J Neurosurg75:766-773, 1991
Impairment of helper T-cell function and lymphokineactivated killer cytotoxicity following severe head injury KEITH B. QUATYROCCHI, M.D., EDMUND H. FRANK, M.D., CLARAMAE H. MILLER, PH.D., ASIM AMIN, M.D., PH.D., BERNARDO W. ISSEL, AND FRANKLIN C. WAGNER JR., M.D. Departments of Neurosurgery and Pathology, University of California Davis Medical Center, Sacramento, and Department of lmmunology, University of California, Davis, California t,, Infection is a major complication of severe head injury, occurring in 50% to 75% of patients who survive to hospitalization. Previous investigations of immune activity following head injury have demonstrated suppression of helper T-cell activation. In this study, the in vitro production of interferon-gamma (INF-3,), interleukin-1 (IL-1), and interleukin-2 (IL-2) was determined in 25 head-injured patients following incubation of peripheral blood lymphocytes (PBL's) with the lymphocyte mitogen phytohemagglutinin (PHA). In order to elucidate the functional status of cellular cytotoxicity, lymphokine-activated killer (LAK) cell cytotoxicity assays were performed both prior to and following incubation of PBL's with IL-2 in five patients with severe head injury. The production of INF-7 and IL-2 by PHA-stimulated PBL's was maximally depressed within 24 hours of injury (p < 0.001 for INF-% p = 0.035 for 1L-2) and partially normalized within 21 days of injury. There was no change in the production of IL-1. When comparing the in vitro LAK cell cytotoxicity of PBL's from headinjured patients and normal subjects, there was a significant depression in LAK cell cytotoxicity both prior to (p = 0.010) and following (p < 0.001) incubation of PBL's with IL-2. The results of this study indicate that IL-2 and INF--r production, normally required for inducing cellmediated immunity, is suppressed following severe head injury. The failure of IL-2 to enhance LAK cell cytotoxicity suggests that factors other than decreased IL-2 production, such as inhibitory soluble mediators or suppressor lymphocytes, may be responsible for the reduction in cellular immune activity following severe head injury. These findings may have significant implications in designing clinical studies aimed at reducing the incidence of infection following severe head injury.
KEY WORDS '
head injury
9 immunity
EARLY 500,000 cases of head trauma occur annually in the United States. 23'5~'59Of the 80,000 patients with moderate to severe head injury, the incidence of infection is 50% to 75%, accounting for 40,000 to 60,000 infectious complications annually. 23'32'53Sepsis, as a cause of mortality, occurs in 10% to 25% of cases, even in the absence of significant systemic injury. 3~ Despite this, the effect of severe head injury on immune function has only recently been investigated and, in many studies, the degree of associated systemic injury has not been described.~7'18'32'53'67 The importance of evaluating immune function in patients with head injury in the absence of systemic injury has been underscored by neuroimmunological reports on animal models, which have demonstrated central nervous system modulation of cellular immune function.6'~4'~5.2z24,34.35.56
N
766
9 cytotoxicity
9 interferon
9 interleukin
In a previous study, we have shown that severe head injury in the absence of significant systemic injury results in anergy to delayed-type hypersensitivity skin testing; in addition, there is a decrease in the expression of helper T-cell activation markers, such as interleukin2 (IL-2) receptors, following peripheral blood lymphocyte (PBL) incubation with the lymphocyte mitogen, phytohemagglutinin (PHA)? 3 These findings were confirmed in a report by Hoyt, eta[. 32 The purpose of this study was to further investigate the effects of severe head injury on cellular immune function. Specifically, we investigated the effect of severe head injury on: l) the ability of PBL's from headinjured patients to produce specific cytokines when stimulated in vitro with the T-cell mitogen PHA, and 2) the ability of PBL's from head-injured patients to elicit a cytotoxic response upon stimulation with IL-2.
J, Neurosurg. / Volume 75/November, 1991
Immunosuppression after severe head injury TABLE 1 Admission clinical data and operativetreatment Factor* Finding age mean 24 yrs range 18-54 yrs admission GCS score mean 7 range 4-8 mode of injury motor-vehicleaccident 85% gunshot wound t 5% admission CT findings epidural hematoma 10% subdural hematoma 40% cerebralcontusion 25% intracerebralhemorrhage 10% gunshot wound 15% operativeprocedure evacuationof hematoma 15% partial lobectomy 30% debridement 10% ventriculostomy 10% intracranial pressuremonitor 20% none 15% * GCS = GlasgowComaScale;CT = computerizedtomography.
In vitro PHA-stimulated production of the cytokines interleukin-1 (IL-1) (primarily produced by monocytes) or IL-2 and interferon-gamma (INF--y) (both primarily produced by activated T cells) has been used to assess cellular immune function in multiple traumatic injury and in a variety of disease states with immune dysfunction. t-4'vHO'20'33,36`45,66'68 In vitro measurements of cellular cytotoxicity, such as lymphokine-activated killer (LAK) cell cytotoxicity, have been used to determine cytotoxic competence in a number of immune disorders. 16.33-35.45.66.68By definition, LAK cell cytotoxicity is generated when PBL's, stimulated with IL-2, result in lysis of natural killer (NK) cell-resistant ceUs. 16'34'35'54' s~,6o-~2To our knowledge, the relationship between severe head injury and alterations in either in vitro PBL cytokine production or LAK cell cytotoxicity has not been previously investigated. The results of this study indicate that IL-2 and INF3' production, normally required for inducing cell-mediated immunity, is suppressed following severe head injury. The failure of IL-2 to enhance LAK cell cytotoxicity suggests that factors other than decreased IL-2 production, such as inhibitory soluble mediators or suppressor lymphocytes, may be responsible for the reduction of cellular immune activity following severe head injury. Materials and Methods Patient Selection Between August 1, 1989, and June 30, 1990, 20 men and five women with a mean age of 24 years (range 18 to 54 years) with severe head injury were prospectively investigated. Twenty healthy men and five healthy J. Neurosurg. / Volume 75 / November, 1991
women with a mean age of 28 years (range 18 to 50 years) were used as control subjects. Criteria for admission to the study were an admission Glasgow Coma Scale 63 score of less than 9 following closed head injury (85% of patients) or gunshot wound to the brain (15% of patients). Patients with significant systemic injury, defined by an Injury Severity Scale I ~,~3score of greater than 27, were excluded. Patients with spinal cord injury, posttraumatic hypotension, or anoxia were also excluded, as were patients with risk factors for acquired immunodeficiency syndrome, with a history of aspirin or nonsteroidal anti-inflammatory medication use, chemotherapy, radiation therapy, or malignancy. Patients treated with corticosteroids and those requiring blood transfusions were also excluded from the study. On admission, all patients were intubated for hyperventilation: 10 patients received mannitol (0.5 gm/kg intravenous loading dose) and 14 received Dilantin (phenytoin, 15 mggkg intravenous loading dose). The admission clinical data are summarized in Table 1. Analysis of variance with multiple comparisons was used to evaluate the data. Significance was defined by a probability of greater than 95%. The study was evaluated and approved by the Human Subject Review Committee at the University of California, Davis, Medical Center, Sacramento, California. Isolation of Peripheral Blood Lyrnphocytes Peripheral blood lymphocytes were isolated as described by Miller and Levy?2 Heparinized peripheral venous blood was diluted in phosphate-buffered saline and the PBL's were isolated by gradient centrifugation. Cytocentrifuge preparations were stained with WrightGiemsa stain and evaluated to determine the percent of mononuelear cells. Viability of the PBL's was determined by trypan blue exclusion at this point and throughout the study. Mitogen Incubation
]'he PBL's were incubated in the presence and absence of PHA as described by Hornicek, et al. 31 Lymphocytes (10 6 cells/ml) were incubated for 72 hours at 37~ in 10% CO2 in culture medium alone or in culture medium containing PHA, 4 pg/ml.* Culture medium consisted of RPMI 1640 with 15% fetal calf serum, 25 mM HEPES buffer, 1 mM L-glutamine, 100 U/ml of penicillin, and 100 pg/ml of streptomycin. Following incubation, the cells were washed and viability was determined. Cytokine Production
Cytokine production was determined following incubation of PBL's for 72 hours in the presence of PHA, as described above. The supernatants were obtained by centrifugation and frozen at -80~ for 3 months prior to analysis. * Culture medium supplied by Sigma Chemical Co., St. Louis, Missouri. 767
K. B. Quattrocchi, et al. InterJi, ron-3, Production. Levels of INF-~r were determined in duplicate by solid-phase radioimmunoassay as described by Woloszczuk. 65 Sample or standard controls were incubated in microtiter wells containing a polystyrene bead coated with monoclonal antibodies specific for human INF-'r.t The wells were then counterstained with monoclonal (L251)-anti-INF-3,.~ The polystyrene beads were washed and gamma emissions were determined. To investigate whether any change in the production of INF--y could be due to the decrease in the absolute number of helper T cells, INF-'r levels were determined following incubation of PHA-stimulated PBL's from head-injured patients at a concentration of 1 • 10 6 and 2 • 10 6 cells/mE Interleukin-1 Production. Measurements of IL-1/3 (beta subunit of IL- 1) levels were made by competitive ~2~I-radioimmunoassay as described by Falkoff, et al. 21 Duplicate sample or standard controls were incubated with anti-IL- 1/3 antibody, after which (~25I)-IL-1/3tracer was added.w Anti-immunoglobulin G antibody covalently bound to magnetic particles was centrifuged and the supernatant gamma emissions were determined. Interleukin-2Production. Duplicate IL-2 levels were determined by an enzyme-linked immunoassay as described by Moriya, et al. 44 Sample or standard controls were incubated in microtiter wells precoated with monoclonal anti-IL-2 antibody, and IL-2 antibody was added.ll Horseradish peroxidase-conjugated immunoglobulin, which bound to the immunoglobulin/IL-2 complex, was added followed by O-phenylenediamine (enzyme substrate); the absorbance of the wells at 490 nm was then determined. Determination of L A K Cytotoxicity Preparation of Effector Cells. Isolated PBL's were adjusted to 5 • 106 PBL/ml and incubated with 500 U/ ml of recombinant IL-2* for 72 hours at 37~ in 5% CO2. The cells were washed and adjusted to a concentration of 5 • 106 PBL/ml. Preparation of Target Cells. Lymphokine-activated killer cell target cell lines were prepared as described by Klimpel, et al. 37 Raji cells, a human NK cell-insensitive Burkitt's lymphoma cell line, were maintained in long-term suspension cultures. The Raji target cells were labeled with 5~Crby incubating 2 • 10 6 Raji cells with 150 tzCi of 5~Cr in 200 ul of culture medium for 2 hours at 370C and 5% CO2. The cells 1"Monoclonal antibody-coated beads manufactured by Centocor, Inc., Malvern, Pennsylvania. ~tMonoclonal (t25I)-anti-INF--rsupplied by Centocor, Inc., Malvern, Pennsylvania. wAnti-IL-l~ antibody and (~25I)-IL-lt/tracersupplied by Advanced Magnetics, Inc., Cambridge, Massachusetts. II Monoclonal anti-IL-2 and IL-2 antibody supplied by Advanced Magnetics, Inc., Cambridge, Massachusetts. * Recombinant IL-2 supplied by Cetus Corp., Emeryville, California. 768
were then washed, viability was determined, and the cells were adjusted to a concentration of 5 x 104 Raji cells/ml. L ymphokine-Activated Killer Cell Cytotoxicity Assay. The LAK cell cytotoxicity was determined in a standard 4-hour 5~Cr release microcytotoxicity assay as described by Klimpel, et al. 37The assay was performed with PBL's from normal subjects and head-injured patients both prior to and following incubation with Ig-2. In either instance, the PBL's were incubated with 51Cr-labeled Raji cells at an effector:target ratio of 100:1. The total release of 5~Cr was determined by incubating 5~Cr-labeled Raji cells with 0.1 N HCI, and the spontaneous release was determined by incubating 5~Cr-labeled Raji cells with culture medium. The microtiter plates were incubated for 4 hours at 37~ and 5% CO2 and the supernatants were evaluated for gamma emissions. The percent of cytotoxicity was determined by the following formula: (ER - SR)/(TR - SR) x 100, where ER = experimental release (supernatant from radiolabeled Raji cells incubated with the PBL sample from a normal subject or head-injured patient), SR = spontaneous release (supernatant from Raji cells incubated with culture medium), and TR = total release (supernatant from Raji cells incubated with 0.1 N HC1). All assays were performed in triplicate. Criteria for Assessment of Infections The hospital day of clinical diagnosis of infection, site of infection, and primary agen t of infection were recorded for all patients. Pneumonia was defined by the presence of fever, leukocytosis, new infiltrates on chest roentgenograms, diagnostic Gram stains, and cultures following bronchoscopy. Sinusitis was diagnosed by the presence of fever, leukocytosis, purulent nasal drainage with new opacification of the involved sinus on sinus series roentgenograms or computerized tomography scans, diagnostic Gram stains, and cultures. The diagnosis of meningitis was made in cases of fever, leukocytosis, typical clinical signs with low glucose levels in the cerebrospinal fluid (CSF), and diagnostic CSF cultures. Septicemia was diagnosed by the presence of fever, leukocytosis, unstable vital signs with a high cardiac output index and low systemic vascular resistance, and diagnostic blood cultures. Results
Peripheral Blood Lymphocyte Production of INF-'y, IL-1, and IL-2 The INF-'r, IL-1, and IL-2 production in vitro by PHA-stimulated PBL's from head-injured patients phlebotomized within 24 hours of injury and from normal subjects is outlined in Table 2. On the 1st day following severe head injury there was an eightfold decrease in the ability of PBL's from head-injured patients to produce INF--y (p < 0.001). Although there was no significant change in the production of IL-1, J. Neurosurg. / Volume 75/November, 1991
Immunosuppression after severe head injury TABLE 2 In Vitro production of INF-'r, 1L-l, and IL-2 following PHA incubation of PBL's* Factor
INF-7 (U/ml) 20 102 4- 27 845 4- 48
IL-1 (pg/0.1ml) 20 470 4- 77 510 4- 75
IL-2 (pg/0.1 ml) 10 6 4- 4 241 4- 80
no. tested head-injuredpatients normal subjects statisticalanalysis p value < 0.00It 0.732 0.035 F-testvalue 172.58~ 0.128 5.221 * Peripheral blood lymphocyteswere obtained from head-injured patients and normal subjects.All patients were phlebotomizedwithin 24 hours of head injury. INF-3,= interferon-v,IL-1 and 2 = interleukin-I and 2; PHA = phytohemagglutinin.Mean values are expressed 4- standard errors of the mean. t Analysis&variance with multiplecomparisons,comparingheadinjured patients to normal subjects:significanceat 95%. z~Scheffi F-test values:significanceat 95%.
there was a significant decrease in the production of IL-2 (p = 0.035). The time course for the decrease in PHA-stimulated INF-y and IL-2 production is illustrated in Fig. 1. The greatest depression in both INF-3' and IL-2 production occurred within 24 hours of injury. At 3 weeks following injury, the PHA-stimulated in vitro production of INF-~, and IL-2 had not returned to normal. In a previous study, we have shown that the in vitro expression of CD4 and IL-2 receptors following PHA stimulation decreased by approximately 30% to 50% following head injury? 3 We therefore investigated the production of INF-3, in PHA-stimulated PBL cultures containing 2 x 106 PBL/ml in order to investigate the possibility that the decreased production of INF-3, was an artifact of the reduced absolute number of helper T cells. The results of this experiment, illustrated in Table 3, indicate that the reduced production of INF-3, was due to factors other than a reduction in the percent of activated helper T cells present in the culture.
TABLE 3 Production of lNF-3,from PHA-stimulated PBL incubated at two concentrations* Subjects & Culture
INF-3, (U/ml)
19Valuer
normal subjects 845 + 48 NA (1 X 10 6 PBL/ml) head-injuredpatients 1 • 106 PBL/ml 102 _+27 < 0.001 2 x 106 PBL/ml 110 _+85 < 0.001 * INF--y = interferon-v; PHA = phytohemagglutinin;PBL = peripheral blood lymphocytes;NA = not applicable.Mean values are expressed _ standard errors of the mean. t Analysisof variancewith multiplecomparisons,comparingPHAstimulated INF-3,production from normal subjectsto both cultures from head-injuredpatients.
There was no significant change in the depression in IL-2 or INF-7 production when factors such as the timing of phlebotomy (prior to or following surgery) or treatment with mannitol or Dilantin were considered (p > 0.05). L ymphokine-Activated Killer Cytotoxicity The effects of severe head injury on L A K cell cytotoxicity are illustrated in Table 4. In vitro viability of both target and effector cells was always greater than 95%. Within 24 hours of severe head injury there was a significant decrease in spontaneous L A K cell cytotoxicity (prior to IL-2 incubation). The mean percent of target cell lysis prior to IL-2 incubation was 11% for normal subjects compared to 3% for head-injured patients (p = 0.010). Following incubation with IL-2 there was an expected increase in L A K cell cytotoxicity in PBL's from all control subjects (average increase in target cell lysis of 21%). In contrast, there was no significant change in L A K cell cytotoxicity when PBL's of severely head-injured patients were incubated with IL-2 (average increase in target cell lysis of only 0.2%).
FIG. 1. Time course of in vitro phytohemagglutinin (PHA)-stimulated production by peripheral blood lymphocytes of interferon-~, (left) and interleukin-2 (right) in patients following severe head injury. All data are presented as mean + standard error of the mean (SE). n = number of cases studied. J. Neurosurg. / Volume 75/November, 1991
769
K. B. Quattrocchi, et al. TABLE 4 LAK cytoloxicity prior to and/hi[owing incuhali(m with interleukim 2 (IL- 22*
Subject
control group 1 group 2 group 3 group 4 group 5 mean patient group 1 group 2 group 3 group4 group 5 mean statistical analysis:~ p value F test
LAK Cytotoxicity Before Incubation With IL-2t
LAK Cytotoxicity After Incubation With IL-2"~
Change in LAK Cytotoxicity
3 2 1 2 3 2
26 + 3 28 ,+ 2 30 _+ 3 42 _+ 2 31 + 3 31_+3
+7 +16 +27 +32 +22 21 _+4
5 ,+ 2 0 5 .+ 1 1 +0 3 .+ 1 3 ,+ 1
3 _+ I 3 _+ I 0 6_+2 9 _+ 2 4 + 1
-2 -3 -5 +5 +6 0.2 _+ 2
19 • 12 ,+ 3 .+ 10 ,+ 9 .+ 11 ,+
0.010 10.50
< 0.001 73.25
0.003 17.77
* Lymphokine-activated killer (LAK) cytotoxicity was determined by standard 4-hour 5~Cr microassay release. All patients were phlebotomized within 24 hours of injury. Mean values are expressed _+ standard errors of the mean. t Data are expressed as %lysis. :~Analysis of variance with multiple comparisons; Scheffe F-test values included. Significance was set at 95%.
The mean percent of target cell lysis following IL-2 incubation for head-injured patients and control subjects was 4% and 31%, respectively (p < 0.001).
Infectious Complications The overall rate of infection was 65%, with pneumonia representing the majority of infections (Table 5). The most common isolates were Staphylococcus aureus (30%) and Haemophilus influenzae (15%). Sepsis was the cause of death in one case. Discussion
Cytokine Production Infection remains a frequent and important complication of severe head injury. Data from animal model studies indicate that central nervous system perturbations adversely affect the immune r e s p o n s e . 6'j4'15'22'24' 3o,34,35.53.56However, in man the relationship between immune response status and the incidence of infection following head injury has not been well documented. 5,17,27,50,67 Earlier investigations have demonstrated a decrease in the number of circulating T cells and anergy to delayed-type hypersensitivity skin testing following severe head injury; however, these did not describe the degree of concomitant systemic injury. 17,67Recent studies have demonstrated that severe head injury, in the 770
TABLE 5 ln~'ctious complications* Infection
% of Cases
pneumonia tracheobronchitis meningitis sinusitis septicemia none
~5% 5% 5% 5% 5% 35%
Infectious Agents
Staph.vlococcusaureus ftaemophilus influenzae Enterococcus species Escherichia coli Propionibacterium species none
% of Cases 30% 15% 10% 5% 5% 35%
* Twenty-five patients were evaluated.
absence of significant systemic injury, results in suppression in the ability of PHA-stimulated PBL's to express classic signs of lymphocyte activation, such as deoxyribonucleic acid production and IL-2 receptor expression. 3z53 This study was therefore designed to further elucidate the immunocompetence of patients sustaining severe head injury (in the absence of significant systemic injury) by assessing the in vitro production of lymphokines and IL-2-enhanced cellular cytotoxicity. Depression of in vitro PHA-stimulated PBL lymphokine production has been used to document defects in cellular immunity in a variety of disease states that adversely affect immune function. 8'92125'36'38'39"41"44'45" 49,52,57.5~.6~,68We have found that severe head injury in the absence of significant systemic injury results in a decrease in the in vitro ability of PBL's to produce INF-3, and IL-2 when stimulated with PHA. The ability of mononuclear cells (presumable monocytes) to produce IL-1 following in vitro stimulation with PHA does not appear to be affected. These findings, in conjunction with previous studies delineating suppression of helper T-cell activation, suggest that severe head injury results in a helper T-cell functional defect. This defect does not appear to resolve within 3 weeks of injury, as opposed to the defect in helper T-cell activation (expression of IL-2 receptors), which normalizes within 3 weeks of injury. 32's3
Cellular Cytotoxicity Lymphokine-activated killer cell cylotoxicity has been used to quantify functional deficits in cellular immunity in a variety of diseases.33'36'4s'66"68 In this assay, IL-2-stimulated PBL's (LAK cells) lyse 5~Cr-labeled NK cell-insensitive target cells. 34'3s The population of cytotoxic lymphocytes (LAK cells) mediating this response may be heterogeneous and could include not only activated cytotoxic T cells but also NK cells, monocytes, helper T cells, and B cells. 16 As a result, suppression of LAK cell cytotoxicity may be to some extent mediated by a number of lymphocyte subpopulations and will therefore occur only under conditions adversely affecting multiple mononuclear cell populations. The marked suppression of LAK cell cytotoxicity prior to incubation with IL-2 in head-injured patients J. Neurosurg. / Volume 75/November, 1991
Immunosuppression after severe head injury, in this study was not entirely unexpected in view of the depressed levels of IL-2 and INF-y produced following in vitro stimulation of their PBL's. However, the failure of IL-2 incubation to enhance LAK cell cytotoxicity suggests that factors other than depressed IL-2 production may be responsible for the depression in cellmediated immunity and cytotoxicity following severe head injury. The possible mechanisms involved include lack of production of other soluble mediators, inherent functional defects in LAK cells, or the release or activation of a suppressor lymphocyte.~226.r4 Experiments are currently in progress to define whether specific PBL population(s) or soluble mediator(s) are responsible for the observed suppression in LAK cell cytotoxicity.
Infectious Complications Although classically believed to be most effective against viral and fungal infections, cell-mediated immunity has recently been implicated as playing an important role against bacterial infections other than those caused by bacteria dwelling intracellularly.16"19~37 This response is presumed to occur following activation of helper T cells by an antigen-presenting cell (usually a monocyte), which in turn stimulates the production of IL-2 and INF-y by activated helper T cells. In a classic cell-mediated immune response, these mediators would activate cytotoxic T cells, monocytes, K cells, and NK ceils, which would destroy viral, fungal, and intracellular bacterial pathogens. Several studies, however, have implicated these mechanisms in the destruction of certain nonintracelIular bacterial pathogens, including Gram-negative enteric organisms and Staphylococcus aureus.1937"4~ Other studies indicate that NK cells may play a significant role in this cellmediated response against certain pyogenic and enteric bacterial pathogens. 37'43 In summary, severe head injury results in suppression of helper T-cell function, based on the suppression of in vitro IL-2 and INF-7 production and LAK cell cytotoxicity. The lack of enhancement of in vitro cellular cytotoxicity following incubation of PBL's with IL-2 indicates that factors other than the decrease in helper T-cell IL-2 production may be responsible for the depression in cell-mediated immunity that occurs following severe head injury. This may have significant implications in designing clinical studies aimed at reducing the incidence of infection following severe head injury. It is therefore recommended that further studies be conducted to test for the presence of inhibitory soluble mediators or suppressor lymphocytes. References
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tance in sepsis and trauma. Ann Surg 182:207-217, 1975 4 I. Merrill JT, Slade SG, Weissmann G, et al: Two pathways of CD11B/CD 18-mediated neurophil aggregation with different involvement of protein kinase C-dependent phosphorylation. J Immunol 145:2608-2611, 1990 42. Miller CH, Levy NB: Effects of storage conditions on lymphocyte phenotypes from healthy and diseased persons. J Clin Lab Anal 3:296-300, 1989 43. Morgan DR, DuPont HL, Gonik B, et al: Cytotoxicity of human peripheral blood and colostral leukocytes against Shigella species. Infect Immuno146:25-33, 1984 44. Moriya N, Kato K, Suzuki N, et al: An enzyme-immunoassay for human interleukin-2. J Immunoassay 8: 131-143, 1987 45. Nagel JE, Chopra RK, Chrest FJ, et al: Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutinin-stimulated cells from elderly donors. J Clin Invest 81:1096-1102, 1988 46. Nencioni L, Villa L, Boraschi D, etal: Natural and antibody-dependent cell-mediated activity against Salmonella typhimurium by peripheral and intestinal lymphoid cells in mice. J Immunol 130:903-907, 1983 47. O'Mahony JB, Wood J J, Rodrick ML, et al: Changes in T lymphocyte subsets following injury. Ann Surg 202: 580-586, 1985 48. Palmblad J, Cantell K, Strander H, et al: Stressor exposure and immunological response in man: interferon-producing capacity and phagocytosis. J Psychosom Res 20: 193-199, 1976 49. Palutke M, KuKuruga D, Tabaczka P: A flow cytometric method for measuring lymphocyte proliferation directly from tissue culture plates using Ki-67 and propidium iodide. J Immunol Methods 105:97-105, 1987 50. Pishel IV, Grigorova IA: [Immune response following head injury.] Zh Nevropatol Psikhiatr 84:672-675, 1984 (Rus) 51. Polk HC, WeUhausen SR, Regan MP, et al: A systematic study of host defense processes in badly injured patients. Ann Surg 204:282-299, 1986 52. Prince HE, Arens L, Kleinman SH: CD4 and CD8 subsets defined by dual-color cytofluorometry which distinguish symptomatic from asymptomatic blood donors seropositire for human immunodeficiency virus. Diagn Clin Immunol 5:188-193, 1987 53. Quattrocchi KB, Frank EH, Miller CH, etal: Suppression of cellular immune activity following severe head injury. J Neurotrauma 7:77-87, 1990 54. Raedter A, Fraenkel S, Klose G, et al: Elevated numbers of peripheral T cells in inflammatory bowel diseases displaying T9 antigen and Fc ~ receptors. Clin Exp Immunol 60:518-524, 1985 55. Reinherz EL, Kung PC, Goldstein G, et al: Discrete stages of human intrathymic differentiation: analysis of normal thymocytes and leukemic lymphoblasts of T-cell lineage. Proc Natl Acad Sci USA 77:1588-1592, 1980 56. Roszman TL, Cross R J, Brooks WH, et al: Hypothalamicimmune interactions. Part II. The effect of hypothalamic lesions on the ability of adherent spleen cells to limit lymphocyte blastogenesis. Immunology 45:737-742, 1982 57. Searles RP, Okudaira K, Savage SM, et al: Ia specific antilymphocyte antibodies in rheumatoid arthritis. Arthritis Rheum 26:486-493, 1983 58. Set Y, Tsang PH, Petrella RJ, et al: Multiple dysfunctions in developmental and activational stages of T lymphocytes, B lymphocytes and monocytes in ARC and AIDS
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Immunosuppression after severe head injury patients. Immunol Lett 16:l 57-162, 1987 59. Shires GT, Jones RC, Perry MO, et al: Trauma, in Schwartz SI (ed): Principles of Surgery, ed 3. New York: McGraw-Hill, 1979, pp 219-231 (see pp 219-223) 60. Stelzer GT, Robinson JP: Flow cytometric evaluation of leukocyte function. Diagn Clin Immunol 5:223-231, 1988 61. Stites DP: Clinical laboratory methods for detection of cellular immune function, in Stites DP, Stobo JD, Wells JV (eds): Basic and Clinical Immunology, ed 5. Norwalk, Corm: Appleton & Lange, 1987, pp 293-297 62. Strander H, Cantell K, Leisti J, et al: Interferon response of lymphocytes in disorders with decreased resistance to infections. Clin Exp Immunol 6:263-272, 1970 63. Teasdale G, Jennett B: Assessment of coma and impaired consciousness. A practical scale. Lancet 2:81-84, 1974 64. Teodorczyk-lnjeyan JA, Sparkes BG, Peters WJ: Serum interleukin-2 receptor as a possible mediator of immunosuppression after burn injury. J Burn Care Rehabil 10: 112-118, 1989 65. Woloszczuk W: A sensitive immunoradiometric assay for
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gamma interferon, suitable for its measurement in serum. Clin Chem 31:1090, 1985 (Letter) 66. Yamaguchi E, Okazaki N, Tsuneta Y, et al: Interleukins in pulmonary sarcoidosis. Dissociative correlations of lung interleukin 1 and 2 with intensity of alveolitis. Am Rev Respir Dis 138:645-651, 1988 67. Young AB, Ott LG, Thompson JS, et al: The cellular immune depression of non-steroid treated severely headinjured patients. Neurosurgery 16:722-723, 1985 (Abstract) 68. Zamkoff KW, Reeves WG, Paolozzi FP, et al: Impaired interleukin regulation of the phytohemagglutinin response to Hodgkin's disease. Clin Immunol lmmunopathoi 35:111-124, 1985 Manuscript received October 3, 1990. Accepted in final form April 1, 1991. Address reprint requests to: Keith B. Quattrocehi, M.D., Department of Neurosurgery, 2516 Stockton Boulevard, Sacramento, California 95816.
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Impairment of helper T-cell function and lymphokineactivated killer cytotoxicity following severe head injury KEITH B. QUATYROCCHI, M.D., EDMUND H. FRANK, M.D., CLARAMAE H. MILLER, PH.D., ASIM AMIN, M.D., PH.D., BERNARDO W. ISSEL, AND FRANKLIN C. WAGNER JR., M.D. Departments of Neurosurgery and Pathology, University of California Davis Medical Center, Sacramento, and Department of lmmunology, University of California, Davis, California t,, Infection is a major complication of severe head injury, occurring in 50% to 75% of patients who survive to hospitalization. Previous investigations of immune activity following head injury have demonstrated suppression of helper T-cell activation. In this study, the in vitro production of interferon-gamma (INF-3,), interleukin-1 (IL-1), and interleukin-2 (IL-2) was determined in 25 head-injured patients following incubation of peripheral blood lymphocytes (PBL's) with the lymphocyte mitogen phytohemagglutinin (PHA). In order to elucidate the functional status of cellular cytotoxicity, lymphokine-activated killer (LAK) cell cytotoxicity assays were performed both prior to and following incubation of PBL's with IL-2 in five patients with severe head injury. The production of INF-7 and IL-2 by PHA-stimulated PBL's was maximally depressed within 24 hours of injury (p < 0.001 for INF-% p = 0.035 for 1L-2) and partially normalized within 21 days of injury. There was no change in the production of IL-1. When comparing the in vitro LAK cell cytotoxicity of PBL's from headinjured patients and normal subjects, there was a significant depression in LAK cell cytotoxicity both prior to (p = 0.010) and following (p < 0.001) incubation of PBL's with IL-2. The results of this study indicate that IL-2 and INF--r production, normally required for inducing cellmediated immunity, is suppressed following severe head injury. The failure of IL-2 to enhance LAK cell cytotoxicity suggests that factors other than decreased IL-2 production, such as inhibitory soluble mediators or suppressor lymphocytes, may be responsible for the reduction in cellular immune activity following severe head injury. These findings may have significant implications in designing clinical studies aimed at reducing the incidence of infection following severe head injury.
KEY WORDS '
head injury
9 immunity
EARLY 500,000 cases of head trauma occur annually in the United States. 23'5~'59Of the 80,000 patients with moderate to severe head injury, the incidence of infection is 50% to 75%, accounting for 40,000 to 60,000 infectious complications annually. 23'32'53Sepsis, as a cause of mortality, occurs in 10% to 25% of cases, even in the absence of significant systemic injury. 3~ Despite this, the effect of severe head injury on immune function has only recently been investigated and, in many studies, the degree of associated systemic injury has not been described.~7'18'32'53'67 The importance of evaluating immune function in patients with head injury in the absence of systemic injury has been underscored by neuroimmunological reports on animal models, which have demonstrated central nervous system modulation of cellular immune function.6'~4'~5.2z24,34.35.56
N
766
9 cytotoxicity
9 interferon
9 interleukin
In a previous study, we have shown that severe head injury in the absence of significant systemic injury results in anergy to delayed-type hypersensitivity skin testing; in addition, there is a decrease in the expression of helper T-cell activation markers, such as interleukin2 (IL-2) receptors, following peripheral blood lymphocyte (PBL) incubation with the lymphocyte mitogen, phytohemagglutinin (PHA)? 3 These findings were confirmed in a report by Hoyt, eta[. 32 The purpose of this study was to further investigate the effects of severe head injury on cellular immune function. Specifically, we investigated the effect of severe head injury on: l) the ability of PBL's from headinjured patients to produce specific cytokines when stimulated in vitro with the T-cell mitogen PHA, and 2) the ability of PBL's from head-injured patients to elicit a cytotoxic response upon stimulation with IL-2.
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Immunosuppression after severe head injury TABLE 1 Admission clinical data and operativetreatment Factor* Finding age mean 24 yrs range 18-54 yrs admission GCS score mean 7 range 4-8 mode of injury motor-vehicleaccident 85% gunshot wound t 5% admission CT findings epidural hematoma 10% subdural hematoma 40% cerebralcontusion 25% intracerebralhemorrhage 10% gunshot wound 15% operativeprocedure evacuationof hematoma 15% partial lobectomy 30% debridement 10% ventriculostomy 10% intracranial pressuremonitor 20% none 15% * GCS = GlasgowComaScale;CT = computerizedtomography.
In vitro PHA-stimulated production of the cytokines interleukin-1 (IL-1) (primarily produced by monocytes) or IL-2 and interferon-gamma (INF--y) (both primarily produced by activated T cells) has been used to assess cellular immune function in multiple traumatic injury and in a variety of disease states with immune dysfunction. t-4'vHO'20'33,36`45,66'68 In vitro measurements of cellular cytotoxicity, such as lymphokine-activated killer (LAK) cell cytotoxicity, have been used to determine cytotoxic competence in a number of immune disorders. 16.33-35.45.66.68By definition, LAK cell cytotoxicity is generated when PBL's, stimulated with IL-2, result in lysis of natural killer (NK) cell-resistant ceUs. 16'34'35'54' s~,6o-~2To our knowledge, the relationship between severe head injury and alterations in either in vitro PBL cytokine production or LAK cell cytotoxicity has not been previously investigated. The results of this study indicate that IL-2 and INF3' production, normally required for inducing cell-mediated immunity, is suppressed following severe head injury. The failure of IL-2 to enhance LAK cell cytotoxicity suggests that factors other than decreased IL-2 production, such as inhibitory soluble mediators or suppressor lymphocytes, may be responsible for the reduction of cellular immune activity following severe head injury. Materials and Methods Patient Selection Between August 1, 1989, and June 30, 1990, 20 men and five women with a mean age of 24 years (range 18 to 54 years) with severe head injury were prospectively investigated. Twenty healthy men and five healthy J. Neurosurg. / Volume 75 / November, 1991
women with a mean age of 28 years (range 18 to 50 years) were used as control subjects. Criteria for admission to the study were an admission Glasgow Coma Scale 63 score of less than 9 following closed head injury (85% of patients) or gunshot wound to the brain (15% of patients). Patients with significant systemic injury, defined by an Injury Severity Scale I ~,~3score of greater than 27, were excluded. Patients with spinal cord injury, posttraumatic hypotension, or anoxia were also excluded, as were patients with risk factors for acquired immunodeficiency syndrome, with a history of aspirin or nonsteroidal anti-inflammatory medication use, chemotherapy, radiation therapy, or malignancy. Patients treated with corticosteroids and those requiring blood transfusions were also excluded from the study. On admission, all patients were intubated for hyperventilation: 10 patients received mannitol (0.5 gm/kg intravenous loading dose) and 14 received Dilantin (phenytoin, 15 mggkg intravenous loading dose). The admission clinical data are summarized in Table 1. Analysis of variance with multiple comparisons was used to evaluate the data. Significance was defined by a probability of greater than 95%. The study was evaluated and approved by the Human Subject Review Committee at the University of California, Davis, Medical Center, Sacramento, California. Isolation of Peripheral Blood Lyrnphocytes Peripheral blood lymphocytes were isolated as described by Miller and Levy?2 Heparinized peripheral venous blood was diluted in phosphate-buffered saline and the PBL's were isolated by gradient centrifugation. Cytocentrifuge preparations were stained with WrightGiemsa stain and evaluated to determine the percent of mononuelear cells. Viability of the PBL's was determined by trypan blue exclusion at this point and throughout the study. Mitogen Incubation
]'he PBL's were incubated in the presence and absence of PHA as described by Hornicek, et al. 31 Lymphocytes (10 6 cells/ml) were incubated for 72 hours at 37~ in 10% CO2 in culture medium alone or in culture medium containing PHA, 4 pg/ml.* Culture medium consisted of RPMI 1640 with 15% fetal calf serum, 25 mM HEPES buffer, 1 mM L-glutamine, 100 U/ml of penicillin, and 100 pg/ml of streptomycin. Following incubation, the cells were washed and viability was determined. Cytokine Production
Cytokine production was determined following incubation of PBL's for 72 hours in the presence of PHA, as described above. The supernatants were obtained by centrifugation and frozen at -80~ for 3 months prior to analysis. * Culture medium supplied by Sigma Chemical Co., St. Louis, Missouri. 767
K. B. Quattrocchi, et al. InterJi, ron-3, Production. Levels of INF-~r were determined in duplicate by solid-phase radioimmunoassay as described by Woloszczuk. 65 Sample or standard controls were incubated in microtiter wells containing a polystyrene bead coated with monoclonal antibodies specific for human INF-'r.t The wells were then counterstained with monoclonal (L251)-anti-INF-3,.~ The polystyrene beads were washed and gamma emissions were determined. To investigate whether any change in the production of INF--y could be due to the decrease in the absolute number of helper T cells, INF-'r levels were determined following incubation of PHA-stimulated PBL's from head-injured patients at a concentration of 1 • 10 6 and 2 • 10 6 cells/mE Interleukin-1 Production. Measurements of IL-1/3 (beta subunit of IL- 1) levels were made by competitive ~2~I-radioimmunoassay as described by Falkoff, et al. 21 Duplicate sample or standard controls were incubated with anti-IL- 1/3 antibody, after which (~25I)-IL-1/3tracer was added.w Anti-immunoglobulin G antibody covalently bound to magnetic particles was centrifuged and the supernatant gamma emissions were determined. Interleukin-2Production. Duplicate IL-2 levels were determined by an enzyme-linked immunoassay as described by Moriya, et al. 44 Sample or standard controls were incubated in microtiter wells precoated with monoclonal anti-IL-2 antibody, and IL-2 antibody was added.ll Horseradish peroxidase-conjugated immunoglobulin, which bound to the immunoglobulin/IL-2 complex, was added followed by O-phenylenediamine (enzyme substrate); the absorbance of the wells at 490 nm was then determined. Determination of L A K Cytotoxicity Preparation of Effector Cells. Isolated PBL's were adjusted to 5 • 106 PBL/ml and incubated with 500 U/ ml of recombinant IL-2* for 72 hours at 37~ in 5% CO2. The cells were washed and adjusted to a concentration of 5 • 106 PBL/ml. Preparation of Target Cells. Lymphokine-activated killer cell target cell lines were prepared as described by Klimpel, et al. 37 Raji cells, a human NK cell-insensitive Burkitt's lymphoma cell line, were maintained in long-term suspension cultures. The Raji target cells were labeled with 5~Crby incubating 2 • 10 6 Raji cells with 150 tzCi of 5~Cr in 200 ul of culture medium for 2 hours at 370C and 5% CO2. The cells 1"Monoclonal antibody-coated beads manufactured by Centocor, Inc., Malvern, Pennsylvania. ~tMonoclonal (t25I)-anti-INF--rsupplied by Centocor, Inc., Malvern, Pennsylvania. wAnti-IL-l~ antibody and (~25I)-IL-lt/tracersupplied by Advanced Magnetics, Inc., Cambridge, Massachusetts. II Monoclonal anti-IL-2 and IL-2 antibody supplied by Advanced Magnetics, Inc., Cambridge, Massachusetts. * Recombinant IL-2 supplied by Cetus Corp., Emeryville, California. 768
were then washed, viability was determined, and the cells were adjusted to a concentration of 5 x 104 Raji cells/ml. L ymphokine-Activated Killer Cell Cytotoxicity Assay. The LAK cell cytotoxicity was determined in a standard 4-hour 5~Cr release microcytotoxicity assay as described by Klimpel, et al. 37The assay was performed with PBL's from normal subjects and head-injured patients both prior to and following incubation with Ig-2. In either instance, the PBL's were incubated with 51Cr-labeled Raji cells at an effector:target ratio of 100:1. The total release of 5~Cr was determined by incubating 5~Cr-labeled Raji cells with 0.1 N HCI, and the spontaneous release was determined by incubating 5~Cr-labeled Raji cells with culture medium. The microtiter plates were incubated for 4 hours at 37~ and 5% CO2 and the supernatants were evaluated for gamma emissions. The percent of cytotoxicity was determined by the following formula: (ER - SR)/(TR - SR) x 100, where ER = experimental release (supernatant from radiolabeled Raji cells incubated with the PBL sample from a normal subject or head-injured patient), SR = spontaneous release (supernatant from Raji cells incubated with culture medium), and TR = total release (supernatant from Raji cells incubated with 0.1 N HC1). All assays were performed in triplicate. Criteria for Assessment of Infections The hospital day of clinical diagnosis of infection, site of infection, and primary agen t of infection were recorded for all patients. Pneumonia was defined by the presence of fever, leukocytosis, new infiltrates on chest roentgenograms, diagnostic Gram stains, and cultures following bronchoscopy. Sinusitis was diagnosed by the presence of fever, leukocytosis, purulent nasal drainage with new opacification of the involved sinus on sinus series roentgenograms or computerized tomography scans, diagnostic Gram stains, and cultures. The diagnosis of meningitis was made in cases of fever, leukocytosis, typical clinical signs with low glucose levels in the cerebrospinal fluid (CSF), and diagnostic CSF cultures. Septicemia was diagnosed by the presence of fever, leukocytosis, unstable vital signs with a high cardiac output index and low systemic vascular resistance, and diagnostic blood cultures. Results
Peripheral Blood Lymphocyte Production of INF-'y, IL-1, and IL-2 The INF-'r, IL-1, and IL-2 production in vitro by PHA-stimulated PBL's from head-injured patients phlebotomized within 24 hours of injury and from normal subjects is outlined in Table 2. On the 1st day following severe head injury there was an eightfold decrease in the ability of PBL's from head-injured patients to produce INF--y (p < 0.001). Although there was no significant change in the production of IL-1, J. Neurosurg. / Volume 75/November, 1991
Immunosuppression after severe head injury TABLE 2 In Vitro production of INF-'r, 1L-l, and IL-2 following PHA incubation of PBL's* Factor
INF-7 (U/ml) 20 102 4- 27 845 4- 48
IL-1 (pg/0.1ml) 20 470 4- 77 510 4- 75
IL-2 (pg/0.1 ml) 10 6 4- 4 241 4- 80
no. tested head-injuredpatients normal subjects statisticalanalysis p value < 0.00It 0.732 0.035 F-testvalue 172.58~ 0.128 5.221 * Peripheral blood lymphocyteswere obtained from head-injured patients and normal subjects.All patients were phlebotomizedwithin 24 hours of head injury. INF-3,= interferon-v,IL-1 and 2 = interleukin-I and 2; PHA = phytohemagglutinin.Mean values are expressed 4- standard errors of the mean. t Analysis&variance with multiplecomparisons,comparingheadinjured patients to normal subjects:significanceat 95%. z~Scheffi F-test values:significanceat 95%.
there was a significant decrease in the production of IL-2 (p = 0.035). The time course for the decrease in PHA-stimulated INF-y and IL-2 production is illustrated in Fig. 1. The greatest depression in both INF-3' and IL-2 production occurred within 24 hours of injury. At 3 weeks following injury, the PHA-stimulated in vitro production of INF-~, and IL-2 had not returned to normal. In a previous study, we have shown that the in vitro expression of CD4 and IL-2 receptors following PHA stimulation decreased by approximately 30% to 50% following head injury? 3 We therefore investigated the production of INF-3, in PHA-stimulated PBL cultures containing 2 x 106 PBL/ml in order to investigate the possibility that the decreased production of INF-3, was an artifact of the reduced absolute number of helper T cells. The results of this experiment, illustrated in Table 3, indicate that the reduced production of INF-3, was due to factors other than a reduction in the percent of activated helper T cells present in the culture.
TABLE 3 Production of lNF-3,from PHA-stimulated PBL incubated at two concentrations* Subjects & Culture
INF-3, (U/ml)
19Valuer
normal subjects 845 + 48 NA (1 X 10 6 PBL/ml) head-injuredpatients 1 • 106 PBL/ml 102 _+27 < 0.001 2 x 106 PBL/ml 110 _+85 < 0.001 * INF--y = interferon-v; PHA = phytohemagglutinin;PBL = peripheral blood lymphocytes;NA = not applicable.Mean values are expressed _ standard errors of the mean. t Analysisof variancewith multiplecomparisons,comparingPHAstimulated INF-3,production from normal subjectsto both cultures from head-injuredpatients.
There was no significant change in the depression in IL-2 or INF-7 production when factors such as the timing of phlebotomy (prior to or following surgery) or treatment with mannitol or Dilantin were considered (p > 0.05). L ymphokine-Activated Killer Cytotoxicity The effects of severe head injury on L A K cell cytotoxicity are illustrated in Table 4. In vitro viability of both target and effector cells was always greater than 95%. Within 24 hours of severe head injury there was a significant decrease in spontaneous L A K cell cytotoxicity (prior to IL-2 incubation). The mean percent of target cell lysis prior to IL-2 incubation was 11% for normal subjects compared to 3% for head-injured patients (p = 0.010). Following incubation with IL-2 there was an expected increase in L A K cell cytotoxicity in PBL's from all control subjects (average increase in target cell lysis of 21%). In contrast, there was no significant change in L A K cell cytotoxicity when PBL's of severely head-injured patients were incubated with IL-2 (average increase in target cell lysis of only 0.2%).
FIG. 1. Time course of in vitro phytohemagglutinin (PHA)-stimulated production by peripheral blood lymphocytes of interferon-~, (left) and interleukin-2 (right) in patients following severe head injury. All data are presented as mean + standard error of the mean (SE). n = number of cases studied. J. Neurosurg. / Volume 75/November, 1991
769
K. B. Quattrocchi, et al. TABLE 4 LAK cytoloxicity prior to and/hi[owing incuhali(m with interleukim 2 (IL- 22*
Subject
control group 1 group 2 group 3 group 4 group 5 mean patient group 1 group 2 group 3 group4 group 5 mean statistical analysis:~ p value F test
LAK Cytotoxicity Before Incubation With IL-2t
LAK Cytotoxicity After Incubation With IL-2"~
Change in LAK Cytotoxicity
3 2 1 2 3 2
26 + 3 28 ,+ 2 30 _+ 3 42 _+ 2 31 + 3 31_+3
+7 +16 +27 +32 +22 21 _+4
5 ,+ 2 0 5 .+ 1 1 +0 3 .+ 1 3 ,+ 1
3 _+ I 3 _+ I 0 6_+2 9 _+ 2 4 + 1
-2 -3 -5 +5 +6 0.2 _+ 2
19 • 12 ,+ 3 .+ 10 ,+ 9 .+ 11 ,+
0.010 10.50
< 0.001 73.25
0.003 17.77
* Lymphokine-activated killer (LAK) cytotoxicity was determined by standard 4-hour 5~Cr microassay release. All patients were phlebotomized within 24 hours of injury. Mean values are expressed _+ standard errors of the mean. t Data are expressed as %lysis. :~Analysis of variance with multiple comparisons; Scheffe F-test values included. Significance was set at 95%.
The mean percent of target cell lysis following IL-2 incubation for head-injured patients and control subjects was 4% and 31%, respectively (p < 0.001).
Infectious Complications The overall rate of infection was 65%, with pneumonia representing the majority of infections (Table 5). The most common isolates were Staphylococcus aureus (30%) and Haemophilus influenzae (15%). Sepsis was the cause of death in one case. Discussion
Cytokine Production Infection remains a frequent and important complication of severe head injury. Data from animal model studies indicate that central nervous system perturbations adversely affect the immune r e s p o n s e . 6'j4'15'22'24' 3o,34,35.53.56However, in man the relationship between immune response status and the incidence of infection following head injury has not been well documented. 5,17,27,50,67 Earlier investigations have demonstrated a decrease in the number of circulating T cells and anergy to delayed-type hypersensitivity skin testing following severe head injury; however, these did not describe the degree of concomitant systemic injury. 17,67Recent studies have demonstrated that severe head injury, in the 770
TABLE 5 ln~'ctious complications* Infection
% of Cases
pneumonia tracheobronchitis meningitis sinusitis septicemia none
~5% 5% 5% 5% 5% 35%
Infectious Agents
Staph.vlococcusaureus ftaemophilus influenzae Enterococcus species Escherichia coli Propionibacterium species none
% of Cases 30% 15% 10% 5% 5% 35%
* Twenty-five patients were evaluated.
absence of significant systemic injury, results in suppression in the ability of PHA-stimulated PBL's to express classic signs of lymphocyte activation, such as deoxyribonucleic acid production and IL-2 receptor expression. 3z53 This study was therefore designed to further elucidate the immunocompetence of patients sustaining severe head injury (in the absence of significant systemic injury) by assessing the in vitro production of lymphokines and IL-2-enhanced cellular cytotoxicity. Depression of in vitro PHA-stimulated PBL lymphokine production has been used to document defects in cellular immunity in a variety of disease states that adversely affect immune function. 8'92125'36'38'39"41"44'45" 49,52,57.5~.6~,68We have found that severe head injury in the absence of significant systemic injury results in a decrease in the in vitro ability of PBL's to produce INF-3, and IL-2 when stimulated with PHA. The ability of mononuclear cells (presumable monocytes) to produce IL-1 following in vitro stimulation with PHA does not appear to be affected. These findings, in conjunction with previous studies delineating suppression of helper T-cell activation, suggest that severe head injury results in a helper T-cell functional defect. This defect does not appear to resolve within 3 weeks of injury, as opposed to the defect in helper T-cell activation (expression of IL-2 receptors), which normalizes within 3 weeks of injury. 32's3
Cellular Cytotoxicity Lymphokine-activated killer cell cylotoxicity has been used to quantify functional deficits in cellular immunity in a variety of diseases.33'36'4s'66"68 In this assay, IL-2-stimulated PBL's (LAK cells) lyse 5~Cr-labeled NK cell-insensitive target cells. 34'3s The population of cytotoxic lymphocytes (LAK cells) mediating this response may be heterogeneous and could include not only activated cytotoxic T cells but also NK cells, monocytes, helper T cells, and B cells. 16 As a result, suppression of LAK cell cytotoxicity may be to some extent mediated by a number of lymphocyte subpopulations and will therefore occur only under conditions adversely affecting multiple mononuclear cell populations. The marked suppression of LAK cell cytotoxicity prior to incubation with IL-2 in head-injured patients J. Neurosurg. / Volume 75/November, 1991
Immunosuppression after severe head injury, in this study was not entirely unexpected in view of the depressed levels of IL-2 and INF-y produced following in vitro stimulation of their PBL's. However, the failure of IL-2 incubation to enhance LAK cell cytotoxicity suggests that factors other than depressed IL-2 production may be responsible for the depression in cellmediated immunity and cytotoxicity following severe head injury. The possible mechanisms involved include lack of production of other soluble mediators, inherent functional defects in LAK cells, or the release or activation of a suppressor lymphocyte.~226.r4 Experiments are currently in progress to define whether specific PBL population(s) or soluble mediator(s) are responsible for the observed suppression in LAK cell cytotoxicity.
Infectious Complications Although classically believed to be most effective against viral and fungal infections, cell-mediated immunity has recently been implicated as playing an important role against bacterial infections other than those caused by bacteria dwelling intracellularly.16"19~37 This response is presumed to occur following activation of helper T cells by an antigen-presenting cell (usually a monocyte), which in turn stimulates the production of IL-2 and INF-y by activated helper T cells. In a classic cell-mediated immune response, these mediators would activate cytotoxic T cells, monocytes, K cells, and NK ceils, which would destroy viral, fungal, and intracellular bacterial pathogens. Several studies, however, have implicated these mechanisms in the destruction of certain nonintracelIular bacterial pathogens, including Gram-negative enteric organisms and Staphylococcus aureus.1937"4~ Other studies indicate that NK cells may play a significant role in this cellmediated response against certain pyogenic and enteric bacterial pathogens. 37'43 In summary, severe head injury results in suppression of helper T-cell function, based on the suppression of in vitro IL-2 and INF-7 production and LAK cell cytotoxicity. The lack of enhancement of in vitro cellular cytotoxicity following incubation of PBL's with IL-2 indicates that factors other than the decrease in helper T-cell IL-2 production may be responsible for the depression in cell-mediated immunity that occurs following severe head injury. This may have significant implications in designing clinical studies aimed at reducing the incidence of infection following severe head injury. It is therefore recommended that further studies be conducted to test for the presence of inhibitory soluble mediators or suppressor lymphocytes. References
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