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Increased levels of circulating interleukin 6 in burn patients
CLINICAL
IMMUNOLOGY
Increased
AND
54, 361-371 (19%)
IMMUNOPATHOLOGY
Levels of Circulating
lnterleukin
6 in Burn Patients
YING Guo,* CAMILLE DICKERSON,* FRANCIS J. CHRITST,~ WILLIAM H. ADLER,? ANDREW M. MUNSTER,* AND RICHARD A. WINCHURCH* *Department of Surgery, Johns Hopkins University School of Medicine, and fClinica1 Immunology Section, National Institute on Aging, Baltimore, Maryland 21224 The serum levels of interleukin 6 (IL-6) were determined in a population of bum patients. In all patients, IL-6 levels were increased over a 3-week interval with peak concentrations reached during the first week after injury. Patients receiving intravenous polymyxin B therapy according to a regimen designed to reduce endotoxemia manifested greatly reduced levels of both circulating endotoxins and IL-6. Certain patients not treated with polymyxin B showed extraordinarily large increases in IL-6 which were associated with lethal or life-threatening clinical complications. Increased IL-6 levels were also associated with decreased percentage of circulating T cells and corresponding increases in B cells. However, IL-6 did not produce any direct inhibitory effects in vitro on T cell representation or function. 0 1990 Academic Press, Inc.
INTRODUCTION
Infectious diseases are a frequent consequence of severe traumatic injury. Following acute thermal injury; opportunistic infections are a leading cause of mortality in those patients who survive the initial phases of injury. The increased incidence of infection following burns is largely due to impairment of the host defense network. While nonspecific defenses such as neutrophil bacteriocidal activity are known to contribute to infectious sequelae in the burn patient, it is generally accepted that failure of specific immune responses plays a crucial role in eventual recovery. Much effort has been devoted toward defining the causes of immunosuppression following traumatic injury. A variety of factors have been described which may contribute to the loss of function. Notable among these factors is bacterial endotoxin. Increases in serum endotoxin levels have been associated with thermal injury and the magnitude of these increases is related to the extent of the injury (1). Endotoxins are associated with immunologic dysfunction (2) and therapy designed to reduce the endotoxin burden improves immune responsiveness in both man (3) and in the laboratory mouse (4). Despite the apparent relationship between endotoxin and immunologic failure, the mechanism(s) by which endotoxins impair function in the burn victim is unknown. Several investigators have studied the production of immunoregulatory factors in the serum of burn victims (5-7). It has been shown that interleukin (IL) 2mediated responses are diminished in burn patients (8) and that burn sera contain increases in soluble, IL-2 receptors (IL-2R) (9). Bum sera also contain factors which inhibit IL-2-induced responses (10) and inhibit lymphocyte proliferation (11). In order to establish whether abnormalities of immune function are due to changes in the levels of endogeneous regulatory factors, we studied the appear361 0090-1229/90 $1.50 Copyright 8 1990 by Academtc Press, lnc All rights of reproduction in any form reserved.
362
GUO
ET AL.
ante of IL-6 in serum from burn patients. The results show that burn sera contain large increases in IL-6 and that treatment with endotoxin-neutralizing regimens of polymyxin B reduces the level of this monokine. The data also show that IL-6 increases are correlated with decreases in the percentage of circulating T cells but do not appear to be causally related to functional changes in T cell activity. Finally, we have observed that large increases in serum IL-6 levels are associated with severe clinical complications and mortality in the bum patients. MATERIALS
AND METHODS
Patient Population Patients entering the Baltimore Regional Bum Center for treatment were admitted to the study. The protocols for the study were approved by the Joint Committee for Clinical Investigation of Johns Hopkins University and F.S. Key Medical Center. Only those patients providing informed consent were entered into the study. At specified intervals after injury, samples of peripheral blood were drawn. Serum, plasma, and peripheral white cells were collected. Serum and plasma samples were aliquoted and frozen at - 20°C until assay. Assays for the expression of lymphocyte phenotypic markers were performed within 6 hr of collection. Samples of blood from healthy volunteers were also collected at the same time and assayed along with patients’ samples. Polymyxin B Treatment Patients were randomized into two groups. In addition to normal therapy, patients in the treatment group were also given polymyxin B according to the following schedule: Day 1,300O units/kg/day; Day 2,400O units/kg/day; Day 3,550O units/kg/day; Day 4,550O units/kg/day; Day 5,400O units/kg/day; and Day 6,300O units/kg/day. The doses were prepared by reconstituting vials of 500,000 units of polymyxin B sulfate in pryrogen-free water and were administered intravenously in two equally divided daily doses. Endotoxin Assay Circulating endotoxin levels were assayed using the chromogenic limulus lysate assay (Whittaker M.A. Bioproducts, Walkersville, MD) according to the instructions supplied by the manufacturer. Briefly, lOO+l volumes of either plasma samples or endotoxin standards were incubated with 50 ~1 of freshly reconstituted limulus lysate in 96-well microtiter plates for 13 min at 37°C. Fifty microliters of diluted substrate was added and the incubation continued for an additional 3 min. The reaction was stopped by the addition of 25% acetic acid and the plates were read at 405 ti. Endotoxin in the plasma samples was corrected for dilution and calculated from a standard curve derived from assay of standards. IL-4 Determination The levels of IL-6 were determined using the B9 assay as described by Aarden (12). The IL-6 dependent B9 cells were obtained from Dr. Rick Nordan, NCI, National Institutes of Health, and were maintained by serial passage in culture.
INTERLEUKIN
6 IN
BURN
PATIENTS
363
For the assay, 10 units of standard IL-6 (Endogen Laboratories, Boston, MA) or serum samples were serially diluted in log, increments in microtiter plates in duplicate. Log-phase B9 cells were added at a final concentration of 1 x lo4 in 5Oql volumes and the plates were incubated for 3 days at 37°C. Tritiate thymidine was added for the final 4 hr of incubation and proliferation was determined by scintillation counts. The data were analyzed by weighted probit analysis using the computer program described by Sette et al. (13). Flow Cytometric
Analysis
Whole blood samples were prepared for cytometric analysis by incubation with ammonium chloride lysing buffer (Ortho Diagnostics, Raritan, NJ) washed with cold PBS and stained with an abbreviated panal of FITC and PE-conjugated monoclonal antibodies. Staining was accomplished by incubation for 30 min at 4°C followed by one wash with staining buffer and a second wash with cold PBS. The cells were fixed in a 1% paraformaldehyde solution and kept at 4°C until analysis. Stained preparations were examined using a FACScan flow cytometer (BectonDickinson Immunocytometry Systems, Mountain View, CA). Lymphocyte gating was accomplished using the leucogate reagent containing FITC-conjugated antiCD45 and PE-conjugated anti-CD14. The percentage and absolute numbers of each cell type examined were determined by computer analysis. Statistical
Analyses
Differences between groups were determined by Student’s t test. The relationships between IL-6 levels and the expression of surface phenotypes were examined by regression analyses. RESULTS
Patient
Population
A total of 21 burn patients were studied. Of these, 7 were treated with polymyxin B in addition to routine postbum therapy. There was no significant difference in the ages or in the degree of injury (total bum surface area) between the control and the polymyxin B-treated groups. Analysis of Endotoxin
Levels
As previously shown, treatment of bum patients with polymyxin B according to the regimen described under Materials and Methods is effective in reducing the endotoxin burden that commonly occurs following thermal injury (14). The profile of endotoxin increases in the control group was roughy bell-shaped with peak endotoxin levels occurring at 3 days postinjury (1). Circulating endotoxins were again increased at 1 week and this increase was sustained for 3 weeks (data not shown). As shown by the data in Table 1, polymyxin B therapy was effective in reducing the levels of circulating endotoxin. Polymyxin B-treated patients showed only small increases in circulating endotoxins, 0.12 + 0.12 units/ml vs 3.5 + 1.08 units/ml for the control group (P < 0.01).
364
GUO ET AL. IL-6 AND ENDOTOXIN
Number of Age Number of Mean peak Mean peak
patients deaths B-6 (units/ml) endotoxin (units/ml)
TABLE
1
LEVELS
IN
BURN PATIENT
SERA
Control
Polymyxin B
P value
14 35 k 4 3 1271 2 692 3.5 5 1.08
I 29 + 4 1 4427 0.12 2 0.12
NS NS <0.05 co.01
Analyses of Circulating Cytokines in the Burn Patient Initially it was observed that burn patient sera contained low but consistent levels of anti-viral activity. In an assay system which measured cytopathic effects of VSV on human embryonic lung cells (MRC 5), this activity was not abrogated by antisera specific for any of the three interferon species. In further attempts to identify the anti-viral factors, sera from both the control and the polymyxin B groups were analyzed for both tumor necrosis factor (TNF a) and IL-6. Using the L929 cytopathic assay, we could not show any evidence of increases in TNF over a 3-week interval. In contrast, all bum patients showed increases in IL-6 levels (Table 1). In the control group the average maximum activity of IL-6 was 1272 units/ml. In contrast, patients treated with polymyxin B showed maximum peak levels of only 44 units/ml. Despite a wide variation in the levels of IL-6 produced by individual patients, the difference between the groups was statistically significant (P = 0.05). The profiles of IL-6 production are illustrated in Fig. 1. The mean peak IL-6 activity in the polymyxin B group occurred on Day 2 and decreased thereafter. By 3 weeks, no serum IL-6 was detectable. In the control group, IL-6 increased to peak levels on Day 6. The mean IL-6 activity for the control patients at Day 6 and the average peak for each individual patient were comparable in magnitude. In contrast to the polymyxin B group the control group showed residual activity of 60 units/ml 21 days after injury. IL-6 as an Indicator
of Morbidity
The levels of IL-6 at all time points were compared in the control and polymyxin B groups (Fig. 2). For comparison, IL-6 levels were assayed in sera from normal volunteers and were below the level of detection of the assay. As indicated in the scatter plot the control group showed significant increases in IL-6 when compared to the polymyxin B group. When the data for the control group were subdivided on the basis of survival, it was apparent that IL-6 levels were much higher in the patients who did not survive. IL-6 and Changes in Lymphocyte Subsets Burn patients suffer from varying degrees of immune impairment. Therefore, it was of interest to determine if increases in IL-6 levels were related to alterations of peripheral blood lymphocyte representation or function. Patients were selected at random and both IL-6 levels and the representation of lymphocyte subsets were
INTERLEUKIN 2000
6 IN BURN PATIENTS
r
36.5
(14)
T m m
1000:
(14)
Control Polymyxin B
I
500400300-
(12)
200-
T
(14) T
100:
z302010: 5432
1
2
3
4
5
6
Days after Burn FIG. 1. Profiles of serum IL-6 levels in control and polymyxin B-treated bum patients t SE are shown for the days indicated. The number of patients assayed on each day are shown in parentheses.
determined. Despite the decreases in the number of circulating lymphocytes in burn patients, there was no apparent relationship between the absolute number or the percentage representation of lymphocytes and the levels of IL-6 (P > 0.2). However, as shown by the data in Fig. 3, there is an inverse relationship between the percentage of T cells (CD3+) and the level of circulating IL-6 (P > 0.01). Conversely, there was a positive correlation between the percentage of B cells (CD19+) and IL-6 (P < 0.01) (Fig. 4). The data in Fig. 5 and 6 indicate that neither helper (CD4 + ) nor suppressor (CD8 + ) cells were predominantly responsible for the decreases in the percentage of CD3-bearing cells. If IL-6 was directly responsible for either an impairment of T cell activity or for a reduction in viable T cells it might be expected that IL-6 would produce a similar effect on normal lymphocytes in vitro. When IL-6 was added to cultures of peripheral blood lymphocytes from normal volunteers and when proliferative responses to both mitogens and recall antigens were measured, there was no effect on either the number of surviving CD3 + cells or in the ability of the cells to respond. Similarly IL-6 did not alter the expression or shedding of IL-2 receptors (IL-2R) and did not interfere with IL-Zinduced proliferation (data not shown).
366
GUO ET AL.
8 0
110
Normals Survivors
Mortalities
Polymyxin B
Control Group FIG. 2. Scatter plot showing IL-6 activity at all time points for control and polymyxin B-treated patients.
DISCUSSION
In this report we describe increases in serum IL-6 levels which are a major consequence of acute thermal injury in man. These data confii the preliminary findings of Nijsten et al. (15) in which IL-6 was shown to be associated with the generation of acute phase reactants in bum patients. The increases in IL-6 are inhibited by treatment of bum patients with polymyxin B. Similarly the increased burden of circulating endotoxins which routinely accompany thermal injury is also
INTERLEUKIN
loo0
6 IN
BURN
367
PATIENTS
I
I
I
I
20
40
60
60
PERCENT
OF TOTAL
LYMPHOCYTES
3. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD3+ T cells. r = -0.606, P = < 0.01. FIG.
inhibited by polymyxin B therapy. This suggests that increases in IL-6 are a result of endotoxin burden. Indeed, a number of reports have shown that endotoxin is a major activator of IL-6 production in a variety of tissues (16, 17). However, endotoxin is known to activate a variety of biological response modifiers, including tumor necrosis factor and interleukin 1, and 11-l is also known to induce increases in IL-6 (18). Currently, it is a matter of conjecture as to whether the increases in IL-6 are a direct consequence of endotoxin or are induced by other mediators such as IL-l. Although polymyxin B may act directly on IL6-producing cells, there is no evidence to support such a direct inhibitory effect. In a number of patients, very large increases in IL-6 were associated with
‘04i /
lo2 I
I.
10’
CDlS+ CELLS
0
0 r
0
p < 0.01
I
I
I
I
12
24
36
46
PERCENT
OF TOTAL
LYMPHOCYTES
FIG. 4. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD19+ B cells. r = 0.652, P = < 0.01.
368
GUO ET AL. 104t
I
I
I
I CD4+ CELLS
n 103F+-
PERCENT
OF TOTAL
LYMPHOCYTES
FIG. 5. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD4+ helper/inducer cells. r = 0.533, P = < 0.01.
life-threatening clinical complications. The profiles of IL-6 increases for three individual patients are illustrated in Fig. 7. Two of these patients showed progressive increases until Day 6 and both patients died on Day 7 (Figs. 7A and 7B). The third patient (Fig. 7C) demonstrated IL-6 increases following an episode of acute respiratory distress (Day 2) and renal failure (Day 3). Other patients also showed increases in IL-6 which were coincident with changes in clinical status. Of these, one patient died 24 hr after an increase of 594 units/ml, while a second patient demonstrated a level of 2360 units/ml 36 hr after suffering cardiac arrest. In the polymyxin B group, one patient died but did not show any extraordinary
J
CELLS
p < 0.05
,000 0
10
PERCENT
20 OF TOTAL
30
40
LYMPHOCYTES
FIG. 6. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD8 + suppressorkytotoxic cells. r = -0.471, P = < 0.05.
INTERLEUKIN
-0
1
369
6 IN BURN PATIENTS
2
3
4
5
6
7
Days after Burn FIG. 7. Profiles of serum IL-6 levels for three control group patients. Clinical complications for each patient are described in the text.
increase in IL-6. IL-6 increases are often coincident with infection (19, 20), but there was no apparent correlation between increases in IL-6 and infectious sequelae in the bum patients. These findings are remarkable in that IL-6 may be either a cause of multiorgan failure or may exacerbate organ failure due to other causes. Alternatively, IL-6 may be a consequence of cell death and, as such, might only serve as a predictor of organ failure. In either case, analyses of serum IL-6 levels may provide a basis for prognosis in bum or trauma victims. However, if IL-6 contributes to multiorgan failure, the use of specific antagonists could provide an important new therapeutic approach. Currently, several potential IL-6 antagonists are being investigated. The mechanism(s) by which IL-6 might contribute to the pathogenesis of burn injury is presently unknown. However, IL-6 is directly responsible for the induction of acute phase protein synthesis and release (21, 22). IL-6 levels in cerebral spinal fluid are increased in patients with bacterial and viral meningitis (19). Similarly, serum IL-6 levels are increased in renal transplant patients shortly after receiving grafts and during episodes of rejection (23). IL-6 may also contribute to the pathogenesis of HIV infection since HIV induces IL-6 synthesis in normal human mononuclear cells (24). In the bum victim, there is no indication that increases in IL-6 are a consequence of infection. Thermal injury is usually associated with varying degrees of immune dysfunc-
GUO ET AL.
tion. When patients are treated with polymyxin B according to the regimen described in this communciation, there is a consistent improvement in the profile of immune responses (3,4). This suggests that endotoxins contribute to the immune dysfunction following burns. Since polymyxin B therapy also abrogates increases in IL-6 levels, we considered the possibility that IL-6 might be causally related to the depressed immune responses. Thus we examined the effects of recombinant IL-6 and bum serum containing large amounts of IL-6 on various parameters of lymphocyte reactivity. Bum sera inhibit the proliferation induced by IL-2 in IL2-dependent CTLL cell lines. However, IL-6 induced no similar effect. Bum sera also contain large concentrations of soluble IL-2R but IL-6 had no effect on either the expression of or rate of shedding of IL-2R. Finally, when the proliferative responses of normal cells to recall antigens were tested in the presence of IL-6, there was no apparent inhibitory effect. In contrast, the data in this report indicate that IL-6 is associated with decreases in the proportion of circulating T cells and corresponsing increases in B cells. Although there are shifts in the subset representation, the percentage of total lymphocytes is unchanged and is independent of IL-6 concentration. The relationship between IL-6 and percentage of T cells is shared equally by both CD4 and CDS. The fact that we could not show any effect of IL-6 on T cell respones suggests that increases in IL-6 and bum-induced immune inhibition are concurrent events induced both directly or indirectly by endotoxin. The question which remains is what is the physiologic role of IL-6 in the bum patient and as an acute phase reactant. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Winchurch, R. A., Thupari, J. N., and Munster, A. M., Surgery 102, 808, 1987. Persson, U., J. Immunol. 118, 78, 1977. Munster, A. M., Winchurch, R. A., Thupari, J. N., and Ernst, C. B., J. Trauma 2.6, 995, 1986. Chiccone, T. G., Munster, A. M., Birmingham, W., and Winchurch, R. A., J. Burn Care Rehabil. 4, 153, 1983. Freeman, T. R., and Shelby, J., J. Trauma 28, 190, 1988. Wolfe, J. H. N., Saporoschetz, I., Young, A. E. O’Connor, N.E., and Mannick, J. A., Ann. Surg. 193, 513-520, 1981. Ozkan, A. N., and Ninnemann, J. L., J. Clin. Immunol. 5, 172, 1985. Teodorczyk-Injeyan, J., Sparkes, B. G., Mills, G. B., Peters, W. J., and Falk, R. E., Clin Exp. Immunol. 65, 570, 1986. Xiao, G.-X., Chopra, R. K., Adler, W. H., Munster, A. M., and Winchurch, R. A., J. Trauma 28, 1669, 1988. Rodrick, M. L., Saporoschetz, I., Wood, J., David, C. F., and Mannick, J. A., Surg. Forum 36, 98, 1986. Munster, A. M., Winchurch, R. A., Birmingham, W., and Keeling, P., Ann. Surg. 192,772, 1980. Aarden, L. A., DeGrot, E. R., Schaap, 0. L., and Lansdorp, P. M., Eur. J. Immunol. 17, 1411, 1987. Sette, A., Adorini, L., Marubini, E., and Doria, G., J. Immunol. Methods 86, 265, 1986. Bender, B. S., Winchurch, R. A., Thupari, J. N., Proust, J. J., Adler, W. H., and Munster, A. M., Clin. Exp. Immunol. 71, 120, 1988. Nijsten, M. W. N., DeGroot, E. R., Ten Duis, H. J., Klasen, H. J., Hack, C. E., and Aarden, L. A., Lancer 2, 921, 1987. Jirik, F. R., Podor, T. J., Hirano, T., Kishimoto, T., Loskutoff, D. J., Carson, D. A., and Lotz, M., .I. Immunol. 142, 144, 1989.
INTERLEUKIN
6 IN BURN PATIENTS
371
17. Fang, Y., Moldawer, L. L., Marano, M., Wei, H., Tatter, S. B., Clarick, R. H., Santhanum, U., Sherris, D., May, L. T., Sehgal, P. B., and Lowry, S. F., J. Immunol. 142, 2321, 1989. 18. Walther, Z., May, L. T., and Sehgal, P., J. Immunol. 140, 974, 1988. 19. Houssiau, F. A., Bukasa, K., Sindic, C. J. M., Van Damme, J., and Van Snick, J., C/in. Exp. Immunol. 71, 320, 1988. 20. Helfgott, D. C., Tatter, S. B., Santhanam, U., Clarick, R. H., Bhardwaj, N., May, L. T., and Sehgal, P. B., .I. Immunol. 142, 948, 1989. 21. Ramadoti, G., Van Damme, J., Rieder, H., and Meyer, K.-H., Eur. J. Immunol. 18, 1259, 1988. 22. Geiger, T., Andus, T., Klaproth, J., Hirano, T., Kishimoto, T., and Heimich, P. C., Eur. J. Immunol. 18, 717, 1988. 23. Van Oers, M. H. J., Van Der Heyden, A. A. P. A. M., and Aarden, L. A., C&n. Exp. Immunol. 71, 314, 1988. 24. Nakajima, K., Martinez-Maza, O., Hirano, T., Breen, E. C., Nishanian, P. G.. Salazar-Gonzalez, J. F., Fahey, J. L., and Kishimoto, T., J. Immunol. 142, 531, 1989. Received July 10, 1989; accepted with revision September 29, 1989
IMMUNOLOGY
Increased
AND
54, 361-371 (19%)
IMMUNOPATHOLOGY
Levels of Circulating
lnterleukin
6 in Burn Patients
YING Guo,* CAMILLE DICKERSON,* FRANCIS J. CHRITST,~ WILLIAM H. ADLER,? ANDREW M. MUNSTER,* AND RICHARD A. WINCHURCH* *Department of Surgery, Johns Hopkins University School of Medicine, and fClinica1 Immunology Section, National Institute on Aging, Baltimore, Maryland 21224 The serum levels of interleukin 6 (IL-6) were determined in a population of bum patients. In all patients, IL-6 levels were increased over a 3-week interval with peak concentrations reached during the first week after injury. Patients receiving intravenous polymyxin B therapy according to a regimen designed to reduce endotoxemia manifested greatly reduced levels of both circulating endotoxins and IL-6. Certain patients not treated with polymyxin B showed extraordinarily large increases in IL-6 which were associated with lethal or life-threatening clinical complications. Increased IL-6 levels were also associated with decreased percentage of circulating T cells and corresponding increases in B cells. However, IL-6 did not produce any direct inhibitory effects in vitro on T cell representation or function. 0 1990 Academic Press, Inc.
INTRODUCTION
Infectious diseases are a frequent consequence of severe traumatic injury. Following acute thermal injury; opportunistic infections are a leading cause of mortality in those patients who survive the initial phases of injury. The increased incidence of infection following burns is largely due to impairment of the host defense network. While nonspecific defenses such as neutrophil bacteriocidal activity are known to contribute to infectious sequelae in the burn patient, it is generally accepted that failure of specific immune responses plays a crucial role in eventual recovery. Much effort has been devoted toward defining the causes of immunosuppression following traumatic injury. A variety of factors have been described which may contribute to the loss of function. Notable among these factors is bacterial endotoxin. Increases in serum endotoxin levels have been associated with thermal injury and the magnitude of these increases is related to the extent of the injury (1). Endotoxins are associated with immunologic dysfunction (2) and therapy designed to reduce the endotoxin burden improves immune responsiveness in both man (3) and in the laboratory mouse (4). Despite the apparent relationship between endotoxin and immunologic failure, the mechanism(s) by which endotoxins impair function in the burn victim is unknown. Several investigators have studied the production of immunoregulatory factors in the serum of burn victims (5-7). It has been shown that interleukin (IL) 2mediated responses are diminished in burn patients (8) and that burn sera contain increases in soluble, IL-2 receptors (IL-2R) (9). Bum sera also contain factors which inhibit IL-2-induced responses (10) and inhibit lymphocyte proliferation (11). In order to establish whether abnormalities of immune function are due to changes in the levels of endogeneous regulatory factors, we studied the appear361 0090-1229/90 $1.50 Copyright 8 1990 by Academtc Press, lnc All rights of reproduction in any form reserved.
362
GUO
ET AL.
ante of IL-6 in serum from burn patients. The results show that burn sera contain large increases in IL-6 and that treatment with endotoxin-neutralizing regimens of polymyxin B reduces the level of this monokine. The data also show that IL-6 increases are correlated with decreases in the percentage of circulating T cells but do not appear to be causally related to functional changes in T cell activity. Finally, we have observed that large increases in serum IL-6 levels are associated with severe clinical complications and mortality in the bum patients. MATERIALS
AND METHODS
Patient Population Patients entering the Baltimore Regional Bum Center for treatment were admitted to the study. The protocols for the study were approved by the Joint Committee for Clinical Investigation of Johns Hopkins University and F.S. Key Medical Center. Only those patients providing informed consent were entered into the study. At specified intervals after injury, samples of peripheral blood were drawn. Serum, plasma, and peripheral white cells were collected. Serum and plasma samples were aliquoted and frozen at - 20°C until assay. Assays for the expression of lymphocyte phenotypic markers were performed within 6 hr of collection. Samples of blood from healthy volunteers were also collected at the same time and assayed along with patients’ samples. Polymyxin B Treatment Patients were randomized into two groups. In addition to normal therapy, patients in the treatment group were also given polymyxin B according to the following schedule: Day 1,300O units/kg/day; Day 2,400O units/kg/day; Day 3,550O units/kg/day; Day 4,550O units/kg/day; Day 5,400O units/kg/day; and Day 6,300O units/kg/day. The doses were prepared by reconstituting vials of 500,000 units of polymyxin B sulfate in pryrogen-free water and were administered intravenously in two equally divided daily doses. Endotoxin Assay Circulating endotoxin levels were assayed using the chromogenic limulus lysate assay (Whittaker M.A. Bioproducts, Walkersville, MD) according to the instructions supplied by the manufacturer. Briefly, lOO+l volumes of either plasma samples or endotoxin standards were incubated with 50 ~1 of freshly reconstituted limulus lysate in 96-well microtiter plates for 13 min at 37°C. Fifty microliters of diluted substrate was added and the incubation continued for an additional 3 min. The reaction was stopped by the addition of 25% acetic acid and the plates were read at 405 ti. Endotoxin in the plasma samples was corrected for dilution and calculated from a standard curve derived from assay of standards. IL-4 Determination The levels of IL-6 were determined using the B9 assay as described by Aarden (12). The IL-6 dependent B9 cells were obtained from Dr. Rick Nordan, NCI, National Institutes of Health, and were maintained by serial passage in culture.
INTERLEUKIN
6 IN
BURN
PATIENTS
363
For the assay, 10 units of standard IL-6 (Endogen Laboratories, Boston, MA) or serum samples were serially diluted in log, increments in microtiter plates in duplicate. Log-phase B9 cells were added at a final concentration of 1 x lo4 in 5Oql volumes and the plates were incubated for 3 days at 37°C. Tritiate thymidine was added for the final 4 hr of incubation and proliferation was determined by scintillation counts. The data were analyzed by weighted probit analysis using the computer program described by Sette et al. (13). Flow Cytometric
Analysis
Whole blood samples were prepared for cytometric analysis by incubation with ammonium chloride lysing buffer (Ortho Diagnostics, Raritan, NJ) washed with cold PBS and stained with an abbreviated panal of FITC and PE-conjugated monoclonal antibodies. Staining was accomplished by incubation for 30 min at 4°C followed by one wash with staining buffer and a second wash with cold PBS. The cells were fixed in a 1% paraformaldehyde solution and kept at 4°C until analysis. Stained preparations were examined using a FACScan flow cytometer (BectonDickinson Immunocytometry Systems, Mountain View, CA). Lymphocyte gating was accomplished using the leucogate reagent containing FITC-conjugated antiCD45 and PE-conjugated anti-CD14. The percentage and absolute numbers of each cell type examined were determined by computer analysis. Statistical
Analyses
Differences between groups were determined by Student’s t test. The relationships between IL-6 levels and the expression of surface phenotypes were examined by regression analyses. RESULTS
Patient
Population
A total of 21 burn patients were studied. Of these, 7 were treated with polymyxin B in addition to routine postbum therapy. There was no significant difference in the ages or in the degree of injury (total bum surface area) between the control and the polymyxin B-treated groups. Analysis of Endotoxin
Levels
As previously shown, treatment of bum patients with polymyxin B according to the regimen described under Materials and Methods is effective in reducing the endotoxin burden that commonly occurs following thermal injury (14). The profile of endotoxin increases in the control group was roughy bell-shaped with peak endotoxin levels occurring at 3 days postinjury (1). Circulating endotoxins were again increased at 1 week and this increase was sustained for 3 weeks (data not shown). As shown by the data in Table 1, polymyxin B therapy was effective in reducing the levels of circulating endotoxin. Polymyxin B-treated patients showed only small increases in circulating endotoxins, 0.12 + 0.12 units/ml vs 3.5 + 1.08 units/ml for the control group (P < 0.01).
364
GUO ET AL. IL-6 AND ENDOTOXIN
Number of Age Number of Mean peak Mean peak
patients deaths B-6 (units/ml) endotoxin (units/ml)
TABLE
1
LEVELS
IN
BURN PATIENT
SERA
Control
Polymyxin B
P value
14 35 k 4 3 1271 2 692 3.5 5 1.08
I 29 + 4 1 4427 0.12 2 0.12
NS NS <0.05 co.01
Analyses of Circulating Cytokines in the Burn Patient Initially it was observed that burn patient sera contained low but consistent levels of anti-viral activity. In an assay system which measured cytopathic effects of VSV on human embryonic lung cells (MRC 5), this activity was not abrogated by antisera specific for any of the three interferon species. In further attempts to identify the anti-viral factors, sera from both the control and the polymyxin B groups were analyzed for both tumor necrosis factor (TNF a) and IL-6. Using the L929 cytopathic assay, we could not show any evidence of increases in TNF over a 3-week interval. In contrast, all bum patients showed increases in IL-6 levels (Table 1). In the control group the average maximum activity of IL-6 was 1272 units/ml. In contrast, patients treated with polymyxin B showed maximum peak levels of only 44 units/ml. Despite a wide variation in the levels of IL-6 produced by individual patients, the difference between the groups was statistically significant (P = 0.05). The profiles of IL-6 production are illustrated in Fig. 1. The mean peak IL-6 activity in the polymyxin B group occurred on Day 2 and decreased thereafter. By 3 weeks, no serum IL-6 was detectable. In the control group, IL-6 increased to peak levels on Day 6. The mean IL-6 activity for the control patients at Day 6 and the average peak for each individual patient were comparable in magnitude. In contrast to the polymyxin B group the control group showed residual activity of 60 units/ml 21 days after injury. IL-6 as an Indicator
of Morbidity
The levels of IL-6 at all time points were compared in the control and polymyxin B groups (Fig. 2). For comparison, IL-6 levels were assayed in sera from normal volunteers and were below the level of detection of the assay. As indicated in the scatter plot the control group showed significant increases in IL-6 when compared to the polymyxin B group. When the data for the control group were subdivided on the basis of survival, it was apparent that IL-6 levels were much higher in the patients who did not survive. IL-6 and Changes in Lymphocyte Subsets Burn patients suffer from varying degrees of immune impairment. Therefore, it was of interest to determine if increases in IL-6 levels were related to alterations of peripheral blood lymphocyte representation or function. Patients were selected at random and both IL-6 levels and the representation of lymphocyte subsets were
INTERLEUKIN 2000
6 IN BURN PATIENTS
r
36.5
(14)
T m m
1000:
(14)
Control Polymyxin B
I
500400300-
(12)
200-
T
(14) T
100:
z302010: 5432
1
2
3
4
5
6
Days after Burn FIG. 1. Profiles of serum IL-6 levels in control and polymyxin B-treated bum patients t SE are shown for the days indicated. The number of patients assayed on each day are shown in parentheses.
determined. Despite the decreases in the number of circulating lymphocytes in burn patients, there was no apparent relationship between the absolute number or the percentage representation of lymphocytes and the levels of IL-6 (P > 0.2). However, as shown by the data in Fig. 3, there is an inverse relationship between the percentage of T cells (CD3+) and the level of circulating IL-6 (P > 0.01). Conversely, there was a positive correlation between the percentage of B cells (CD19+) and IL-6 (P < 0.01) (Fig. 4). The data in Fig. 5 and 6 indicate that neither helper (CD4 + ) nor suppressor (CD8 + ) cells were predominantly responsible for the decreases in the percentage of CD3-bearing cells. If IL-6 was directly responsible for either an impairment of T cell activity or for a reduction in viable T cells it might be expected that IL-6 would produce a similar effect on normal lymphocytes in vitro. When IL-6 was added to cultures of peripheral blood lymphocytes from normal volunteers and when proliferative responses to both mitogens and recall antigens were measured, there was no effect on either the number of surviving CD3 + cells or in the ability of the cells to respond. Similarly IL-6 did not alter the expression or shedding of IL-2 receptors (IL-2R) and did not interfere with IL-Zinduced proliferation (data not shown).
366
GUO ET AL.
8 0
110
Normals Survivors
Mortalities
Polymyxin B
Control Group FIG. 2. Scatter plot showing IL-6 activity at all time points for control and polymyxin B-treated patients.
DISCUSSION
In this report we describe increases in serum IL-6 levels which are a major consequence of acute thermal injury in man. These data confii the preliminary findings of Nijsten et al. (15) in which IL-6 was shown to be associated with the generation of acute phase reactants in bum patients. The increases in IL-6 are inhibited by treatment of bum patients with polymyxin B. Similarly the increased burden of circulating endotoxins which routinely accompany thermal injury is also
INTERLEUKIN
loo0
6 IN
BURN
367
PATIENTS
I
I
I
I
20
40
60
60
PERCENT
OF TOTAL
LYMPHOCYTES
3. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD3+ T cells. r = -0.606, P = < 0.01. FIG.
inhibited by polymyxin B therapy. This suggests that increases in IL-6 are a result of endotoxin burden. Indeed, a number of reports have shown that endotoxin is a major activator of IL-6 production in a variety of tissues (16, 17). However, endotoxin is known to activate a variety of biological response modifiers, including tumor necrosis factor and interleukin 1, and 11-l is also known to induce increases in IL-6 (18). Currently, it is a matter of conjecture as to whether the increases in IL-6 are a direct consequence of endotoxin or are induced by other mediators such as IL-l. Although polymyxin B may act directly on IL6-producing cells, there is no evidence to support such a direct inhibitory effect. In a number of patients, very large increases in IL-6 were associated with
‘04i /
lo2 I
I.
10’
CDlS+ CELLS
0
0 r
0
p < 0.01
I
I
I
I
12
24
36
46
PERCENT
OF TOTAL
LYMPHOCYTES
FIG. 4. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD19+ B cells. r = 0.652, P = < 0.01.
368
GUO ET AL. 104t
I
I
I
I CD4+ CELLS
n 103F+-
PERCENT
OF TOTAL
LYMPHOCYTES
FIG. 5. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD4+ helper/inducer cells. r = 0.533, P = < 0.01.
life-threatening clinical complications. The profiles of IL-6 increases for three individual patients are illustrated in Fig. 7. Two of these patients showed progressive increases until Day 6 and both patients died on Day 7 (Figs. 7A and 7B). The third patient (Fig. 7C) demonstrated IL-6 increases following an episode of acute respiratory distress (Day 2) and renal failure (Day 3). Other patients also showed increases in IL-6 which were coincident with changes in clinical status. Of these, one patient died 24 hr after an increase of 594 units/ml, while a second patient demonstrated a level of 2360 units/ml 36 hr after suffering cardiac arrest. In the polymyxin B group, one patient died but did not show any extraordinary
J
CELLS
p < 0.05
,000 0
10
PERCENT
20 OF TOTAL
30
40
LYMPHOCYTES
FIG. 6. Correlation analysis showing the relationship between serum IL-6 levels and the representation of circulating CD8 + suppressorkytotoxic cells. r = -0.471, P = < 0.05.
INTERLEUKIN
-0
1
369
6 IN BURN PATIENTS
2
3
4
5
6
7
Days after Burn FIG. 7. Profiles of serum IL-6 levels for three control group patients. Clinical complications for each patient are described in the text.
increase in IL-6. IL-6 increases are often coincident with infection (19, 20), but there was no apparent correlation between increases in IL-6 and infectious sequelae in the bum patients. These findings are remarkable in that IL-6 may be either a cause of multiorgan failure or may exacerbate organ failure due to other causes. Alternatively, IL-6 may be a consequence of cell death and, as such, might only serve as a predictor of organ failure. In either case, analyses of serum IL-6 levels may provide a basis for prognosis in bum or trauma victims. However, if IL-6 contributes to multiorgan failure, the use of specific antagonists could provide an important new therapeutic approach. Currently, several potential IL-6 antagonists are being investigated. The mechanism(s) by which IL-6 might contribute to the pathogenesis of burn injury is presently unknown. However, IL-6 is directly responsible for the induction of acute phase protein synthesis and release (21, 22). IL-6 levels in cerebral spinal fluid are increased in patients with bacterial and viral meningitis (19). Similarly, serum IL-6 levels are increased in renal transplant patients shortly after receiving grafts and during episodes of rejection (23). IL-6 may also contribute to the pathogenesis of HIV infection since HIV induces IL-6 synthesis in normal human mononuclear cells (24). In the bum victim, there is no indication that increases in IL-6 are a consequence of infection. Thermal injury is usually associated with varying degrees of immune dysfunc-
GUO ET AL.
tion. When patients are treated with polymyxin B according to the regimen described in this communciation, there is a consistent improvement in the profile of immune responses (3,4). This suggests that endotoxins contribute to the immune dysfunction following burns. Since polymyxin B therapy also abrogates increases in IL-6 levels, we considered the possibility that IL-6 might be causally related to the depressed immune responses. Thus we examined the effects of recombinant IL-6 and bum serum containing large amounts of IL-6 on various parameters of lymphocyte reactivity. Bum sera inhibit the proliferation induced by IL-2 in IL2-dependent CTLL cell lines. However, IL-6 induced no similar effect. Bum sera also contain large concentrations of soluble IL-2R but IL-6 had no effect on either the expression of or rate of shedding of IL-2R. Finally, when the proliferative responses of normal cells to recall antigens were tested in the presence of IL-6, there was no apparent inhibitory effect. In contrast, the data in this report indicate that IL-6 is associated with decreases in the proportion of circulating T cells and corresponsing increases in B cells. Although there are shifts in the subset representation, the percentage of total lymphocytes is unchanged and is independent of IL-6 concentration. The relationship between IL-6 and percentage of T cells is shared equally by both CD4 and CDS. The fact that we could not show any effect of IL-6 on T cell respones suggests that increases in IL-6 and bum-induced immune inhibition are concurrent events induced both directly or indirectly by endotoxin. The question which remains is what is the physiologic role of IL-6 in the bum patient and as an acute phase reactant. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Winchurch, R. A., Thupari, J. N., and Munster, A. M., Surgery 102, 808, 1987. Persson, U., J. Immunol. 118, 78, 1977. Munster, A. M., Winchurch, R. A., Thupari, J. N., and Ernst, C. B., J. Trauma 2.6, 995, 1986. Chiccone, T. G., Munster, A. M., Birmingham, W., and Winchurch, R. A., J. Burn Care Rehabil. 4, 153, 1983. Freeman, T. R., and Shelby, J., J. Trauma 28, 190, 1988. Wolfe, J. H. N., Saporoschetz, I., Young, A. E. O’Connor, N.E., and Mannick, J. A., Ann. Surg. 193, 513-520, 1981. Ozkan, A. N., and Ninnemann, J. L., J. Clin. Immunol. 5, 172, 1985. Teodorczyk-Injeyan, J., Sparkes, B. G., Mills, G. B., Peters, W. J., and Falk, R. E., Clin Exp. Immunol. 65, 570, 1986. Xiao, G.-X., Chopra, R. K., Adler, W. H., Munster, A. M., and Winchurch, R. A., J. Trauma 28, 1669, 1988. Rodrick, M. L., Saporoschetz, I., Wood, J., David, C. F., and Mannick, J. A., Surg. Forum 36, 98, 1986. Munster, A. M., Winchurch, R. A., Birmingham, W., and Keeling, P., Ann. Surg. 192,772, 1980. Aarden, L. A., DeGrot, E. R., Schaap, 0. L., and Lansdorp, P. M., Eur. J. Immunol. 17, 1411, 1987. Sette, A., Adorini, L., Marubini, E., and Doria, G., J. Immunol. Methods 86, 265, 1986. Bender, B. S., Winchurch, R. A., Thupari, J. N., Proust, J. J., Adler, W. H., and Munster, A. M., Clin. Exp. Immunol. 71, 120, 1988. Nijsten, M. W. N., DeGroot, E. R., Ten Duis, H. J., Klasen, H. J., Hack, C. E., and Aarden, L. A., Lancer 2, 921, 1987. Jirik, F. R., Podor, T. J., Hirano, T., Kishimoto, T., Loskutoff, D. J., Carson, D. A., and Lotz, M., .I. Immunol. 142, 144, 1989.
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17. Fang, Y., Moldawer, L. L., Marano, M., Wei, H., Tatter, S. B., Clarick, R. H., Santhanum, U., Sherris, D., May, L. T., Sehgal, P. B., and Lowry, S. F., J. Immunol. 142, 2321, 1989. 18. Walther, Z., May, L. T., and Sehgal, P., J. Immunol. 140, 974, 1988. 19. Houssiau, F. A., Bukasa, K., Sindic, C. J. M., Van Damme, J., and Van Snick, J., C/in. Exp. Immunol. 71, 320, 1988. 20. Helfgott, D. C., Tatter, S. B., Santhanam, U., Clarick, R. H., Bhardwaj, N., May, L. T., and Sehgal, P. B., .I. Immunol. 142, 948, 1989. 21. Ramadoti, G., Van Damme, J., Rieder, H., and Meyer, K.-H., Eur. J. Immunol. 18, 1259, 1988. 22. Geiger, T., Andus, T., Klaproth, J., Hirano, T., Kishimoto, T., and Heimich, P. C., Eur. J. Immunol. 18, 717, 1988. 23. Van Oers, M. H. J., Van Der Heyden, A. A. P. A. M., and Aarden, L. A., C&n. Exp. Immunol. 71, 314, 1988. 24. Nakajima, K., Martinez-Maza, O., Hirano, T., Breen, E. C., Nishanian, P. G.. Salazar-Gonzalez, J. F., Fahey, J. L., and Kishimoto, T., J. Immunol. 142, 531, 1989. Received July 10, 1989; accepted with revision September 29, 1989