A. Nuri Ozkan

Suppression ofin 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.

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.

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-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.

Immunosuppression in Trauma Patients

An immunosuppressed state develops following traumatic injury, which makes patients more prone to develop infection. A variety of disturbances accompany injury that affect both specific and nonspecific components of host defense. Many clinical studies have attempted to evaluate the many deficits that follow injury and place the patient at a higher risk for infection. Several components of host defense are affected simultaneously and include (1) cellular changes (decreased activation of T-lymphocyte subsets with decreased helper cells, increased suppressor T-cell function, increased but abnormal activity of macrophages, activation of polymorphonuclear leukocytes with depressed chemotaxis and killing); (2) depressed nonspecific and specific serum immunity (e.g., depressed fibronectin and immunoglobulin levels); (3) the presence of altered cytokine levels (interleukin-1 [IL-1], IL-2, IL-6, tumor necrosis factor) levels; (4) ongoing serum proteolytic activity; and (5) the generation of serum suppressive peptides. An in-depth understanding of the deficits that occur following injury in host defense will provide the basis for therapeutic intervention.

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.

A rapid solid phase assay for the detection of circulating immune complexes

A simplified process, which we have termed Enzyme Immune Complex Assay (EICATM) for the detection of circulating immune complexes (CICs), is described herein. The method utilizes readily available reagents, small quantities of serum, and can be performed quickly with a minimal amount of equipment. Serum from 38 normal controls, 98 burn patients, 36 frostbite injury patients, and 21 patients with elevated rheumatoid factor (RF) were tested for CICs in an immune function study. Elevated immune complex levels were found in the group of patients with frostbite injury, and in the group with elevated RF. Serum from thermally injured patients had slightly depressed yet normal CIC levels. The detection of elevated CICs by the EICATM method compared favorably with the more cumbersome Raji cell method, with the added advantage of simplicity, speed, and the ability to detect non-IgG immune complexes.

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.