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The human leukocyte antigens and their relationship to infection
The Human Leukocyte Antigens and Their Relationship to Infection William G. Cheadle, MD, Louisville, Kentucky
H u m a n leukocyte antigen DR (HLA-DR) expression on p e r i p h e r a l blood monocytes has b e e n found to correlate highly with infection in m a n y clinical scenarios. This is particularly true for the t r a u m a patient, where changes in HLA-DR expression p r e d a t e and t h e r e f o r e often predict development of infection. Expression of this antigen is limited to immun o c o m p e t e n t cells, such as B lymphocytes, m a c r o phages, and activated T cells. The HLA-DR h e t e r o d i m e r is required for m a j o r histocompatibflity complex restricted antigen presentation, a key step in the development of a specific immune response. The degree of m o n o c y t e HLA-DR expression may reflect the ability eventually to present antigen, since close correlation has been found between the two. T h e r e was r e m a r k a b l e reproducibility of m o n o c y t e HLA-DR expression among > 1 0 0 asymptomatic volunteers without regard to age, gender, race, and sampling time. I m m u n o s u p p r e s sive medication had no effect. Incubation of m o n o cytcs f r o m severely infected patients with endotoxin distinguished survivors f r o m those who died by enhanced HLA-DR expression in the survivors. Of several agents that enhance HLA-DR expression, interferon-~/has received the most attention in experimental models as well as humans. Although promising in selected patients, further clinical trials will be needed to define its specific role. Identification of the patient at high risk for infection, particularly following trauma, will be crucial for the efficient evaluation of future therapeutic interventions. Monocyte HLA-DR expression is the first simple assessment of the host immune response to play an important role in this endeavor.
From the Department of Surgery and Price Institute of Surgical Research, University of Louisville School of Medicine, Louisville, Kentucky. Requests for reprints should be addressed to William G. Cheadle, MD, Department of Surgery, University of Louisville School of Medicine, Louisville,Kentucky40292.
nfection continues to be a common clinical problem, particularly in the surgical patient, and with the Iincreasing age and immunodepressive states in which we find our patient population, is likely to continue to be so. Despite improvements in surgical techniques, antibiotic spectra, monitored anesthesia, availability of blood products, and modern critical care, mortality for many surgical infectious diseases has not changed in the last four decades. Infection and multiple organ failure are the most common causes of late death after severe injury [1,2] and the cost of infectious morbidity is enormous in terms of patient suffering and utilization of critical care resources. For the most part, the surgeon assumes an immediate therapeutic role in terms of early intervention in diseases that require resection, debridement, diversion of the fecal stream, and/or intensive critical care. The trauma patient, however, offers a unique opportunity to study the high-risk patient who does not present immediately with infection. Major abdominal and wound infections, as well as pneumonia, generally present after the first 5 days following injury. This time period before the onset of infection allows the study of such patients to determine various risk factors for infection and, ultimately, intervention designed to prevent infection during this phase of clinical illness. We have focused on the severely injured patient over the last decade and have been particularly interested in the derangements of the host immune system that predispose to major infection. MAJOR H I S T O C O M P A T I B I L I T Y LOCUS AND T H E HLA SYSTEM The human leukocyte antigens (HLA) were initially identified on peripheral blood lymphocytes and designated the HLA system. There are four regions, HLA-A, B, C, and D, on the sixth chromosome, which encode their respective glycoprotein cell surface antigens. The immune response genes of the major histocompatibility locus are actually located immediately adjacent to those for certain complement components [3]. HLA-A, B, and C antigens are expressed on the cell surface in association with beta2-microglobulin, a structure similar to the immunoglobulin G (IgG) molecule. These antigens are expressed on all nucleated cells and platelets. HLA-DR is expressed on the cell surface as a heterodimer with alpha and 13chains encoded by major histocompatibility complex (MHC) genes. Expression of the HLA-DR antigen is largely restricted to B lymphocytes, activated T lymphocytes, and antigen-presenting cells, such as macrophages. Gene Dausset, George Davis Snell, and Baruj Benacerraf won the Nobel Prize in 1980 for discovery of the histocompatibility antigens on both human and animal
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cells and for their respective genetic control. Antibodies that agglutinated leukocytes were initially found in the serum of patients who had received multiple transfusions, as well as in multiparous women. This led to the prediction of the existence of such white cell antigens that were highly diverse, because individual response was quite variable. Their clinical prominence, however, has been related to antigenicity leading to graft rejection and human organ transplantation. Indeed, the term "major histocompatibility complex" relates to this, and modern tissue typing involves all of the aforementioned HLA antigens. These are the most highly immunogenic cell surface molecules. It was subsequently shown that the HLA-DR antigens are intimately involved in MHCrestricted antigen presentation, which is particularly important for defense against infectious disease. This initial recognition process is required for subsequent specific immune responses in terms of development of specific clones of cytotoxic T cells, as well as B-cell differentiation into plasma cells and clonally specific antibody production. At present, there are actually >100 specifically recognized HLA antigens. The major subtypes are HLA-A, B, C, D, DR, DQ, and DP. These are divided into class I which includes HLA-A, B, and C antigens, and class 11 antigens D, DR, DQ, and DP. Whereas the class I antigens are distributed on virtually all cells, the class II antigens are generally restricted to the previously described immunocompetent cells. All class I antigens, as well as DR and DQ antigens, can be detected by a microcytotoxicity test utilizing specific antibodies, and in the case of tissue typing, to recipient serum. HLA-D types of antigens were initially described by use of the mixed lymphocyte reaction in which daughter cells did not respond to parent cells and, therefore, were considered to be homozygous for a mixed lymphocyte reaction gene. With the advent of monoclonal antibodies, detection by flow cytometry has greatly simplified detection for the HLA-DR antigen. ROLE OF HLA-DR DURING ANTIGEN PRESENTATION
Antigen presentation is now recognized as a key initial event in the development of a specific immune response [4,5]. This requires an HLA-DR molecule in order to proceed, hence the term "MHC restricted." After phagocytosis of foreign antigen and partial digestion in the cytoplasm of the antigen-presenting cell, the digests are then carried to the cell surface and expressed in contiguity with a DR molecule itself. This is seen in Figure 1 and is much akin to a baseball in a catcher's glove. The complex is then recognized by a particular clone of a T-helper cell via a specific receptor in conjunction with the cluster designation 4 (CD4) on the T cell. Indeed, MHC-2 restricted antigen presentation occurs almost exclusively to the T-helper cell. In genetically engineered MHC-deficient mice, CD4 T-helper cells were found to be depleted, indicating that this process can occur very e~rly in embryonic maturation [6]. 76S
It appears that the process of antigen presentation is of most importance in transplant rejection in a nonMHC restricted fashion involving class I antigens. However, in development of an immune response to infectious agents, MHC restriction is important. Specific cytotoxic T-cell expansion that results from this process is involved in lysis of virally infected host cells. In addition, antibody production from plasma cells is involved in antibody-mediated bacterial lysis as well as antibody-mediated phagocytosis. These responses are of paramount importance in host defense against bacterial disease. After an initial encounter with a foreign antigen, both IgM and IgG antibodies are produced for several weeks after exposure. However, after a second exposure, a memory response occurs in which rapid production of antibodies occurs within days. This opsonic response is responsible for immunity to a variety of pathogenic organisms and their toxins following immunization. The memory response also results in markedly increased amounts of IgG antibody. MEASUREMENT OF MONOCYTE HLA-DR ANTIGEN EXPRESSION
HLA-DR expression on peripheral blood monocytes has been measured by a dual monoclonal antibody staining technique using whole blood [7]. The results can be made available within 4 hours of venipuncture. Fluorescent labeled anti-Mo2 (CD14) and anti-HLA-DR monoclonal antibodies were used to stain whole blood. Flow cytometric two-color analysis was then carried out, and only cells that costained with Mo2 and HLA-DR were regarded as monocytes that expressed HLA-DR. Mo2 (CD14), a human myeloid antigen, is expressed only on mature peripheral blood monocytes and not on lymphocytes or neutrophils, thus yielding high specificity for the monocyte population [8]. We examined both the percentage of monocytes that expressed HLA-DR antigen as well as the density of antigen expression, termed "mean fluorescence intensity." Similar results were obtained with both measurements, and there was an inverse correlation with the percentage and mean fluorescence intensity of HLA-DR expression on peripheral blood monocytes and development of infection (Figure 2). There was an initial reduction in monocyte HLA-DR expression in all patients immediately following injury, but in patients who recovered without infection, there was return to < 1 standard deviation of our cohort of asymptomatic volunteers by 1 week. In patients who developed major infection, return of HLA-DR expression was delayed until after recovery from infection, generally occurring about 3 weeks after injury. In patients who died, usually with major infection, there was a consistent downward trend, leveling off at only about 20% of peripheral blood monocytes that expressed this antigen. Whole blood was also incubated with 10 ixg/mL of lipopolysaccharide and analyzed for HLA-DR expression as well. The results distinguished those patients who survived from those who died; all survivors had percentages of monocytes
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HLA SYSTEM AND INFECTION
expressing HLA-DR < 1 standard deviation from our volunteer cohort mean, whereas the values of patients who died were significantly below this level [9].
IMMUNE-ASSOCIATED ANTIGEN EXPRESSION IN ANIMAL MODELS Both Ia expression, the murine analog of HLA-DR expression in humans, and antigen presentation have been found to be defective following hemorrhage [10]. This correlated with increased risk of infection following
hemorrhage in these animals, and treatment aimed at restoration of antigen presentation capacity was associated with enhanced survival [11]. We found reduced Ia antigen expression in several murine models of surgical infection, including thigh suture and peritonitis, which returned to normal coincident with survival from the infection. The depression seen in Ia antigen expression was related to proximity of the site of infection. For example, peritoneal macrophages were found to have the greatest depression in Ia compared with cells from other organs in an experimental peritonitis model.
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and 10th postoperative days, compared with the 19 patients without infection. This observation was coincident with development of the wound infections. It was interesting to note that mean fluorescence intensity in these patients was also significantly less preoperatively and this may indicate that certain patients are indeed at risk because of constitutively low DR expression. This was further borne out by the fact that asymptomatic volunteers who identified themselves as having recurrent minor infections tended to have lower monocTte HLA-DR expression.
HLA-DR EXPRESSION IN ASYMPTOMATIC
VOLUNTEERS The pronounced correlation between monocyte H L A - D R expression and infection in the trauma patients prompted us to study > 100 asymptomatic volunteers to determine reproducibility and whether any variance occurred with patient demographics [9]. We also studied patients in a variety of clinical scenarios who were believed to be at increased risk or to have developed major infection. We found that 85% of peripheral blood monocytes from the volunteers expressed the H L A - D R antigen and this was not affected by age, gender, race, or time at which the blood sample was taken. There was also no diurnal variation and in particular no seasonal variation during the time of year. The assay was remarkably reproducible among individuals. No correlation between blood alcohol and HLA-DR expression was found in the series of trauma patients nor any correlation in a volunteer who was followed serially while obtaining a maximum alcohol level of 150 mg/dL. In studies of family pedigrees, we noticed that there were some families of low expressors that tended to carry this trait through several generations. In patients who were fairly high expressors of HLA- DR, the trait was carried on through several generations as well.
ASSOCIATION OF HLA-DR W I T H I N F E C T I O N AND O T H E R DISEASE A group of 21 patients with major infection, including bacteremia, pneumonia, and necrotizing fasciitis, were then studied at a single point when overt clinical infection was present [9]. The mean value of 38% of monocytes expressing HLA- D R antigen for these patients was significantly below the mean of 85% previously obtained from the volunteers. Nine of these patients died and their mean fluorescence intensity was significantly lower than that in the 12 patients who survived. As seen in the trauma patients, monocyte HLA-DR expression from those patients who survived increased to mean control levels after incubation with lipopolysaccharide, whereas those from patients who died only increased to slightly > 50% (Figure 3). We also examined 26 patients who had undergone renal transplantation and were maintained on a variety of immunosuppressive regimens utilizing prednisolone, cyclosporine, and azathioprine [9]. These patients were not undergoing treatment for acute rejection and did not have any obvious clinical infection. Their level of monocyte HLA-DR expression was similar to that of the asymptomatic volunteers, with no effect of the exogenously administered immunosuppressive drugs.
HLA-DR EXPRESSION AFTER ELECTIVE SURGERY
We subsequently examined 24 patients who underwent laparotomy and found that the mean percentage of monocytes with H L A - D R expression in 19 patients without infection was significantly reduced from their own preoperative values 1 and 3 days following their surgical procedures, but rapidly returned to the preoperative level by postoperative day 5 [9]. In five patients who developed wound infections, there were significantly fewer monocytes that expressed D R antigen on the 7th
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Figure 2. Percentages of monocytes that expressed HLA-DR antigen after severe injury: 9 = uneventful recovery group; [ ] = major sepsis group; /k = group of patients who died. Error bars refer to the standard error of the mean. *Statistically significant difference from uneventful recovery group; tstatistically significant difference from major sepsis group. (Reprinted with permission from Br J Surg [30] .)
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HLA SYSTEM AND INFECTION
Specific disease states have been associated with a variety of human leukocyte antigens. In particular, HLA-DR antigen expression on monocytes has been found to be reduced in several diseases, including tuberculosis, psoriasis, Hodgkin's disease, and systemic lupus erythematosus [12-15]. Reduced levels of HLA-DR expression have also been found in congenital and acquired immunodeficiency syndromes [16,17].
STUDY OF TRAUMA PATIENTS AT RISK FOR
INFECTION The emergency surgical services at Humana Hospital, University of Louisville receive about 1,500 admissions a year for major trauma, providing ample opportunity to study the critical care of these patients. In a systematic study designed to assess the response of the host immune system [7], we examined a multitude of parameters representative of both nonspecific and specific components of the immune response over time after injury. The subsequent clinical courses of these patients were carefully followed and it was found that age, severity of injury, and presence of bacterial contamination at the time of injury all greatly influenced the risk of infection. Most parameters of the host immune response were depressed immediately after injury, including delayed hypersensitivity, immunoglobulin production, serum opsonic capacity, as well as many aspects of macrophage, lymphocyte, and neutrophil function [18-20]. Other facets of the immune response have been found to be defective as well, prompting us to wonder if this indeed might be beneficial shortly after injury. Indeed, initial restoration of the immune response only after complete resuscitation 48-72 hours after injury may be most appropriate [21]. We classified our patients according to clinical outcome with respect to infection and believe this is crucial to interpretation of results. Three categories were developed:
MEANS TO ENHANCE MONOCYTE HLA-DR
EXPRESSION It is logical to look for agents to enhance HLA-DR expression and several have been found to do so. Such agents include lipopolysaccharide, interferon (IFN)-~/, muramyl dipeptide, monophosphoryl lipid A, and the complement fragment C5a [22,23]. They occur via several different mechanisms. Lipopolysaccharide is considered to cause externalization of preformed antigen to the cell surface, whereas muramyl dipeptide may be involved in enhancement of transport through the cell membrane [24]. IFN--/has been shown to increase de novo protein synthesis by enhanced messenger RNA production [25]. On incubation of low HLA-DR expressing monocytes from patients following trauma, enhanced expression was seen after 24 48 hours of in vitro culture with IFN-~/ [26]. A number of animal experiments in which IFN--/was administered both prior to and/or after bacterial challenge demonstrated significantly enhanced survival. After proper dose-response experiments were carried out, an initial pilot study of 15 patients was conducted. Two doses of IFN-~ were given, 0.5 and 1 mg, for 7-10 days. We found a marked enhancement in HLA-DR expression for the first week compared with historical controls, as well as fewer infections than expected. This led to a major clinical trial of >200 severely injured patients randomized to receive either a placebo or IFN-',/for 10 days [27]. Although no overall benefit was seen in regard to development of major infection and death, there appeared to be benefit in patients who developed major abdominal and wound infection. Indeed, 17 patients in the IFN group required reoperation or computer tomographic-directed drainage, compared with 22 in the placebo group. Only 10 patients in the IFN group developed these infections after 1 week, versus 19 in the placebo group. There was significant enhancement in both percentages of monocytes that expressed HLA-DR as well as their mean fluorescence intensity in the IFN-treated patients,
1. Uneventful recovery in which only minor infections might occur. 2. Major infections that impacted adversely on the clinical course and prolonged hospital stay. These included bacteremia not associated with central lines, pneumonia, major wound or soft tissue infections, body cavity abscesses or infection, and the need for reoperation or percutaneous drainage of collections.
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. Patients who died were grouped together; most of these died of infection. Of the parameters measured, only one, expression of the class II major histocompatibility cell surface antigen HLA-DR on peripheral blood monocytes, correlated consistently with clinical outcome with regard to development of major infection and death from such infection [7].
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Figure 3, Monocyte HLA-DR expression with and without iipopolysaccharide (LPS) incubation and its relation to clinical outcome in 21 patients with severe infection. There was significant difference in the LPS groups between those who survived and those who died. (Reprinted with permission from [9].) VOLUME 165 NO. 2A (SUPPL)
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Figure 4. Density of monocyte HLA-DR expression declines toward the placebo level on cessation of interferon--/therapy in those patients who recovered without major infection. (Reprinted with permission from [27].) which rapidly declined after the drug was withdrawn (Figure 4). A second study of 416 patients from nine centers demonstrated significant reduction in death and major infection in blunt trauma patients (D.J. Dries, unpublished data, 1991). However, most of the benefit occurred in one center. A further trial focusing on those patients most likely to benefit with a longer duration of treatment will be necessary to prove the efficacy of IFN-~ in this clinical setting. THE OUTCOME PREDICTIVE SCORE
An important clinical goal, of course, is reduction in infectious morbidity and mortality. Identification of such patients at high risk is imperative for them to be entered into trials of agents designed to reduce such risk. We developed an outcome predictive score that has been found to be quite reliable in the prediction of major infection following major trauma [28]. The score was reasonably specific in that few patients with low outcome predictive scores developed infection. Most trauma scores have been constructed to predict immediate mortality from hemorrhage and head injury or to compare the severity of illness among patients, but not to assess risk of infectious morbidity and mortality. We constructed the outcome predictive score based on age, severity of injury as calculated by the injury severity score, amount of bacterial contamination, and percentage of monocytes that expressed H L A - D R within 24 hours of admission. In our initial study of 61 trauma patients, the mean score of each clinical outcome group was significantly different from the other within 24 hours of hospitalization. The score was again calculated and its validity further tested in the first clinical trial of IFN--y. In the placebo cohort of patients, the outcome predictive score on day 1 was not helpful in predicting those patients who were at risk for infection, but by day 3 it was highly predictive and reasonably specific. It may well be that utilization of the outcome predictive score to enter patients into clinical trials 48-72 hours after injury is more appropriate. This would allow more accurate
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prognosis of severe head injuries, so those with fatal injuries are not entered, and one could begin administration of agents aimed at immune restoration following the initial resuscitation period. This would be applicable to any immunomodulatory agent but, of course, most relevant to those known to enhance D R expression. CONCLUSION The H L A system is becoming increasingly associated with a wide variety of disease, indicating that constitutional genetic abnormalities predispose to risk of acquiring disease. There appear to be individuals with low monocyte H L A - D R expression who are indeed at increased risk of infection because of this inherent abnormality. H L A - D R expression and many other components of the immune response have been found to be depressed after major injury. The strong correlation between monocyte H L A - D R expression and clinical outcome with respect to infection suggests that restoration of such expression may help prevent or abrogate the severity of infection. Only one agent known to enhance H L A - D R expression, IFN-% has been extensively studied. Although promising in selected patients, further work will be needed to define its role. Proper conduct of such trials requires that a truly at-risk population be studied; monocyte H L A - D R expression will help to identify such patients at high risk for infection following major injury. Because there is good correlation between Ia expression and antigen presentation experimentally, agents that enhance the latter should hold promise for future clinical trials. REFERENCES
1. Baker CC, Oppenheimer, Lewis FR, et al. The epidemiology of trauma deaths. Am J Surg 1980; 140: 144-50. 2. Goris RJA, Draaisma J. Causes of death after blunt trauma. J Trauma 1982; 22: 141-6. 3. McDevitt HO. Current concepts in immunology: regulation of the immune response by the major histocompatibility system. N Engl J Med 1980;303: 1514-7. 4. Unanue ER, Beller DI, Lu CY et al. Antigen presentation: comments on its regulation and mechanism. J Immunol 1984; 132: 1-7. 5. Harding CV, Unanue ER. Cellular mechanisms of antigen processing and the function of class I and II major histocompatibility complex molecules. Cell Regul 1990 1: 499-509. 6. Grusby MJ, Johnson RS, Papaioannou VE, Glimcher LH. Depletion of CD4 + T Cells in major histocompatibility complex class II-deficient mice. Science 1991;253: 1417-20. 7. Polk HC Jr, George CD, Wellhausen SR, et al. A systematic study of host defence processes in badly injured patients. Ann Surg 1986; 204: 282-99. 8. Todd RF III, Nadler LM, Schlossman SF. Antigens on human monocytes identified by monoclonal antibodies, J Immunol 1981; 126: 1435-42. 9. Cheadle WG, Hershman MJ, Wellhausen SR, Polk HC Jr. HLA-DR expression on peripheral blood monocytes correlates with surgical infection. Am J Surg 1991; 161: 639-45. 10. Ayala A, Perrin MM, Chaudry IH. Defective macrophage antigen presentation following haemorrhage is associated with the loss of MHC class II (Ia) antigens. Immunology 1990; 70: 33-9. I 1. Ertel W, Meldrum Dr, Morrison MH, et al. Immunoprotec-
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rive effect of a calcium channel blocker on macrophage antigen presentation function, Ia expression and interleukin-1 synthesis following hemorrhage. Surgery 1990; 108; 154-60. 12. Volk H-D, Koyka I, Goan S-R, et al. Serum-mediated inhibition of the interferon-gamma-induced HLA-DR expression on monocytes in patients with psoriasis. J Invest Dermatol 1986; 87: 524-7. 13. Shirakawa F, Yamashita U, Suzuki H. Decrease in HLA-DRpositive monocytes in patients with systemic lupus erythematosus (SLE). J Immunol 1985; 134: 3560-2. 14. Tweardy DJ, Schacter BZ, Ellner JJ. Association of altered dynamics of monocyte surface expression of human leukocyte antigen DR with immuno-suppression in tuberculosis. J Infect Dis 1984; 149: 3l-7. 15. Nagai H, Fisher RI, Cossman J, Oppenheim JJ. Decreased expression of class II major histocompatibility antigens on monocytes from patients with Hodgkin's disease. J Leukocyte Biol 1986; 39: 313-21. 16. De Pr6val C, Hadam MR, Mach B. Regulation of genes for HLA class II antigens in cell lines from patients with severe combined immunodeficiency. N Engl J Med 1988; 318: 1295-300. 1 7. Heagy W, Kelley VE, Strom TB, et al. Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome: increased expression with interferon-gamma. J Clin Invest 1984; 74: 2089-96. 18. Faist E, Mewes A, Strasser T, et al. Alteration of monocyte function following major injury. Arch Surg 1988; 123: 287-92. 19. Faist E, Kupper T, Baker CC, et al. Depression of cellular immunity after major injury: its association with post-traumatic complications and its reversal with immunomodulation. Arch Surg 1986; 121: 1000-5. 20. Lanser ME, Mao P, Brown G, et al. Serum-mediated depres-
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sion of neutrophil chemiluminescence following blunt trauma. Ann Surg 1985; 202: 111-8. 21. Border JR. Hypothesis: sepsis, multiple systems organ failure, and the macrophage. Arch Surg 1988; 123:285-6 22. Todd RF III, Alvarez PA, Brott DA, Liu DY. Bacterial tipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e. J Immuno11985; 135: 3869-77. 23. Appel SH, Wellhausen SR, Montgomery R, DeWeese RC, Polk HC Jr. Experimental and clinical significance of endotoxindependent HLA-DR expression on monocytes. J Surg Res 1989; 47: 39-44. 24. Gonwa TA, Stobo JD. Differential expression of Ia molecules by human monocytes. J Clin Invest 1984; 74: 859-66. 25. Sonnenfeld G, Meruelo D, McDevitt HO, Merigan TC. Effect of type 1 and type II interferons on murine thymocyte surface antigen expression: induction or selection? Cell Immunol 1981; 57: 427-39. 26. Hershman MI, Appel SH, Wellhausen SR, et al. Interferongamma treatment increases HLA-DR expression on monocytes in severely injured patients. Clin Exp Immuno11989; 77: 67-70. 27. Polk HC Jr, Cheadle WG, Livingston DH, et al. A randomized prospective clinical trial to determine the efficacy of interferon--r in severely injured patients. Am J Surg 1992 163:191-96 28. Hershman MJ, Cheadle WG, George CD, Kuftinec D, Polk HC Jr. An outcome predictive score for sepsis and death following trauma. Injury 1988; 19: 263-6. 29. Sinha AA, Lopez MT, McDevitt HO. Autoimmune diseases: the failure of self-tolerance. Science 1990; 248: 1380-7. 30. Hershman MJ, Cheadle WG, Wellhausen SR, Davidson PF, and Polk HC Jr. Br J Surg 1990; 77: 204-7.
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H u m a n leukocyte antigen DR (HLA-DR) expression on p e r i p h e r a l blood monocytes has b e e n found to correlate highly with infection in m a n y clinical scenarios. This is particularly true for the t r a u m a patient, where changes in HLA-DR expression p r e d a t e and t h e r e f o r e often predict development of infection. Expression of this antigen is limited to immun o c o m p e t e n t cells, such as B lymphocytes, m a c r o phages, and activated T cells. The HLA-DR h e t e r o d i m e r is required for m a j o r histocompatibflity complex restricted antigen presentation, a key step in the development of a specific immune response. The degree of m o n o c y t e HLA-DR expression may reflect the ability eventually to present antigen, since close correlation has been found between the two. T h e r e was r e m a r k a b l e reproducibility of m o n o c y t e HLA-DR expression among > 1 0 0 asymptomatic volunteers without regard to age, gender, race, and sampling time. I m m u n o s u p p r e s sive medication had no effect. Incubation of m o n o cytcs f r o m severely infected patients with endotoxin distinguished survivors f r o m those who died by enhanced HLA-DR expression in the survivors. Of several agents that enhance HLA-DR expression, interferon-~/has received the most attention in experimental models as well as humans. Although promising in selected patients, further clinical trials will be needed to define its specific role. Identification of the patient at high risk for infection, particularly following trauma, will be crucial for the efficient evaluation of future therapeutic interventions. Monocyte HLA-DR expression is the first simple assessment of the host immune response to play an important role in this endeavor.
From the Department of Surgery and Price Institute of Surgical Research, University of Louisville School of Medicine, Louisville, Kentucky. Requests for reprints should be addressed to William G. Cheadle, MD, Department of Surgery, University of Louisville School of Medicine, Louisville,Kentucky40292.
nfection continues to be a common clinical problem, particularly in the surgical patient, and with the Iincreasing age and immunodepressive states in which we find our patient population, is likely to continue to be so. Despite improvements in surgical techniques, antibiotic spectra, monitored anesthesia, availability of blood products, and modern critical care, mortality for many surgical infectious diseases has not changed in the last four decades. Infection and multiple organ failure are the most common causes of late death after severe injury [1,2] and the cost of infectious morbidity is enormous in terms of patient suffering and utilization of critical care resources. For the most part, the surgeon assumes an immediate therapeutic role in terms of early intervention in diseases that require resection, debridement, diversion of the fecal stream, and/or intensive critical care. The trauma patient, however, offers a unique opportunity to study the high-risk patient who does not present immediately with infection. Major abdominal and wound infections, as well as pneumonia, generally present after the first 5 days following injury. This time period before the onset of infection allows the study of such patients to determine various risk factors for infection and, ultimately, intervention designed to prevent infection during this phase of clinical illness. We have focused on the severely injured patient over the last decade and have been particularly interested in the derangements of the host immune system that predispose to major infection. MAJOR H I S T O C O M P A T I B I L I T Y LOCUS AND T H E HLA SYSTEM The human leukocyte antigens (HLA) were initially identified on peripheral blood lymphocytes and designated the HLA system. There are four regions, HLA-A, B, C, and D, on the sixth chromosome, which encode their respective glycoprotein cell surface antigens. The immune response genes of the major histocompatibility locus are actually located immediately adjacent to those for certain complement components [3]. HLA-A, B, and C antigens are expressed on the cell surface in association with beta2-microglobulin, a structure similar to the immunoglobulin G (IgG) molecule. These antigens are expressed on all nucleated cells and platelets. HLA-DR is expressed on the cell surface as a heterodimer with alpha and 13chains encoded by major histocompatibility complex (MHC) genes. Expression of the HLA-DR antigen is largely restricted to B lymphocytes, activated T lymphocytes, and antigen-presenting cells, such as macrophages. Gene Dausset, George Davis Snell, and Baruj Benacerraf won the Nobel Prize in 1980 for discovery of the histocompatibility antigens on both human and animal
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cells and for their respective genetic control. Antibodies that agglutinated leukocytes were initially found in the serum of patients who had received multiple transfusions, as well as in multiparous women. This led to the prediction of the existence of such white cell antigens that were highly diverse, because individual response was quite variable. Their clinical prominence, however, has been related to antigenicity leading to graft rejection and human organ transplantation. Indeed, the term "major histocompatibility complex" relates to this, and modern tissue typing involves all of the aforementioned HLA antigens. These are the most highly immunogenic cell surface molecules. It was subsequently shown that the HLA-DR antigens are intimately involved in MHCrestricted antigen presentation, which is particularly important for defense against infectious disease. This initial recognition process is required for subsequent specific immune responses in terms of development of specific clones of cytotoxic T cells, as well as B-cell differentiation into plasma cells and clonally specific antibody production. At present, there are actually >100 specifically recognized HLA antigens. The major subtypes are HLA-A, B, C, D, DR, DQ, and DP. These are divided into class I which includes HLA-A, B, and C antigens, and class 11 antigens D, DR, DQ, and DP. Whereas the class I antigens are distributed on virtually all cells, the class II antigens are generally restricted to the previously described immunocompetent cells. All class I antigens, as well as DR and DQ antigens, can be detected by a microcytotoxicity test utilizing specific antibodies, and in the case of tissue typing, to recipient serum. HLA-D types of antigens were initially described by use of the mixed lymphocyte reaction in which daughter cells did not respond to parent cells and, therefore, were considered to be homozygous for a mixed lymphocyte reaction gene. With the advent of monoclonal antibodies, detection by flow cytometry has greatly simplified detection for the HLA-DR antigen. ROLE OF HLA-DR DURING ANTIGEN PRESENTATION
Antigen presentation is now recognized as a key initial event in the development of a specific immune response [4,5]. This requires an HLA-DR molecule in order to proceed, hence the term "MHC restricted." After phagocytosis of foreign antigen and partial digestion in the cytoplasm of the antigen-presenting cell, the digests are then carried to the cell surface and expressed in contiguity with a DR molecule itself. This is seen in Figure 1 and is much akin to a baseball in a catcher's glove. The complex is then recognized by a particular clone of a T-helper cell via a specific receptor in conjunction with the cluster designation 4 (CD4) on the T cell. Indeed, MHC-2 restricted antigen presentation occurs almost exclusively to the T-helper cell. In genetically engineered MHC-deficient mice, CD4 T-helper cells were found to be depleted, indicating that this process can occur very e~rly in embryonic maturation [6]. 76S
It appears that the process of antigen presentation is of most importance in transplant rejection in a nonMHC restricted fashion involving class I antigens. However, in development of an immune response to infectious agents, MHC restriction is important. Specific cytotoxic T-cell expansion that results from this process is involved in lysis of virally infected host cells. In addition, antibody production from plasma cells is involved in antibody-mediated bacterial lysis as well as antibody-mediated phagocytosis. These responses are of paramount importance in host defense against bacterial disease. After an initial encounter with a foreign antigen, both IgM and IgG antibodies are produced for several weeks after exposure. However, after a second exposure, a memory response occurs in which rapid production of antibodies occurs within days. This opsonic response is responsible for immunity to a variety of pathogenic organisms and their toxins following immunization. The memory response also results in markedly increased amounts of IgG antibody. MEASUREMENT OF MONOCYTE HLA-DR ANTIGEN EXPRESSION
HLA-DR expression on peripheral blood monocytes has been measured by a dual monoclonal antibody staining technique using whole blood [7]. The results can be made available within 4 hours of venipuncture. Fluorescent labeled anti-Mo2 (CD14) and anti-HLA-DR monoclonal antibodies were used to stain whole blood. Flow cytometric two-color analysis was then carried out, and only cells that costained with Mo2 and HLA-DR were regarded as monocytes that expressed HLA-DR. Mo2 (CD14), a human myeloid antigen, is expressed only on mature peripheral blood monocytes and not on lymphocytes or neutrophils, thus yielding high specificity for the monocyte population [8]. We examined both the percentage of monocytes that expressed HLA-DR antigen as well as the density of antigen expression, termed "mean fluorescence intensity." Similar results were obtained with both measurements, and there was an inverse correlation with the percentage and mean fluorescence intensity of HLA-DR expression on peripheral blood monocytes and development of infection (Figure 2). There was an initial reduction in monocyte HLA-DR expression in all patients immediately following injury, but in patients who recovered without infection, there was return to < 1 standard deviation of our cohort of asymptomatic volunteers by 1 week. In patients who developed major infection, return of HLA-DR expression was delayed until after recovery from infection, generally occurring about 3 weeks after injury. In patients who died, usually with major infection, there was a consistent downward trend, leveling off at only about 20% of peripheral blood monocytes that expressed this antigen. Whole blood was also incubated with 10 ixg/mL of lipopolysaccharide and analyzed for HLA-DR expression as well. The results distinguished those patients who survived from those who died; all survivors had percentages of monocytes
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HLA SYSTEM AND INFECTION
expressing HLA-DR < 1 standard deviation from our volunteer cohort mean, whereas the values of patients who died were significantly below this level [9].
IMMUNE-ASSOCIATED ANTIGEN EXPRESSION IN ANIMAL MODELS Both Ia expression, the murine analog of HLA-DR expression in humans, and antigen presentation have been found to be defective following hemorrhage [10]. This correlated with increased risk of infection following
hemorrhage in these animals, and treatment aimed at restoration of antigen presentation capacity was associated with enhanced survival [11]. We found reduced Ia antigen expression in several murine models of surgical infection, including thigh suture and peritonitis, which returned to normal coincident with survival from the infection. The depression seen in Ia antigen expression was related to proximity of the site of infection. For example, peritoneal macrophages were found to have the greatest depression in Ia compared with cells from other organs in an experimental peritonitis model.
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and 10th postoperative days, compared with the 19 patients without infection. This observation was coincident with development of the wound infections. It was interesting to note that mean fluorescence intensity in these patients was also significantly less preoperatively and this may indicate that certain patients are indeed at risk because of constitutively low DR expression. This was further borne out by the fact that asymptomatic volunteers who identified themselves as having recurrent minor infections tended to have lower monocTte HLA-DR expression.
HLA-DR EXPRESSION IN ASYMPTOMATIC
VOLUNTEERS The pronounced correlation between monocyte H L A - D R expression and infection in the trauma patients prompted us to study > 100 asymptomatic volunteers to determine reproducibility and whether any variance occurred with patient demographics [9]. We also studied patients in a variety of clinical scenarios who were believed to be at increased risk or to have developed major infection. We found that 85% of peripheral blood monocytes from the volunteers expressed the H L A - D R antigen and this was not affected by age, gender, race, or time at which the blood sample was taken. There was also no diurnal variation and in particular no seasonal variation during the time of year. The assay was remarkably reproducible among individuals. No correlation between blood alcohol and HLA-DR expression was found in the series of trauma patients nor any correlation in a volunteer who was followed serially while obtaining a maximum alcohol level of 150 mg/dL. In studies of family pedigrees, we noticed that there were some families of low expressors that tended to carry this trait through several generations. In patients who were fairly high expressors of HLA- DR, the trait was carried on through several generations as well.
ASSOCIATION OF HLA-DR W I T H I N F E C T I O N AND O T H E R DISEASE A group of 21 patients with major infection, including bacteremia, pneumonia, and necrotizing fasciitis, were then studied at a single point when overt clinical infection was present [9]. The mean value of 38% of monocytes expressing HLA- D R antigen for these patients was significantly below the mean of 85% previously obtained from the volunteers. Nine of these patients died and their mean fluorescence intensity was significantly lower than that in the 12 patients who survived. As seen in the trauma patients, monocyte HLA-DR expression from those patients who survived increased to mean control levels after incubation with lipopolysaccharide, whereas those from patients who died only increased to slightly > 50% (Figure 3). We also examined 26 patients who had undergone renal transplantation and were maintained on a variety of immunosuppressive regimens utilizing prednisolone, cyclosporine, and azathioprine [9]. These patients were not undergoing treatment for acute rejection and did not have any obvious clinical infection. Their level of monocyte HLA-DR expression was similar to that of the asymptomatic volunteers, with no effect of the exogenously administered immunosuppressive drugs.
HLA-DR EXPRESSION AFTER ELECTIVE SURGERY
We subsequently examined 24 patients who underwent laparotomy and found that the mean percentage of monocytes with H L A - D R expression in 19 patients without infection was significantly reduced from their own preoperative values 1 and 3 days following their surgical procedures, but rapidly returned to the preoperative level by postoperative day 5 [9]. In five patients who developed wound infections, there were significantly fewer monocytes that expressed D R antigen on the 7th
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THE AMERICAN JOURNAL OF SURGERY VOLUME 165 NO. 2A (SUPPL) FEBRUARY 1993
HLA SYSTEM AND INFECTION
Specific disease states have been associated with a variety of human leukocyte antigens. In particular, HLA-DR antigen expression on monocytes has been found to be reduced in several diseases, including tuberculosis, psoriasis, Hodgkin's disease, and systemic lupus erythematosus [12-15]. Reduced levels of HLA-DR expression have also been found in congenital and acquired immunodeficiency syndromes [16,17].
STUDY OF TRAUMA PATIENTS AT RISK FOR
INFECTION The emergency surgical services at Humana Hospital, University of Louisville receive about 1,500 admissions a year for major trauma, providing ample opportunity to study the critical care of these patients. In a systematic study designed to assess the response of the host immune system [7], we examined a multitude of parameters representative of both nonspecific and specific components of the immune response over time after injury. The subsequent clinical courses of these patients were carefully followed and it was found that age, severity of injury, and presence of bacterial contamination at the time of injury all greatly influenced the risk of infection. Most parameters of the host immune response were depressed immediately after injury, including delayed hypersensitivity, immunoglobulin production, serum opsonic capacity, as well as many aspects of macrophage, lymphocyte, and neutrophil function [18-20]. Other facets of the immune response have been found to be defective as well, prompting us to wonder if this indeed might be beneficial shortly after injury. Indeed, initial restoration of the immune response only after complete resuscitation 48-72 hours after injury may be most appropriate [21]. We classified our patients according to clinical outcome with respect to infection and believe this is crucial to interpretation of results. Three categories were developed:
MEANS TO ENHANCE MONOCYTE HLA-DR
EXPRESSION It is logical to look for agents to enhance HLA-DR expression and several have been found to do so. Such agents include lipopolysaccharide, interferon (IFN)-~/, muramyl dipeptide, monophosphoryl lipid A, and the complement fragment C5a [22,23]. They occur via several different mechanisms. Lipopolysaccharide is considered to cause externalization of preformed antigen to the cell surface, whereas muramyl dipeptide may be involved in enhancement of transport through the cell membrane [24]. IFN--/has been shown to increase de novo protein synthesis by enhanced messenger RNA production [25]. On incubation of low HLA-DR expressing monocytes from patients following trauma, enhanced expression was seen after 24 48 hours of in vitro culture with IFN-~/ [26]. A number of animal experiments in which IFN--/was administered both prior to and/or after bacterial challenge demonstrated significantly enhanced survival. After proper dose-response experiments were carried out, an initial pilot study of 15 patients was conducted. Two doses of IFN-~ were given, 0.5 and 1 mg, for 7-10 days. We found a marked enhancement in HLA-DR expression for the first week compared with historical controls, as well as fewer infections than expected. This led to a major clinical trial of >200 severely injured patients randomized to receive either a placebo or IFN-',/for 10 days [27]. Although no overall benefit was seen in regard to development of major infection and death, there appeared to be benefit in patients who developed major abdominal and wound infection. Indeed, 17 patients in the IFN group required reoperation or computer tomographic-directed drainage, compared with 22 in the placebo group. Only 10 patients in the IFN group developed these infections after 1 week, versus 19 in the placebo group. There was significant enhancement in both percentages of monocytes that expressed HLA-DR as well as their mean fluorescence intensity in the IFN-treated patients,
1. Uneventful recovery in which only minor infections might occur. 2. Major infections that impacted adversely on the clinical course and prolonged hospital stay. These included bacteremia not associated with central lines, pneumonia, major wound or soft tissue infections, body cavity abscesses or infection, and the need for reoperation or percutaneous drainage of collections.
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. Patients who died were grouped together; most of these died of infection. Of the parameters measured, only one, expression of the class II major histocompatibility cell surface antigen HLA-DR on peripheral blood monocytes, correlated consistently with clinical outcome with regard to development of major infection and death from such infection [7].
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Figure 3, Monocyte HLA-DR expression with and without iipopolysaccharide (LPS) incubation and its relation to clinical outcome in 21 patients with severe infection. There was significant difference in the LPS groups between those who survived and those who died. (Reprinted with permission from [9].) VOLUME 165 NO. 2A (SUPPL)
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Figure 4. Density of monocyte HLA-DR expression declines toward the placebo level on cessation of interferon--/therapy in those patients who recovered without major infection. (Reprinted with permission from [27].) which rapidly declined after the drug was withdrawn (Figure 4). A second study of 416 patients from nine centers demonstrated significant reduction in death and major infection in blunt trauma patients (D.J. Dries, unpublished data, 1991). However, most of the benefit occurred in one center. A further trial focusing on those patients most likely to benefit with a longer duration of treatment will be necessary to prove the efficacy of IFN-~ in this clinical setting. THE OUTCOME PREDICTIVE SCORE
An important clinical goal, of course, is reduction in infectious morbidity and mortality. Identification of such patients at high risk is imperative for them to be entered into trials of agents designed to reduce such risk. We developed an outcome predictive score that has been found to be quite reliable in the prediction of major infection following major trauma [28]. The score was reasonably specific in that few patients with low outcome predictive scores developed infection. Most trauma scores have been constructed to predict immediate mortality from hemorrhage and head injury or to compare the severity of illness among patients, but not to assess risk of infectious morbidity and mortality. We constructed the outcome predictive score based on age, severity of injury as calculated by the injury severity score, amount of bacterial contamination, and percentage of monocytes that expressed H L A - D R within 24 hours of admission. In our initial study of 61 trauma patients, the mean score of each clinical outcome group was significantly different from the other within 24 hours of hospitalization. The score was again calculated and its validity further tested in the first clinical trial of IFN--y. In the placebo cohort of patients, the outcome predictive score on day 1 was not helpful in predicting those patients who were at risk for infection, but by day 3 it was highly predictive and reasonably specific. It may well be that utilization of the outcome predictive score to enter patients into clinical trials 48-72 hours after injury is more appropriate. This would allow more accurate
80S
prognosis of severe head injuries, so those with fatal injuries are not entered, and one could begin administration of agents aimed at immune restoration following the initial resuscitation period. This would be applicable to any immunomodulatory agent but, of course, most relevant to those known to enhance D R expression. CONCLUSION The H L A system is becoming increasingly associated with a wide variety of disease, indicating that constitutional genetic abnormalities predispose to risk of acquiring disease. There appear to be individuals with low monocyte H L A - D R expression who are indeed at increased risk of infection because of this inherent abnormality. H L A - D R expression and many other components of the immune response have been found to be depressed after major injury. The strong correlation between monocyte H L A - D R expression and clinical outcome with respect to infection suggests that restoration of such expression may help prevent or abrogate the severity of infection. Only one agent known to enhance H L A - D R expression, IFN-% has been extensively studied. Although promising in selected patients, further work will be needed to define its role. Proper conduct of such trials requires that a truly at-risk population be studied; monocyte H L A - D R expression will help to identify such patients at high risk for infection following major injury. Because there is good correlation between Ia expression and antigen presentation experimentally, agents that enhance the latter should hold promise for future clinical trials. REFERENCES
1. Baker CC, Oppenheimer, Lewis FR, et al. The epidemiology of trauma deaths. Am J Surg 1980; 140: 144-50. 2. Goris RJA, Draaisma J. Causes of death after blunt trauma. J Trauma 1982; 22: 141-6. 3. McDevitt HO. Current concepts in immunology: regulation of the immune response by the major histocompatibility system. N Engl J Med 1980;303: 1514-7. 4. Unanue ER, Beller DI, Lu CY et al. Antigen presentation: comments on its regulation and mechanism. J Immunol 1984; 132: 1-7. 5. Harding CV, Unanue ER. Cellular mechanisms of antigen processing and the function of class I and II major histocompatibility complex molecules. Cell Regul 1990 1: 499-509. 6. Grusby MJ, Johnson RS, Papaioannou VE, Glimcher LH. Depletion of CD4 + T Cells in major histocompatibility complex class II-deficient mice. Science 1991;253: 1417-20. 7. Polk HC Jr, George CD, Wellhausen SR, et al. A systematic study of host defence processes in badly injured patients. Ann Surg 1986; 204: 282-99. 8. Todd RF III, Nadler LM, Schlossman SF. Antigens on human monocytes identified by monoclonal antibodies, J Immunol 1981; 126: 1435-42. 9. Cheadle WG, Hershman MJ, Wellhausen SR, Polk HC Jr. HLA-DR expression on peripheral blood monocytes correlates with surgical infection. Am J Surg 1991; 161: 639-45. 10. Ayala A, Perrin MM, Chaudry IH. Defective macrophage antigen presentation following haemorrhage is associated with the loss of MHC class II (Ia) antigens. Immunology 1990; 70: 33-9. I 1. Ertel W, Meldrum Dr, Morrison MH, et al. Immunoprotec-
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rive effect of a calcium channel blocker on macrophage antigen presentation function, Ia expression and interleukin-1 synthesis following hemorrhage. Surgery 1990; 108; 154-60. 12. Volk H-D, Koyka I, Goan S-R, et al. Serum-mediated inhibition of the interferon-gamma-induced HLA-DR expression on monocytes in patients with psoriasis. J Invest Dermatol 1986; 87: 524-7. 13. Shirakawa F, Yamashita U, Suzuki H. Decrease in HLA-DRpositive monocytes in patients with systemic lupus erythematosus (SLE). J Immunol 1985; 134: 3560-2. 14. Tweardy DJ, Schacter BZ, Ellner JJ. Association of altered dynamics of monocyte surface expression of human leukocyte antigen DR with immuno-suppression in tuberculosis. J Infect Dis 1984; 149: 3l-7. 15. Nagai H, Fisher RI, Cossman J, Oppenheim JJ. Decreased expression of class II major histocompatibility antigens on monocytes from patients with Hodgkin's disease. J Leukocyte Biol 1986; 39: 313-21. 16. De Pr6val C, Hadam MR, Mach B. Regulation of genes for HLA class II antigens in cell lines from patients with severe combined immunodeficiency. N Engl J Med 1988; 318: 1295-300. 1 7. Heagy W, Kelley VE, Strom TB, et al. Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome: increased expression with interferon-gamma. J Clin Invest 1984; 74: 2089-96. 18. Faist E, Mewes A, Strasser T, et al. Alteration of monocyte function following major injury. Arch Surg 1988; 123: 287-92. 19. Faist E, Kupper T, Baker CC, et al. Depression of cellular immunity after major injury: its association with post-traumatic complications and its reversal with immunomodulation. Arch Surg 1986; 121: 1000-5. 20. Lanser ME, Mao P, Brown G, et al. Serum-mediated depres-
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sion of neutrophil chemiluminescence following blunt trauma. Ann Surg 1985; 202: 111-8. 21. Border JR. Hypothesis: sepsis, multiple systems organ failure, and the macrophage. Arch Surg 1988; 123:285-6 22. Todd RF III, Alvarez PA, Brott DA, Liu DY. Bacterial tipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e. J Immuno11985; 135: 3869-77. 23. Appel SH, Wellhausen SR, Montgomery R, DeWeese RC, Polk HC Jr. Experimental and clinical significance of endotoxindependent HLA-DR expression on monocytes. J Surg Res 1989; 47: 39-44. 24. Gonwa TA, Stobo JD. Differential expression of Ia molecules by human monocytes. J Clin Invest 1984; 74: 859-66. 25. Sonnenfeld G, Meruelo D, McDevitt HO, Merigan TC. Effect of type 1 and type II interferons on murine thymocyte surface antigen expression: induction or selection? Cell Immunol 1981; 57: 427-39. 26. Hershman MI, Appel SH, Wellhausen SR, et al. Interferongamma treatment increases HLA-DR expression on monocytes in severely injured patients. Clin Exp Immuno11989; 77: 67-70. 27. Polk HC Jr, Cheadle WG, Livingston DH, et al. A randomized prospective clinical trial to determine the efficacy of interferon--r in severely injured patients. Am J Surg 1992 163:191-96 28. Hershman MJ, Cheadle WG, George CD, Kuftinec D, Polk HC Jr. An outcome predictive score for sepsis and death following trauma. Injury 1988; 19: 263-6. 29. Sinha AA, Lopez MT, McDevitt HO. Autoimmune diseases: the failure of self-tolerance. Science 1990; 248: 1380-7. 30. Hershman MJ, Cheadle WG, Wellhausen SR, Davidson PF, and Polk HC Jr. Br J Surg 1990; 77: 204-7.
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