John A. Mannick

Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection.

Objective Patients 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‐12. Summary Background Data Perturbations 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‐τ) 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 IL‐12, 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. Methods The 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 I 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 highly suppressed. Finally, burn and sham‐burn animals, 20 per group, were treated in vivo with IL‐12 (25 ng daily for 5 days) and observed for mortality after septic challenge (cecal ligation and puncture [CLP]) performed on day 10 after injury. Results Peripheral blood mononuclear cells from burn and trauma patients produced less IFN‐τ, the index cytokine of Th‐1 cells, than PBMCs from healthy individuals 1 to 14 days after burn injury (SE = 77.6 ± 16 pg/mL patients vs. 141.3 ± 35 pg/mL controls, p < 0.05). However, production of IL‐4, the index cytokine of Th‐2 cells, by patient PBMCs was increased (51.0 ± 13.0 pg/mL patients vs. 26.9 ± 2.5 controls, p < 0.05). Splenocytes from mice 10 days after burn injury, when compared with sham‐burn controls, showed diminished production of IL‐2 (1.04 ± 0.91 units/mL burns vs. 5.8 ± 0.55 units/mL controls, p < 0.05) and IFN‐τ (1.05 ± 0.7 units/mL burns vs. 12.0 ± 8.9 units/ mL controls, p < 0.05). However, burn splenocytes produced more IL‐4 (2492 ± 157.0 pg/mL burns vs. 672.0 ± 22.7 pg/mL controls, p < 0.01) and IL‐10 (695.2 ± 20.8 pg/mL burns vs. 567.0 ± 16.7 pg/mL controls, p < 0.05). Splenocyte production of IL‐12 was also reduced after burn (0.20 ± 0.035 units/mL) as compared with sham burn (0.46 ± 0.08 units/mL, p < 0.05). The reduction in IL‐2, IFN‐τ, and IL‐12 production by burn splenocytes was reflected by a tenfold decrease in expression of their respective cytokine mRNAs. In vivo IL‐12 treatment of burn animals decreased mortality from CLP on day 10 after injury from 85% to 15% (sham‐burn mortality after CLP, 15%, p < 0.05) and increased splenocyte IFN‐τ production to supranormal levels. Conclusions Serious injury induced diminished production of IL‐12 and a shift to the Th‐2 phenotype with increased production of IL‐4 and IL‐10, cytokines known to inhibit Th‐1 function. The ability of exogenous IL‐12 to restore Th‐1 cytokine production and resistance to infection suggests a therapeutic role for IL‐12 in the immune dysfunction seen after major injury.

The effects of injury on the adaptive immune response

For more than thirty years it has been apparent that serious injury in humans and experimental animals is associated with a decrease in immune functions dependent upon T cells, the principal cells involved in initiating adaptive immune responses. This review focuses on more recent evidence that T helper cell function is altered after serious injury with loss of T helper 1 function and cytokine production and with preservation of T helper 2 function and an increased production of T helper 2 cytokines. Emphasis is placed on the importance of interactions between the innate and adaptive immune systems in the perturbed immune responses seen following injury. Immunomodulatory strategies are mentioned that have had success in animal models in ameliorating the diminished resistance to infection commonly seen after major traumatic or thermal injury. Finally, it is emphasized that immunomodulatory treatments that are successful in preventing infection may be contraindicated once infection is manifest.

Injury primes the innate immune system for enhanced Toll-like receptor reactivity.

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-1β, IL-6, and TNF-α 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.

The immunologic response to injury.

Research on the immune consequences of shock and trauma by multiple laboratories over more than 20 years has resulted in the following paradigm, which is currently accepted by most investigators in this field: serious traumatic or thermal injury is quickly followed, after initial resuscitation, by the systemic inflammatory response syndrome (SIRS) which, in a sizeable minority of patients, will lead inexorably to multiple organ dysfunction syndrome (early MODS), with an attendant high mortality. The majority of seriously injured patients survive the initial SIRS response without developing early MODS, and after a period of relative clinical stability, manifest a compensatory antiinflammatory response syndrome (CARS) with suppressed immunity and diminished resistance to infection. Resultant infection and its attendant inflammation in turn can lead to multiple organ dysfunction (late MODS) and death (Fig. 1). This paradigm has several implications of potential importance in interpreting the sometimes conflicting results of research in this area:

Inadequate interleukin 2 production. A fundamental immunological deficiency in patients with major burns.

We studied the production of the two major mediators of cellular immune responses, Interleukin 1 (IL-1) and Interleukin 2 (IL-2), by the peripheral blood mononuclear cells of 23 burn patients (16 men, seven women, mean age 48.9 years) compared with 23 matched controls (16 men, seven women, mean age 46.7 years). Serial measurements were made of IL-1 production by adherent mononuclear cells after stimulation with lipopolysaccharide and of IL-2 production by lymphocytes after stimulation with phytohemagglutinin (PHA). Eighty determinations of IL-2 production by lymphocytes from 12 patients with greater than 30% body surface area burn revealed a mean IL-2 production of 0.71 u as compared with a mean of 1.23 u for patients with less than 30% burns (p = 0.04). Patients with greater than 30% body surface area burns had significantly reduced IL-2 production (p ≤ 0.05) until 60 days after injury, whereas those with smaller burns had reduced IL-2 production only at 20–29 and 30–39 days postburn. Nine burn patients with systemic sepsis showed significantly lower IL-2 production (p = 0.03) at 10–29 days postburn than nonseptic patients, and significantly less IL-2 production during septic episodes. Eight patients with greater than 50% suppression of lymphocyte response to PHA produced less IL-2 (0.4 u) than patients with less than 50% suppression, (1.07 u, p = 0.004). IL-1 production was significantly elevated as compared with controls (4.45 u vs. 3.6 u, p = 0.05) early after injury, but was subsequently within the normal range regardless of burn size. The percentage of circulating helper T-lymphocytes, the principal source of IL-2, was also reduced, although this did not always correlate with IL-2 production, which remained depressed after recovery of the helper T-cell population. These results indicate that failure to produce IL-2, a powerful mediator of cellular immune responses, is an important mechanism underlying the defective cell mediated immunity seen in burn patients.

Aortic aneurysm repair. Reduced operative mortality associated with maintenance of optimal cardiac performance.

Recent advances in the operative management of aortic aneurysms have resulted in a decreased rate of morbidity and mortality. In 1972, we hypothesized that a further reduction in operative mortality might be obtained with controlled perioperative fluid management based on data provided by the thermistor-tipped pulmonary artery balloon catheter. From 1972 to 1979 a flow directed pulmonary artery catheter was inserted in each of 110 consecutive patients prior to elective or urgent repair of nonruptured infrarenal aortic aneurysms. The slope of the left ventricular performance curve was determined preoperatively by incremental infusions of salt-poor albumin and Ringers lactate solution. With each increase in the pulmonary arterial wedge pressure (PAWP), the cardiac index (CI) was measured. The PAWP was then maintained intra- and postoperatively at levels providing optimal left ventricular performance for the individual patient. There were no 30-day operative deaths among the patients in this series and only one in-hospital mortality (0.9%), four months following surgery. The five-year cumulative survival rate for patients in the present series was 84%, a rate which does not differ significantly from that expected for a normal age-corrected population. Since the patient population was unselected and there were no substantial alterations in operative technique during the present period, these improved results support the hypothesis that operative mortality attending the elective or urgent repair of abdominal aortic aneurysm can be minimized by maintenance of optimal cardiac performance with careful attention to fluid therapy during the perioperative period.

Association of anergy with an immunosuppressive peptide fraction in the serum of patients with cancer.

Abstract To study the relation between circulating immunosuppressive factors and anergy in patients with cancer, we tested 53 patients with cancer for hypersensitivity to skin-test antigens and 2,4-dinitrochlorobenzene. Of 41 patients with negative skin tests, 27 (66 per cent) had immunosuppressive serum (≥70 per cent inhibition of lymphocyte stimulation by phytohemagglutinin). None of 12 with positive skin tests had immunosuppressive serum. Thus, anergy and serum immunosuppressive activity were correlated (p<0.05). After ion-exchange chromatography, most of the immunosuppressive activity in cancer serum was in the first peak, fraction I. The same fraction from subjects without cancer contained no detectable immunosuppressive activity. Mean total immunosuppressive activity of cancer serum was 166.0 ± 97.5 (S.D.) units, and that of non-cancer serum was 30.4 ± 9.2 units (p<0.05). Diafiltration of fraction I. from cancer serum yielded a fraction (< 10,000 daltons) that was highly immunosuppressive. Thus, ane...

Depression of cellular immunity after multiple trauma in the absence of sepsis

We have previously reported that severe burn injury was regularly accompanied by impaired lymphocyte responses to T cell mitogens, circulating suppressor lymphocytes, and serum factors suppressive of lymphocyte activation. However, in burned patients it was difficult to determine whether these manifestations of suppressed immunity were predictive of, or the result of, sepsis which was ubiquitous in this population. In an attempt to clarify this issue, we have studied 31 patients with multiple trauma (without burns) mean age, 31 years; average injury severity score, 22; range, 9-56; in whom sepsis was less common. Patients were tested for lymphocyte response to the T cell mitogens PHA and Con A, the percentage of circulating putative suppressor (OKT8) and helper (OKT4) T cells using monoclonal antibodies, circulating suppressor cell activity as revealed by functional assays, and serum suppression of lymphocyte activation. Patients were compared with ten normal volunteers (mean age, 32) studied simultaneously. Significant suppression (greater than 50% compared with controls) in lymphocyte responses to mitogens 1 to 5 days after injury was seen in 12 patients, was accompanied by a shift in the ratio of helper (OKT4) to suppressor (OKT8) T cells (patients, 0.96:1; normals, 1.82:1; p less than 0.01), and was followed by the appearance of significant (greater than 50%) serum suppressive activity in six of the 12 patients. Circulating suppressor cell activity as revealed by functional assays was also seen early after injury in three of 12 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of Human Lymphocytes: Inhibition by Homologous Alpha Globulin

An alpha globulin fraction prepared from normal human plasma by column chromatography prevents homologous lymphocyte transformation and the stimulation of DNA, and also protein synthesis induced by phytohemagglutinin and specific antigens. These observations support the concept of a normal circulating immunosuppressant factor which prevents lymphoid cell proliferation.

CD4+CD25+ Regulatory T Cells Control Innate Immune Reactivity after Injury

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.