic, for example, due to issues associated with instrument platform robustness. The approach will also be useful for more effectively discovering subtle abundance changes in broad systems biology studies.