Immunobiol., vol. 180, pp. 385-394 (1990)
1 Div.
Clinical Immunobiology, Department of Internal Medicine, Univ. Hospital Innsbruck, and 2 Department of Urology, General Hospital Salzburg, Austria
Treatment of Cancer Patients with Recombinant Interferon-y Induces Release of Endogenous Tumor Necrosis Factor-a::WALTERE. AULITZKyl, WOLFGANGK. AULITZKy2,]ULIANFRICK2, MANFRED HEROLD 1, GUNTHER GASTL 1, HERBERT TILd, MANUELA BERGER 1, CHRISTOPH HUBER 1
Received October 24,1989' Accepted in Revised Form January 25,1990
Abstract Study objective: 1) to investigate serum levels of tumor necrosis factor-a in patients treated with recombinant interferon-y and 2) to relate changes in TNF serum levels to other biological responses observed during treatment with interferon gamma (IFN-y). Patients: Five patients suffering from metastasizing renal cell carcinoma. Intervention: Each patient received three treatment cycles of 10 fig, 100 fig and 500 fig IFN-y applied three times at weekly intervals. The treatment cycles were separated by a therapy free interval of two weeks. The order of dose levels was randomly assigned to each patient. Measurements and main results: Tumor necrosis factor alpha (TNF-a), IFN-y and neopterin serum levels, monocyte counts in the peripheral blood and body temperature were measured immediately before and 4, 24, 48, 72, and 168 h after each application of IFN-y. Results indicated that elevated serum levels of TNF-a are induced by 100 fig and 500 fig IFN-y. Repeated application of the same dose led to downregulation of TNF release into the serum. Changes in TNF serum levels did not correlate with the magnitude of febrile reactions, neopterin production or IFN-y serum levels.
Introduction
Cytokines are a class of proteins produced by stimulated leukocytes. The genes of many of these proteins have recently been cloned, and recombinant DNA technology has provided us with sufficient amounts for clinical studies. These studies have demonstrated significant clinical efficacy of certain cytokines in distinct malignant and infectious diseases (1). The mode of action of these molecules, however, is still poorly understood. In 'f This work was financially supported by the Austrian Fund "Zur Fiirderung der wissenschaftlichen Forschung» project nr. 6526, and by grants from «Ernst Boehringer Institut fUr Arzneimittelforschung», Vienna, Austria. Test kits for assessment of TNF-a and IFN-y serum levels were kindly provided by Medgenix Inc. Brussels, Belgium. We gratefully acknowledge Miss Gabi Schiinhuber for excellent technical assistance.
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particular, it is not clear whether they act directly on the malignant cells or indirectly via activation of host effector mechanisms. Nor is it known whether induction or endogenous release of other mediator molecules is essential for the in vivo biological activity of cytokines. Such a view is indirectly supported by the observation that the cytokines tested so far in clinical studies share similar toxic side effects (2, 3). IFN-y represents an immunomodulatory agent with pleiotropic biological activities (4): it plays a key role in MHC-restricted immune recognition and destruction by T cells via up regulation of synthesis of HLA molecules (5, 6); it is a potent activator of monocytes (7) and also exhibits antiproliferative properties against a number of normal and transformed cells. Tumor necrosis factor is a cytokine primarily produced by macrophages/ monocytes and displays significant synergistic activities with IFN-y. TNF is the mediator of monocyte cytotoxicity induced by IFN -y and other cytokines (8). TNF also amplifies HLA DR gene induction by IFN-y (9). Combined treatment with both cytokines was associated with increased anti-tumor activity and toxicity (10). On the other hand, it has been shown that IFN-y itself stimulates the in vitro release of TNF-a from monocytes (11). Whether release of endogenous TNF is induced by therapeutic application of IFN-y to our knowledge has not yet been investigated. The present study aimed 1) to analyze TNF-a serum levels in patients treated with recombinant IFN-y and 2) to relate induction of endogenous TNF-a to various biological responses observed under IFN-y therapy.
Patients, Material and Methods Patients
Five male patients suffering from metastasizing renal cell carcinoma (RCC), age 46-68, median 55 years were treated in a phase IIII trial at the General Hospital in Salzburg, Austria. Tumor nephrectomy was performed on all patients at least three months prior to initiation of IFN therapy. The patients had Karnovsky indices> 80 %. Exclusion criteria were infections and other major concomitant diseases. Each patient had given written consent before start of the study. The clinical results of this trial will be reported elsewhere (12). The clinical characteristics of the patients selected for this study are summarized in Table 1.
Table 1. Clinical Characteristics Patient Number
2 3
4 5
Age
Sex
Site of Metastases
Response
55 68 46 62 55
male male male male male
lymph node lung lung lung, liver lung
PR SD CR PR PRO
Induction of endogenous tumor necrosis factor by interferon-y therapy . 387
Treatment schedule Each patient initially received either 10, 100 or 500 !-Ig IFN-y subcutaneously-injected three times at weekly intervals. After a therapy-free interval of two weeks the next dose level was applied. The third dose was also given in the same manner to each patient. The order of dose levels was randomly assigned to the patients. After this dose-finding phase the patients were further treated with 100 !-Ig IFN-y once weekly. Serum samples were taken immediately before and 4, 24, 48, 72, and 168 h after the first and third application of each dose of IFN-y.
Trial substance Recombinant IFN-y, originally produced by Genentech Inc., with a specific activity of 2 x 10E7 I.U./mg protein was obtained from Boehringer Ingelheim International (Ingelheim, FRG).
Assays Serum levels of TNF-a or IFN-y were determined by commercially available immunoradiometric assays (IRMA) (Medgenix Brussels, Belgium). The lower limit of detection using these tests is > 5 pglml for TNF-a and> 0.2 U/m! (NIH standard) for IFN-y. Neopterin serum levels were determined by a commercially available radioimmunoassay (Henning Inc., Berlin, FRG).
Statistics Wilcoxon matched-pairs signed-rank test and Mann-Whitney's U test were performed using a commercially available statistical software package (SPSS Inc, Chicago, IL, USA).
Results
Tumor necrosis factor-a serum levels after treatment with recombinant interferon-y TNF-a serum levels were measured in 282 serum samples and ranged from ato 81 ng/l (median 0). Two out of five patients had detectable serum levels of TNF-a in all pretreatment samples (Fig. 1). Administration of recombinant IFN-y led to a statistically significant increase of TNF-a serum levels (p < 0.01). TNF levels peaked 24 h after application of rIFN-y and returned to baseline values within four days. The increments in TNF serum levels observed after treatment with IFNy were dose-dependent: No changes in serum TNF levels were observed after 10 fA.g IFN-y. In contrast, after treatment with 100 fA.g all five patients and four of the five after administration of 500 fA.g rIFN -y showed an increase in TNF-a serum levels. The increase after 500 fA.g IFN-y was not greater than that observed after 100 fA.g (Fig. 2). The order of dose levels had no apparent influence on the serum TNF response of the patients (data not shown). Repeated application of the two higher dose levels of rIFN-y, however, led to downregulation of endogenous TNF-a: After the first treatment with 100 fA.g or 500 fA.g rIFN-y a median increase in TNF-a serum levels of 14 ng/l was observed, whereas the third application of the same dose was followed by a median increment of 1 ng/l (Fig. 3a, p = 0.08).
388 .
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500 ug IFN gamma day 1 TNF level (ngll)
50~--~~-------------------'
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o
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100 IIg IFN gamma day 1
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Figure 1. TNF serum levels before and after the first administration of various dose levels of IFN-y (administration day 1). The serum levels of individual patients are shown.
Relationship between TNF and IFN-y serum levels
TNF-a and IFN-y serum levels were measured in parallel in the same samples. IFN-y was not detected in any of the sera tested before the commencement of therapy. After subcutaneous administration of the two higher dose levels, all patients showed a dose dependent increase of IFN-y levels peaking 4 h after administration of the cytokine (Fig. 4). The exogen-
Induction of endogenous tumor necrosis factor by interferon-y therapy . 389
TNF
monocytes
40rTN~F_n~g/~I~(d~'I~I'~)________________- - ,
120
monocyte, (percent reduction)
-.~=-------~,
100
80
80
40
20
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100
500
10
100
500
IFN dose (Ug)
IFN dose (Ug)
neopterin
body temperature
60rNe~o~pe~rt~in~n~m~ol/~I~(d~'I~ta~)____________- - ,
Ceislull (maximum)
--
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39
38
37
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500
10
IFN dose (Ug)
100
500
IFN dose (Ug) pat. nr *- -
1
t-
2
-*
3
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04
)(
5
: median
Figure 2. Dose dependency of the increase of TNF and neopterin release, percent reduction of the number of monocytes in peripheral blood and peak temperatures after treatment with IFN -y. Delta values and percentages are calculated from pretreatment and peak or nadir values, Bars represent median values, dots represent individual patients.
ously administered cytokine disappeared from the blood stream within 48 h. Repeated subcutaneous administration of IFN-y led to comparable serum levels (Fig. 3b). Relationship between endogenous TNF-a serum concentration and induction of fever
None of the patients had an elevated body temperature before administration of IFN-y. After therapy, a dose-dependent increase in temperature
390 .
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et al.
TNF ng/l (delta)
Temp. oC (peak)
mono. (%reduction)
IFN Y U/ml(delta)
Neopterin (delta)
100 _
80
60
40
20
1st administration
0
20
40
60
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E222l3rd administration
Figure 3. Comparison of the response of TNF and neopterin serum levels, body temperature and monocyte counts after the first and the third administration of 100 [!g and 500 [!g IFN-y. For TNF, IFN-y and neopterin the median difference between pretreatment and peak values after treatment are shown. Response of monocytes is shown as the median percent reduction, febrile reactions as the median peak temperature after treatment with the two higher dose levels of IFN -y.
was observed (Fig. 2). These increments were seen prior to the appearance of detectable TNF serum levels (Fig. 4). Accordingly, no statistically significant correlation was observed between temperature and TNF serum levels (n=248, r=-0.12, n.s.), nor was a relationship observed between the increase in temperature and TNF-a serum levels observed after each application of IFN-y (n = 44, r = 0.5, n.s.). Moreover, the only patient who had no detectable TNF serum levels after 500 f!g IFN-y, also suffered febrile reactions comparable to those seen in the other four patients. Relationship between TNF-a serum levels and number of peripheral blood monocytes or neopterin levels
Further, we tested whether downregulation of TNF release by macrophages was accompanied by a downregulation of other IFN -y mediated effects on monocytes. Application of IFN -y leads to a rapid change in the homing properties of circulating monocytes and transiently decreases their numbers after 2 h. The dose response relationship of this phenomenon was similar to that observed for TNF release: 10 f!g IFN -y caused only minor changes, whereas both 100 f!g and 500 f!g resulted in a median reduction of monocytes by approximately 80 % of pretreatment counts (Fig. 2). A significant reduction of monocytes was also observed after the third
administration (median reduction 63 %), although smaller than after the first treatment.
Induction of endogenous tumor necrosis factor by interferon-y therapy . 391 IFN gamma lerum levell (5001lg IFN gamma I.C.) IFN gamma (UtlliO
60,-~---------------------,
50
40
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~
10
o
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80
80 100 120 140 180 180 200
hourI after IFN gamma application neopterin (5001liJ IFN gamma I.C.)
body temperature (5001lg IFN gamma I.C.) CII,luI 40,------------------------.
o
o
'36 '-'----'--"---'---'---'----'--'---'---'---'
20
40
80 .80 100 120 140 180 180 200
hourI after IFN gamma application
0
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80 100 120 140 180 180 200
hourI Ifter IFN gamma appllcatlbn
Figure 4. Kinetics of IFN-y and neopterin serum levels and body temperature after the first treatment with 500 I-Ig IFN-y.
Serum samples were also tested for neopterin content. Neopterin is a metabolic product of monocytes activated by IFN-y (13). Neopterin levels increased in all patients after 100 J.lg and 500 J.lg of IFN-y peaking at 48 h after the application (Fig. 4). The relationship between the IFN-y dose and the induction of neopterin was different from that of TNF-a; while 100 J.lg and 500 J.lg of IFN-yinduced comparable concentrations of TNF-a, 500 J.lg
392 .
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of IFN-y induced significantly higher neopterin levels than 100 !!g (p < 0.05, Fig. 2). Furthermore, neopterin increments after the third administration were within the same range as those observed after the first treatment with IFN-y (Fig. 3).
Discussion The findings presented indicate that increased serum levels of TNF-a are observed in patients treated with rIFN-y. This finding has important implications as the release of endogenous TNF-a may cause or contribute to the toxic side effects of IFN-y therapy which include fever, flu-like syndrome, hypotension and hematologic toxicity. In addition, antitumor activity by IFN -y might be mediated or enhanced by the release of endogenous TNF-a. TNF-a is involved in many potential IFN-dependent mechanisms against malignant cells such as monocyte cytotoxicity or enhancement of MHC antigen expression (8, 9). Thus enhancement of TNF-a production may be of importance in therapeutic success in tumor patients. Due to the fact that the number of patients analyzed in this study is small, the question of whether endogenous release of TNF-a is related to tumor response in these patients has to be answered in further studies. We also failed to demonstrate a relationship between TNF serum levels and the intensity of febrile reactions in these patients. The serum levels of TNF-a did not correlate to body temperature, nor was the increase in TNF serum levels after IFN-y administration related to the degree of fever. Moreover, peak temperatures were already reached before endogenous TNF-a serum levels were detectable. In addition, downregulation of endogenous TNF production after repeated application of IFN-y was not accompanied by the development of tachyphylaxis. Furthermore, we investigated whether the downregulation of TNF response after repeated high-dose application was caused by unresponsiveness of monocytes to the action of IFN-y. We analyzed two different IFNy mediated effects on these cells: 1) the influence of IFN-y treatment on the homing properties of monocytes and 2) the induction of neopterin synthesis. As shown in Figure 3, the effect of IFN-y on both parameters was somewhat lower after the third administration. Nevertheless, significant neopterin induction and decrease of monocyte numbers were observed also after repeated application of IFN -y. Thus monocytes were still capable of responding to IFN-y. This raises the question of whether IFN-y mediated TNF induction is selectively downregulated or whether factors other than endogenous production of the cytokine influence its serum level. Two mechanisms may explain the failure to correlate TNF-a serum levels with other biological responses subsequent to lFN-y. Firstly, the TNF levels reached after IFN-y therapy were considerably lower than those observed after administration of endotoxin or LAK cell therapy (14, 15),
Induction of endogenous tumor necrosis factor by interferon-y therapy . 393
thus, the endogenous TNF-a concentration might be too small to elicit significant biological activity. Nevertheless, TNF serum levels after exogenous treatment with TNF comparable to those observed after IFN-y therapy are accompanied by measurable adverse events such as temperature elevations'~':·). Secondly, IFN-y treatment results in the disappearance of certain peripheral blood cells from the vascular space (16). Experimental animal studies suggest that the cells activated by IFN-y leave the bloodstream and are enriched in the tissues (17). It therefore seems likely that IFN-y-induced TNF-a is produced primarily in the tissues. TNF serum levels may then only represent the excess of the cytokine not locally bound by receptors. Thus, serum content of TNF-a not only depends on production of TNF but also on the density and functional activity of its receptors in the tissues and on the quantity of TNF binding proteins in the serum. The presence of such a TNF binding protein in the serum has recently been demonstrated by OLSSON et al. (18). In addition, it has been shown that IFN-y regulates not only production of TNF-a by monocytes but also expression of TNF receptors on various cells (19). On this basis it is likely that TNF-a serum levels after treatment with IFN-y do not necessarily reflect the actual rate of production or biological activity. The question of whether concurrent upregulation of TNF receptors by IFN-y masks TNF release into the serum remains to be clarified. Weare presently addressing this question by simultaneously measuring both the TNF protein levels in serum and the mRNA expression in peripheral mononuclear blood cells.
References 1. MIHICH, E. 1986. Future perspectives for biological response modifiers. Sem. Oncol. 13: 234. 2. BROWN, T. D., J. KOELLER, K. BEOUGHER, J. GOLANDO, E. M. BONNEM, R. J. SPIEGEL, and D. D. VON HOFF. 1987. A phase I clinical trial of recombinant DNA gamma interferon. J. Clin. One. 5: 790. 3. FEINBERG, B., R. KURZROCK, M. TALPAZ, S. SAKS, and J. U. GUTTERMAN. 1988. A phase I trial of intravenously administered recombinant tumor necrosis factor alpha in cancer patients. J. Clin. One. 6: 1328. 4. TRINCHIERI, G., and B. PERUSSIA. 1985. Immune interferon: a pleiotropic lymphokine with multiple effects. Immunology Today, 6: 131. 5. NIEDERWIESER, D.,J. AUBOCK,J. TROPPMAIR, P. BOCK, P. FRITSCH, and C. HUBER. 1988. IFN mediated induction of MHC expression on human keratinocytes and its influence on in vitro immune response. J. Immunol. 140: 2556. 6. GERRARD, T. L., D. R. DYER, K. C. ZOON, D. ZUR NEDDEN, and J. SIEGEL. 1988. Modulation of class I and II histocompatibility antigens on human T cell lines by IFNgamma. J. Immunol. 140: 3450. 7. NATHAN, C. F., T. J. PRENDERGAST, M. E. WIEBER, R. E. STANLEY, E. PLATZER, H. G. REMOLD, K. WELTE, B. Y. RUBIN, and H. W. MURRAY. 1984. Activation of human macrophages: comparison of other cytokines with interferon-gamma. J. Exp. Med. 160: 600. "". W. E. Aulitzky, unpublished observation
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aI.
8. PH ILIP, R., and L. EpSTEIN. 1986. T umor necrosis factor as imm unomodu latOr and mediator of monocyte cytotoxicity induced by itself, gamma interfe ron and inted eukin 1. Nature 6083: 86. 9. ARENZANA~SEI SDEDOS, F., S. MOGENSEN, F. VUll LlER, W. FIERS, and j. L. VIRELIZIER. 1988. Autocrine secretion of tumor necrosis factor under the influence of imerfero n ~ gamma amplifies H LA-DR gene induction in human monocytes. Proc. Nul. Acad. Sci. USA 85: 6087. 10. ScHNEIDER, M., O. BOWERSOX, andj. E. TALMADGE. 1987. Combination immu notherapy with rTNF and rIFN-y : increased therapy and toxicity . Fed. Proc. 46: 561. 11. NEDWIN, G. E., T. S. SVEDERSKY, T. S. BRI NGMAN, M. A. PALLADINO, and D. V. GOEDDEL. 1985. Effect of interleuki n 2, interferon-y and milogens in the production of tu mor necrosis factor alpha and beta. j. Immuno l. 135: 2492. 12. AULlTZKY, W., G. GASTl , W. E. AULlTZKY, j. FRICK, B. MULL, B. LANSKE, and C. H UBER. 1989. Successful t reatment of metastasizing renal cell carcinoma with biologically active doses of interferon-yo J . Clin. O ncol. 7:1875. 13. H UBER, c., S. R. BATCH'ElOR, D. FUCHS, A. HAUSEN, A. LANG, D. N IEDERWIESER, G. REIBNEGGER, P. SWETLY, j. TRorrM AIR, and H. WACHTER. 1984. Immune response associated p roduction of neopterin. Release from macrophages primarily under control of
IFN·y. j. Exp. Med. 160: 310.
14. GEMLO, B. T., M. A. P AllADINO, H. S. JAFFE, 1. P. EsrEvIK, and A. A. RAYNER. 1988. C irculating cytokines in patients w ith metastatic cancer treated with recombinant interleukin 2 a ndIymphokine activated killer cells. Cancer Res. 48: 5864. 15. MICHIE, H . R., K. R. MANOQUE, D. R. SPRIGGS, M. D. REVHAUG, S. DWYER, C. A. DINARELLO, A. CERAMI, S. M. WOLff:, and D . W. WILMORE. 1988. Detection of circulating tumor necrosis factor after endotoxi n administratio n. N. Eng. j. Med. 318: 1481. 16. AULlT.lKY, W. E., W. AULlTZKY, G. G ASTL, B. LANSKE, H. TILG, j. REIITER, M. BERGER, M. HEROLD, j. FRICK, and CH. H UBER. 1989. Acute effects of single doses of recombinant interferon-yon white blood cell counts and lymphocyte subsets in patients with advanced renal cell cancer. j. Interferon Res. 9: 425. t 7. TALMADGE, ]. E. , H . R.TRIBBLE, R. W. PENNINGTON, H . PHILLIPS, and R. H . WILTROUT. 1987. Immunomodulatory and immunotherapeutic p roperties of recombinant gamma interferon and recombinant tumor necrosis factor in mice. Cancer Res. 47: 2563. 18. PEETRE, c., H . THYSELL, A. GRUBB, and 1. OLSSON. 1988. A tumor necrosis factor binding protein is present in human biological fluids. Eur. J. H ematol. 41 : 4 14. 19. AGGARVAL, B. B., T. E. EESSALU, and P. E. H ASs. 1985. C haracterization of receptors for tu mor necrosis factor-a and thei r regulation by gamma interferon. Nature 318 : 665. Dr. W. E. AULlTZKY, D iv. Clinical l mmunobiology, Dept. Internal Medicine, V niv. H ospital Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria
1 Div.
Clinical Immunobiology, Department of Internal Medicine, Univ. Hospital Innsbruck, and 2 Department of Urology, General Hospital Salzburg, Austria
Treatment of Cancer Patients with Recombinant Interferon-y Induces Release of Endogenous Tumor Necrosis Factor-a::WALTERE. AULITZKyl, WOLFGANGK. AULITZKy2,]ULIANFRICK2, MANFRED HEROLD 1, GUNTHER GASTL 1, HERBERT TILd, MANUELA BERGER 1, CHRISTOPH HUBER 1
Received October 24,1989' Accepted in Revised Form January 25,1990
Abstract Study objective: 1) to investigate serum levels of tumor necrosis factor-a in patients treated with recombinant interferon-y and 2) to relate changes in TNF serum levels to other biological responses observed during treatment with interferon gamma (IFN-y). Patients: Five patients suffering from metastasizing renal cell carcinoma. Intervention: Each patient received three treatment cycles of 10 fig, 100 fig and 500 fig IFN-y applied three times at weekly intervals. The treatment cycles were separated by a therapy free interval of two weeks. The order of dose levels was randomly assigned to each patient. Measurements and main results: Tumor necrosis factor alpha (TNF-a), IFN-y and neopterin serum levels, monocyte counts in the peripheral blood and body temperature were measured immediately before and 4, 24, 48, 72, and 168 h after each application of IFN-y. Results indicated that elevated serum levels of TNF-a are induced by 100 fig and 500 fig IFN-y. Repeated application of the same dose led to downregulation of TNF release into the serum. Changes in TNF serum levels did not correlate with the magnitude of febrile reactions, neopterin production or IFN-y serum levels.
Introduction
Cytokines are a class of proteins produced by stimulated leukocytes. The genes of many of these proteins have recently been cloned, and recombinant DNA technology has provided us with sufficient amounts for clinical studies. These studies have demonstrated significant clinical efficacy of certain cytokines in distinct malignant and infectious diseases (1). The mode of action of these molecules, however, is still poorly understood. In 'f This work was financially supported by the Austrian Fund "Zur Fiirderung der wissenschaftlichen Forschung» project nr. 6526, and by grants from «Ernst Boehringer Institut fUr Arzneimittelforschung», Vienna, Austria. Test kits for assessment of TNF-a and IFN-y serum levels were kindly provided by Medgenix Inc. Brussels, Belgium. We gratefully acknowledge Miss Gabi Schiinhuber for excellent technical assistance.
386 .
WALTERE. AULITZKY
et al.
particular, it is not clear whether they act directly on the malignant cells or indirectly via activation of host effector mechanisms. Nor is it known whether induction or endogenous release of other mediator molecules is essential for the in vivo biological activity of cytokines. Such a view is indirectly supported by the observation that the cytokines tested so far in clinical studies share similar toxic side effects (2, 3). IFN-y represents an immunomodulatory agent with pleiotropic biological activities (4): it plays a key role in MHC-restricted immune recognition and destruction by T cells via up regulation of synthesis of HLA molecules (5, 6); it is a potent activator of monocytes (7) and also exhibits antiproliferative properties against a number of normal and transformed cells. Tumor necrosis factor is a cytokine primarily produced by macrophages/ monocytes and displays significant synergistic activities with IFN-y. TNF is the mediator of monocyte cytotoxicity induced by IFN -y and other cytokines (8). TNF also amplifies HLA DR gene induction by IFN-y (9). Combined treatment with both cytokines was associated with increased anti-tumor activity and toxicity (10). On the other hand, it has been shown that IFN-y itself stimulates the in vitro release of TNF-a from monocytes (11). Whether release of endogenous TNF is induced by therapeutic application of IFN-y to our knowledge has not yet been investigated. The present study aimed 1) to analyze TNF-a serum levels in patients treated with recombinant IFN-y and 2) to relate induction of endogenous TNF-a to various biological responses observed under IFN-y therapy.
Patients, Material and Methods Patients
Five male patients suffering from metastasizing renal cell carcinoma (RCC), age 46-68, median 55 years were treated in a phase IIII trial at the General Hospital in Salzburg, Austria. Tumor nephrectomy was performed on all patients at least three months prior to initiation of IFN therapy. The patients had Karnovsky indices> 80 %. Exclusion criteria were infections and other major concomitant diseases. Each patient had given written consent before start of the study. The clinical results of this trial will be reported elsewhere (12). The clinical characteristics of the patients selected for this study are summarized in Table 1.
Table 1. Clinical Characteristics Patient Number
2 3
4 5
Age
Sex
Site of Metastases
Response
55 68 46 62 55
male male male male male
lymph node lung lung lung, liver lung
PR SD CR PR PRO
Induction of endogenous tumor necrosis factor by interferon-y therapy . 387
Treatment schedule Each patient initially received either 10, 100 or 500 !-Ig IFN-y subcutaneously-injected three times at weekly intervals. After a therapy-free interval of two weeks the next dose level was applied. The third dose was also given in the same manner to each patient. The order of dose levels was randomly assigned to the patients. After this dose-finding phase the patients were further treated with 100 !-Ig IFN-y once weekly. Serum samples were taken immediately before and 4, 24, 48, 72, and 168 h after the first and third application of each dose of IFN-y.
Trial substance Recombinant IFN-y, originally produced by Genentech Inc., with a specific activity of 2 x 10E7 I.U./mg protein was obtained from Boehringer Ingelheim International (Ingelheim, FRG).
Assays Serum levels of TNF-a or IFN-y were determined by commercially available immunoradiometric assays (IRMA) (Medgenix Brussels, Belgium). The lower limit of detection using these tests is > 5 pglml for TNF-a and> 0.2 U/m! (NIH standard) for IFN-y. Neopterin serum levels were determined by a commercially available radioimmunoassay (Henning Inc., Berlin, FRG).
Statistics Wilcoxon matched-pairs signed-rank test and Mann-Whitney's U test were performed using a commercially available statistical software package (SPSS Inc, Chicago, IL, USA).
Results
Tumor necrosis factor-a serum levels after treatment with recombinant interferon-y TNF-a serum levels were measured in 282 serum samples and ranged from ato 81 ng/l (median 0). Two out of five patients had detectable serum levels of TNF-a in all pretreatment samples (Fig. 1). Administration of recombinant IFN-y led to a statistically significant increase of TNF-a serum levels (p < 0.01). TNF levels peaked 24 h after application of rIFN-y and returned to baseline values within four days. The increments in TNF serum levels observed after treatment with IFNy were dose-dependent: No changes in serum TNF levels were observed after 10 fA.g IFN-y. In contrast, after treatment with 100 fA.g all five patients and four of the five after administration of 500 fA.g rIFN -y showed an increase in TNF-a serum levels. The increase after 500 fA.g IFN-y was not greater than that observed after 100 fA.g (Fig. 2). The order of dose levels had no apparent influence on the serum TNF response of the patients (data not shown). Repeated application of the two higher dose levels of rIFN-y, however, led to downregulation of endogenous TNF-a: After the first treatment with 100 fA.g or 500 fA.g rIFN-y a median increase in TNF-a serum levels of 14 ng/l was observed, whereas the third application of the same dose was followed by a median increment of 1 ng/l (Fig. 3a, p = 0.08).
388 .
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500 ug IFN gamma day 1 TNF level (ngll)
50~--~~-------------------'
40
30
20
10
oL-~~~e-~-4~~--~~--~
o
4
6
8
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day of study 10 IIg IFN gamma day 1
100 IIg IFN gamma day 1
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TNF level (ng/ll
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Figure 1. TNF serum levels before and after the first administration of various dose levels of IFN-y (administration day 1). The serum levels of individual patients are shown.
Relationship between TNF and IFN-y serum levels
TNF-a and IFN-y serum levels were measured in parallel in the same samples. IFN-y was not detected in any of the sera tested before the commencement of therapy. After subcutaneous administration of the two higher dose levels, all patients showed a dose dependent increase of IFN-y levels peaking 4 h after administration of the cytokine (Fig. 4). The exogen-
Induction of endogenous tumor necrosis factor by interferon-y therapy . 389
TNF
monocytes
40rTN~F_n~g/~I~(d~'I~I'~)________________- - ,
120
monocyte, (percent reduction)
-.~=-------~,
100
80
80
40
20
10
100
500
10
100
500
IFN dose (Ug)
IFN dose (Ug)
neopterin
body temperature
60rNe~o~pe~rt~in~n~m~ol/~I~(d~'I~ta~)____________- - ,
Ceislull (maximum)
--
40,--------~--------------_,
39
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37
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500
10
IFN dose (Ug)
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IFN dose (Ug) pat. nr *- -
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Figure 2. Dose dependency of the increase of TNF and neopterin release, percent reduction of the number of monocytes in peripheral blood and peak temperatures after treatment with IFN -y. Delta values and percentages are calculated from pretreatment and peak or nadir values, Bars represent median values, dots represent individual patients.
ously administered cytokine disappeared from the blood stream within 48 h. Repeated subcutaneous administration of IFN-y led to comparable serum levels (Fig. 3b). Relationship between endogenous TNF-a serum concentration and induction of fever
None of the patients had an elevated body temperature before administration of IFN-y. After therapy, a dose-dependent increase in temperature
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Figure 3. Comparison of the response of TNF and neopterin serum levels, body temperature and monocyte counts after the first and the third administration of 100 [!g and 500 [!g IFN-y. For TNF, IFN-y and neopterin the median difference between pretreatment and peak values after treatment are shown. Response of monocytes is shown as the median percent reduction, febrile reactions as the median peak temperature after treatment with the two higher dose levels of IFN -y.
was observed (Fig. 2). These increments were seen prior to the appearance of detectable TNF serum levels (Fig. 4). Accordingly, no statistically significant correlation was observed between temperature and TNF serum levels (n=248, r=-0.12, n.s.), nor was a relationship observed between the increase in temperature and TNF-a serum levels observed after each application of IFN-y (n = 44, r = 0.5, n.s.). Moreover, the only patient who had no detectable TNF serum levels after 500 f!g IFN-y, also suffered febrile reactions comparable to those seen in the other four patients. Relationship between TNF-a serum levels and number of peripheral blood monocytes or neopterin levels
Further, we tested whether downregulation of TNF release by macrophages was accompanied by a downregulation of other IFN -y mediated effects on monocytes. Application of IFN -y leads to a rapid change in the homing properties of circulating monocytes and transiently decreases their numbers after 2 h. The dose response relationship of this phenomenon was similar to that observed for TNF release: 10 f!g IFN -y caused only minor changes, whereas both 100 f!g and 500 f!g resulted in a median reduction of monocytes by approximately 80 % of pretreatment counts (Fig. 2). A significant reduction of monocytes was also observed after the third
administration (median reduction 63 %), although smaller than after the first treatment.
Induction of endogenous tumor necrosis factor by interferon-y therapy . 391 IFN gamma lerum levell (5001lg IFN gamma I.C.) IFN gamma (UtlliO
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o
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Figure 4. Kinetics of IFN-y and neopterin serum levels and body temperature after the first treatment with 500 I-Ig IFN-y.
Serum samples were also tested for neopterin content. Neopterin is a metabolic product of monocytes activated by IFN-y (13). Neopterin levels increased in all patients after 100 J.lg and 500 J.lg of IFN-y peaking at 48 h after the application (Fig. 4). The relationship between the IFN-y dose and the induction of neopterin was different from that of TNF-a; while 100 J.lg and 500 J.lg of IFN-yinduced comparable concentrations of TNF-a, 500 J.lg
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of IFN-y induced significantly higher neopterin levels than 100 !!g (p < 0.05, Fig. 2). Furthermore, neopterin increments after the third administration were within the same range as those observed after the first treatment with IFN-y (Fig. 3).
Discussion The findings presented indicate that increased serum levels of TNF-a are observed in patients treated with rIFN-y. This finding has important implications as the release of endogenous TNF-a may cause or contribute to the toxic side effects of IFN-y therapy which include fever, flu-like syndrome, hypotension and hematologic toxicity. In addition, antitumor activity by IFN -y might be mediated or enhanced by the release of endogenous TNF-a. TNF-a is involved in many potential IFN-dependent mechanisms against malignant cells such as monocyte cytotoxicity or enhancement of MHC antigen expression (8, 9). Thus enhancement of TNF-a production may be of importance in therapeutic success in tumor patients. Due to the fact that the number of patients analyzed in this study is small, the question of whether endogenous release of TNF-a is related to tumor response in these patients has to be answered in further studies. We also failed to demonstrate a relationship between TNF serum levels and the intensity of febrile reactions in these patients. The serum levels of TNF-a did not correlate to body temperature, nor was the increase in TNF serum levels after IFN-y administration related to the degree of fever. Moreover, peak temperatures were already reached before endogenous TNF-a serum levels were detectable. In addition, downregulation of endogenous TNF production after repeated application of IFN-y was not accompanied by the development of tachyphylaxis. Furthermore, we investigated whether the downregulation of TNF response after repeated high-dose application was caused by unresponsiveness of monocytes to the action of IFN-y. We analyzed two different IFNy mediated effects on these cells: 1) the influence of IFN-y treatment on the homing properties of monocytes and 2) the induction of neopterin synthesis. As shown in Figure 3, the effect of IFN-y on both parameters was somewhat lower after the third administration. Nevertheless, significant neopterin induction and decrease of monocyte numbers were observed also after repeated application of IFN -y. Thus monocytes were still capable of responding to IFN-y. This raises the question of whether IFN-y mediated TNF induction is selectively downregulated or whether factors other than endogenous production of the cytokine influence its serum level. Two mechanisms may explain the failure to correlate TNF-a serum levels with other biological responses subsequent to lFN-y. Firstly, the TNF levels reached after IFN-y therapy were considerably lower than those observed after administration of endotoxin or LAK cell therapy (14, 15),
Induction of endogenous tumor necrosis factor by interferon-y therapy . 393
thus, the endogenous TNF-a concentration might be too small to elicit significant biological activity. Nevertheless, TNF serum levels after exogenous treatment with TNF comparable to those observed after IFN-y therapy are accompanied by measurable adverse events such as temperature elevations'~':·). Secondly, IFN-y treatment results in the disappearance of certain peripheral blood cells from the vascular space (16). Experimental animal studies suggest that the cells activated by IFN-y leave the bloodstream and are enriched in the tissues (17). It therefore seems likely that IFN-y-induced TNF-a is produced primarily in the tissues. TNF serum levels may then only represent the excess of the cytokine not locally bound by receptors. Thus, serum content of TNF-a not only depends on production of TNF but also on the density and functional activity of its receptors in the tissues and on the quantity of TNF binding proteins in the serum. The presence of such a TNF binding protein in the serum has recently been demonstrated by OLSSON et al. (18). In addition, it has been shown that IFN-y regulates not only production of TNF-a by monocytes but also expression of TNF receptors on various cells (19). On this basis it is likely that TNF-a serum levels after treatment with IFN-y do not necessarily reflect the actual rate of production or biological activity. The question of whether concurrent upregulation of TNF receptors by IFN-y masks TNF release into the serum remains to be clarified. Weare presently addressing this question by simultaneously measuring both the TNF protein levels in serum and the mRNA expression in peripheral mononuclear blood cells.
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14. GEMLO, B. T., M. A. P AllADINO, H. S. JAFFE, 1. P. EsrEvIK, and A. A. RAYNER. 1988. C irculating cytokines in patients w ith metastatic cancer treated with recombinant interleukin 2 a ndIymphokine activated killer cells. Cancer Res. 48: 5864. 15. MICHIE, H . R., K. R. MANOQUE, D. R. SPRIGGS, M. D. REVHAUG, S. DWYER, C. A. DINARELLO, A. CERAMI, S. M. WOLff:, and D . W. WILMORE. 1988. Detection of circulating tumor necrosis factor after endotoxi n administratio n. N. Eng. j. Med. 318: 1481. 16. AULlT.lKY, W. E., W. AULlTZKY, G. G ASTL, B. LANSKE, H. TILG, j. REIITER, M. BERGER, M. HEROLD, j. FRICK, and CH. H UBER. 1989. Acute effects of single doses of recombinant interferon-yon white blood cell counts and lymphocyte subsets in patients with advanced renal cell cancer. j. Interferon Res. 9: 425. t 7. TALMADGE, ]. E. , H . R.TRIBBLE, R. W. PENNINGTON, H . PHILLIPS, and R. H . WILTROUT. 1987. Immunomodulatory and immunotherapeutic p roperties of recombinant gamma interferon and recombinant tumor necrosis factor in mice. Cancer Res. 47: 2563. 18. PEETRE, c., H . THYSELL, A. GRUBB, and 1. OLSSON. 1988. A tumor necrosis factor binding protein is present in human biological fluids. Eur. J. H ematol. 41 : 4 14. 19. AGGARVAL, B. B., T. E. EESSALU, and P. E. H ASs. 1985. C haracterization of receptors for tu mor necrosis factor-a and thei r regulation by gamma interferon. Nature 318 : 665. Dr. W. E. AULlTZKY, D iv. Clinical l mmunobiology, Dept. Internal Medicine, V niv. H ospital Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria
