Alteration in Ca2+ homeostasis by a trauma peptide.

Postinjury tissue inflammation with PMN elastase proteolysis generates immunosuppressive fibronectin peptides (FNDP) impairing chemotaxis, T-cell activation, and proliferation. Excess intracellular Ca2+ ([Ca2+]i) impairs T-cell activation. This study quantifies the changes in [Ca2+]i following exposure to a degradation peptide of fibronectin to determine the mechanism of action of these peptides on calcium homeostasis. Isolated human PBLs were exposed to immunosuppressive concentrations of FNDP after loading with the [Ca2+]i probe FURA-2AM. Resting and sustained [Ca2+]i concentrations were calculated and compared to buffer control. The mechanism of action was determined by pretreatment with: (1) EDTA binding extra cellular Ca2+: [Ca2+]e, (2) the Ca2+ channel blockers verapamil and nifedipine, and (3) inhibition of [Ca2+]i released by dantrolene. Inositol triphosphate (IP3) essential for [Ca2+]i release was measured following T-cell stimulation as well. FNDP caused 200-400% increases in [Ca2+]i concentration relative to buffer control at known suppressive doses. Verapamil and nifedipine partially block [Ca2+]i influx by as much as 50% suggesting the slow Ca2+ (voltage independent) channels are partially responsible for the increased [Ca2+]i seen following FNDP. EDTA completely suppressed [Ca2+]e influx but did not completely inhibit the release of [Ca2+]i although IP3 was 80% suppressed. The increase in [Ca2+]i following FNDP stimulation is due to release of intracellular stores.

Head injury: an immunologic deficit in T-cell activation.

Despite progress in the management of increased pressure following head injury, infection remains the most common complication in survivors. This study attempted to better define the immunologic deficit that occurs immediately and in the early post-recovery period following severe head injury. Twenty-seven patients admitted with the primary diagnosis of severe head injury (mean Glasgow Coma Score, 6.75) were studied within 24 hours of injury and at weekly intervals. T-cell proliferative response to mitogen stimulation was assessed. T-cell antigen expression following PHA stimulation was assessed for helper (CD4) and suppressor (CD8) subpopulations: the early T-cell activation antigens interleukin-2 receptor (IL2R) and transferrin receptor (TFR), and the late antigen (HLA-DR) using flow cytometry. Polymorphonuclear leukocyte function was assessed using oxidative burst. There was a reduction in the proliferative response of T cells to mitogen stimulation. The B-cell response seemed unaffected. The diminished proliferative response was accompanied by diminished expression of early activation antigens (IL2R and TFR) and late (following DNA synthesis) (HLA-DR). This was seen primarily on helper/inducer cells (CD4+). Polymorphonuclear leukocyte function was unaffected.

In vitro inhibition of IL-2 biosynthesis in activated human peripheral blood mononuclear cells by a trauma-induced glycopeptide.

Traumatic injury often results in profound immunopathology that can lead to immunosuppression, thereby increasing the morbidity and mortality due to sepsis. The isolation and partial characterization of an immunosuppressive glycopeptide (SAP) from serum of severely burned patients has previously been reported by our laboratory. Recently, this trauma peptide has also been identified in the serum of patients with multiple blunt trauma. This glycopeptide is capable of suppressing neutrophil chemotaxis, T-cell blastogenesis and the lysis of human erythrocytes. We demonstrate in this report that SAP inhibits interleukin 2 (IL-2) biosynthesis by mitogen-stimulated peripheral blood mononuclear cells. Peptide concentrations of 50 nmol and above significantly inhibited IL-2 production. Inhibition was not reduced by the addition of indomethacin or anti-PGE2 to cultures containing greater than 100 nmol of peptide, suggesting that inhibition is not entirely prostaglandin-mediated. Preliminary studies have shown that IL-2 suppression by SAP can be partially reversed by the addition of calcium ionophore. These results suggest a potential immunosuppressive mechanism of the trauma peptide in which T cell blastogenesis is inhibited by interference in IL-2 biosynthesis.

Trauma peptide induction of lymphocyte changes predictive of sepsis.

Post-trauma immunosuppression is characterized by T-cell subpopulation changes and the presence of a low molecular weight suppressive active peptide (SAP), which suppresses T-cell blastogenesis and neutrophil chemotaxis. This study evaluated post-trauma T-cell antigens and suppressive active peptide/T-cell interactions to determine if the suppressive active peptide concentrations predictive of sepsis can cause changes in antigen expression predictive of sepsis. Human lymphocyte markers and differentiation antigens were analyzed post-trauma using flow cytometry for markers predictive of sepsis. Changes induced by purified suppressive active peptide incubated with normal human lymphocytes were similarly analyzed by flow cytometry. SAP concentrations for incubation were chosen which correlated with concentrations in patients developing clinical sepsis. Significant T-cell changes in patients who developed sepsis include: decreased total T-cells, decreased helper cells, decreased natural killer cells, increased Ia expressing mononuclear cells, increased activated T-cells, (L22) and increased IL-2 expressing cells (TAC). Suppressive active peptide can activate T-cells and cause significant increased expression of IL-2 receptors and natural killer cells. Other T-cell changes following trauma predictive of sepsis seem to occur independent of in vitro incubation with suppressive active peptides. IL-2 expressing cells are known to be more readily suppressed by the suppressive peptide. Suppressive peptide activation and subsequent inhibition of T-cells suggests a potential way to explain suppressive peptide-induced immunosuppression following trauma.

Immunosuppression by a peptide from the gelatin binding domain of human fibronectin.

Circulating immunosuppressive peptides are found in conjunction with elevated protease activity in injured patients' serum. Previous work suggests that fibronectin may be a source of these peptides. Elastase-generated fibronectin degradation products were chromatographically separated and assessed for immunosuppressive capacity in the neutrophil chemotaxis and mixed lymphocyte reaction bioassays. The suppressive fibronectin degradation products fraction 22 inhibited chemotaxis by 53% (P less than 0.01) and mixed lymphocyte reaction by 41% (P less than 0.001). Incubation of the suppressive fraction 22 with gelatin, neuraminidase, or anti-SAP (suppressor active peptide) antibody reverses the chemotaxis inhibition to control values. These results indicate that an elastase-generated fragment of fibronectin, which potently inhibits neutrophil and lymphocyte activity, is located within the sialated gelatin binding portion of the molecule. Immunosuppression reversal by anti-SAP antibody, and the requirement for sialic acid, suggests similarity between fraction 22 and SAP, although other explanations are plausible. This peptide fragment from fibronectin may impact on the clinical immunosuppression seen in patients following severe trauma.

Isolation of an immunosuppressive trauma peptide and its relationship to fibronectin.

The purpose of this study was to characterize a suppressive active glycopeptide (SAP) using affinity chromatography (AFFI) and explore its similarity to fibronectin (FN) degradation products. It is postulated that SAP is a degradation fragment of a large serum-borne protein, possibly FN. Human trauma serum (HTS) and elastase-degraded human FN were run over an AFFI prepared with monoclonal antibody to SAP. Bound protein was eluted with 2M NaCl and dialyzed to remove salt. Immunosuppressive activity of HTS and FN eluates was monitored using inhibition of neutrophil chemotaxis (CTX). Active fractions were compared to the starting material and controls with 15% PAGE. AFFI eluates of HTS revealed a high molecular weight protein band (450 kd) with no inhibitory CTX activity and singular low molecular weight protein (LMW) band (less than 20,000) with 93% +/- 5% CTX suppression. Elastase-digested purified human FN eluates run under identical conditions over the same AFFI revealed two bands with an identical elution profile as HTS eluates. CTX suppression 95 +/- 5% was seen only with the LMW band. Incubation of enriched LWF fragments of digested FN and HTS with whole FN reversed CTX suppression. This suggests affinity purification of a single LMW suppressive glycopeptide which may bind to or be a degradation product of plasma or cellular fibronectin.

Immunosuppressive effects of a trauma-induced suppressor active peptide.

The isolation and partial characterization of an immunosuppressive glycopeptide from sera of severely burned patients has previously been reported. Recently, a monoclonal antibody to this factor and an enzyme linked immunosorbent assay for detection of the peptide have been developed. The presence of the peptide in elevated quantity has been demonstrated in serum of patients with multiple blunt trauma as well as thermally injured patients. It was determined that the peptide is capable of suppressing neutrophil chemotaxis and T-cell blastogenesis as measured by MLR. Inhibition of B-cell blastogenesis induced by the peptide as measured by LPS mitogen-induced proliferation was demonstrated to be less sensitive to suppression. Further, it appears that activated T lymphocytes, those expressing increased IL-2 receptors, are more sensitive to suppression by the peptide at lower concentrations than are nonactivated T lymphocytes.

Elastase and suppressor active peptide activity following burn injury.

Proteolytic enzyme activity following trauma and inflammation plays a key role in the pathophysiology of injury. The precise mechanisms involved in the induction of protease release has not been determined. We show here that sera from burn patients with greater than 40% TBSA have significantly elevated levels of active elastase which correspond with significantly increased levels of suppressor active peptide (SAP) and suppression of neutrophil chemotaxis. The elastase inhibitory capacity of serum from burned or blunt trauma patients was within normal range, suggesting that the primary elastase inhibitor, alpha-1-proteinase inhibitor, is functionally active. Additionally, granulocytes exposed to suppressor active peptide in vitro resulted in a markedly elevated release of elastase into the culture supernatants. These data suggest that the suppressor peptide is capable of not only suppressing immune function but is also a potent mediator for the induction of proteolytic enzyme release from leukocytes.

Trauma peptide-mediated prostaglandin E2 biosynthesis: a potential mechanism for trauma-induced immunosuppression.

In vitro exposure of peripheral-blood-adherent mononuclear cells or amnion cells to nanomolar quantities of a trauma-associated immunosuppressive peptide resulted in an increased biosynthesis of prostaglandin E2 (PGE2). Trauma peptide enhanced prostaglandin E2 biosynthesis by as much as 425% compared to buffer controls. The addition of trauma peptide to mixed lymphocyte cultures significantly inhibited [3H]thymidine incorporation by human peripheral blood lymphocytes. Addition of indomethacin (an inhibitor of prostaglandin biosynthesis) to mixed lymphocyte cultures did not significantly abrogate the immunosuppressive activity of the peptide. These results indicate that suppression of T lymphocyte blastogenesis by trauma peptide is probably mediated by at least two mechanisms: (1) by increased PGE2 biosynthesis, induced by trauma peptide, and (2) through a non-cyclooxygenase-mediated pathway.

Immunologic monitoring of infection risk in trauma patients: research questions and an approach to the problem.

Sepsis due to injury-related immunosuppression is generally accepted to be the leading cause of morbidity and mortality following trauma. A clear relationship between the amount of burn injury and immunosuppression can be demonstrated, but quantitative relationships between the actual amount of blunt or penetrating injury, a "state" of immunosuppression, and the subsequent development of sepsis has not been clearly established. We have studied and attempted to characterize multiply traumatized patients to identify which components or immune parameters suggest the opportunity to predict sepsis. This report briefly reviews the literature in this area and summarizes current work from our laboratory attempting to identify potential markers.

Generation and activity of suppressor peptides following traumatic injury.

Severe trauma is known to produce pathophysiologic changes leading to the generation of immunosuppressive compounds. With recent advances in biotechnology, a number of these factors have been identified and characterized. Many of these substances have been found to be degradation products of normal serum and tissue proteins. These degradation products have profound biologic activity both in vivo and in vitro. This report briefly focuses on a number of these factors and summarizes the current work involved in the determination of the identity and mechanisms of a previously reported suppressor-active peptide isolated from the serum of trauma patients.

The immunosuppressive activity of C1q degradation peptides.

We have previously reported the existence and activity of a collagen-like, low molecular weight, suppressive peptide complex in patients with greater than 40% BSA burns. Since C1q participates in the inflammatory response and contains a collagen-like sequence, we have tested, in vitro, the putative generation and immunologic activity of C1q peptide fragments under physical conditions present in burned patients. Nanogram quantities of heat- and enzyme-generated fragments of C1q were shown to be suppressive in vitro to neutrophil chemotaxis, and the mixed lymphocyte response (MLR). The addition of lymphocytes pretreated with C1q fragments suppressed ongoing MLR, indicating the activation of suppressor cells by the peptides. Rabbit anti-C1q globulin was found to reduce the suppressive activity of the collagen-like suppressor which was isolated from human burn sera. Our results therefore suggest that C1q may be an early source of degradation peptides which have strong nonspecific immunosuppressive activity following thermal injuries.

Reversal of SAP-induced immunosuppression and SAP detection by a monoclonal antibody.

A murine monoclonal IgG antibody (MAb) to column-isolated trauma-induced suppressor active peptide (SAP) was produced and utilized in these studies for the further characterization of SAP. Specificity of the antibody was confirmed by enzyme-linked immunosorbent assay (ELISA), passive immunoblotting, and reversal of SAP-induced neutrophil chemotaxis inhibition. ELISA analysis revealed binding of anti-SAP MAb to a serum protein present in both whole burn and normal serum, but only to burn serum using a less than 25,000-mw serum fraction. This suggests that SAP may be an injury-induced degradation product of a greater than 25,000-mw serum protein. Immunoblotting using a less than 25,000-mw burn serum fraction demonstrated MAb binding to a single low molecular weight protein band. Using the MAb in an ELISA immunodiagnostic procedure, it appeared that SAP levels were significantly elevated in the sera of burned patients who died from their injuries compared to levels in sera of controls or patients who survived.

Comparison of local and systemic immunity after intratracheal, intraperitoneal, and intravenous immunization of mice exposed to either aerosolized or ingested lead.

The humoral immunity of newborn mice exposed for 28 days to 2.5 mg/m3 aerosolized Pb(NO3)2 (Pb28-aero) or of 2-week-old mice similarly exposed for 14 days (Pb14-aero) was compared with that of both 2-week-old mice given 125 micrograms Pb(NO3)2/day by gastric intubation for 14 days (Pb14-oral) and of 4-week-old nonexposed controls. Mice from each group were immunized with 10(8) sheep red blood cells by intravenous (i.v.), intraperitoneal (ip), or intratracheal (it) routes of immunization. Immunity was assessed by both hemagglutination and the enumeration of antibody-forming cells from the spleen and thoracic lymph nodes. All treatment groups had decreased thymus/body weight and spleen/body weight ratios whereas only Pbaero groups had enlarged livers. The most significant immunosuppression occurred in the ip-immunized Pb28-aero group. A significant suppression of humoral immunity was also observed in thoracic lymph node samples from Pbaero groups immunized it or iv. There was no apparent immunosuppression in any treatment group after iv immunization. These results indicate that aerosolized lead is more immunosuppressive than equivalent amounts of ingested lead. This is most likely due to the greater absorption of inhaled lead and the subsequent cytotoxicity of lead for cells in the draining lymph nodes.

Suppression of in vitro lymphocyte and neutrophil responses by a low molecular weight suppressor active peptide from burn-patient sera.

Thermal injury produces profound pathophysiological changes in the severely burned patient. Primary among these is the modulation of immunity, leading to episodes of immunosuppression and thus increasing the risk of sepsis and possible death. We herein report the isolation of a low molecular weight suppressor active peptide (SAP) which appears to be responsible for many of the observed immunologic changes in burned patients. SAP suppressed T-lymphocyte blastogenesis in the mixed lymphocyte reaction (MLR) and inhibited neutrophil chemotaxis (CTX) in vitro. Characterization of SAP revealed a complex structure comprised of (1) a peptide component rich in glycine, serine, and alanine; (2) a carbohydrate component containing sialic acid; and (3) a fatty acid component, tentatively identified as prostaglandin E. The immunosuppressive activity of SAP is dependent upon the presence of all three structural components. The molecular weight of SAP was estimated to be 3654 as determined by Amicon cell ultrafiltration and amino acid analysis. The isoelectric point of SAP was estimated by chromatofocusing and ion-exchange chromatography to be between 3.2 and 3.6. We hypothesize that the suppressor active peptide may be comprised of cellular or tissue components released into the circulation at the time of injury.

Circulating toxins in human disease: a viable concept?

Many clinical and biological effects have been attributed to the presence of mediators in the general circulation of patients after injury, a major operation, or the onset of certain diseases. Often, the term toxin is applied in an attempt to describe these circulating mediators. In the discussion that follows, cutaneous burn toxin is cited as an example and an opinion is ventured concerning the continued acceptance of both the term toxin and the toxic concept in modern medicine.

Definition of a burn injury-induced immunosuppressive serum component.

We and others have previously observed that immunologic activity can often be restored to both lymphocytes and neutrophils by removing them from the burn environment, leading to the conclusion that burn serum contains substances capable of suppressing immunologic function. The present studies were initiated to better define the serum component(s) responsible for this immunosuppression. The majority of immunosuppressive activity vs. both neutrophil chemotaxis and mixed lymphocyte cultures contained in large-volume serum samples obtained from three patients with greater than 40% body surface area flame burns was found to reside in a less than 25,000 mw fraction of serum obtained by Amicon ultrafiltration. A single suppressive serum component was isolated by precipitation and resuspension, followed by ion-exchange chromatography using an SP Sephadex C-25 column. Purity of the samples was verified by SDS slab-gel electrophoresis, and immunosuppressive activity was confirmed vs. both lymphocytes and neutrophils. Analysis of this isolated burn-associated suppressor indicates: a) a molecular weight of between 1,000 and 5,000 daltons; b) a complex composition containing a protein component, a lipid component, and a carbohydrate component; c) a structure which is heat stable, pH stable and unaffected by treatment with trypsin, proteinase K, DNAse, and RNAse; and d) a noncytotoxic immunosuppressive mode of action. It appears that the suppressive activity is dependent upon a prostaglandin portion of this low molecular weight complex.

Hemolysis and suppression of neutrophil chemotaxis by a low molecular weight component of human burn patient sera.

During the past year, we have reported the isolation and characterization of a low molecular mass (less than 5000 Da) complex (SAP), having profound immunosuppressive activity, found in the plasma of patients with major thermal injuries. Our continued studies have revealed that non-cytotoxic inhibition of neutrophil chemotaxis by SAP is paralleled by erythrocyte hemolysis by the same compound. Both neutrophil inhibitory and hemolytic activities appear to be a function of the peptide portion of SAP (determined by sensitivity to trypsin and pronase), which is augmented by the presence of sialic acid in the complex (activity eliminated by the addition of neuraminidase). The lipid portion of SAP, which is critical to its neutrophil immunosuppressive activity, does not appear to participate in erythrocyte hemolysis. Hemolytic activity of SAP is not due to protease activity, and does not appear to be receptor dependent. However, it is completely dependent upon the presence of calcium. The hemolytic activity of SAP appears to be abrogated by the addition of cerium nitrate, leading to a hypothesized relationship of SAP to the immunological activity of "burn toxin", previously described in detail (Schoenenberger, G.A. (1975) Monogr. Allergy 9, 72-139).