Leukemia and Lymphoma, Vol. 17. pp. 169-173
`Reprints available directly from the publisher
`Photocopying permitted by license only
`
`0 1995 Harwood Academic Publishers GmbH
`Printed in the United States of America
`
`Cytokine Dysregulation in Chronic Graft Versus
`Host Disease
`
`V. BARAK.1 F. LEVI-SCHAFFER.? B. NISMANl and A. NAGLER3
`
`Departments of Oncology,' Pharmacology? and Bone Marrow Transplantation.3 Hadassah University Hospital and Hebrew University,
`Jerusalem, Israel
`
`(Received March 14. 1994)
`
`Cytokines play a key role in the pathogenesis of chronic Graft versus Host Disease (cGVHD) and
`various studies have shown abberant production of cytokines by immune cells from GVHD patients.
`Based on these findings and others showing that high TNF levels precede the development of CVHD,
`we evaluated inflammatory cytokine levels following BMT and during the development of cGVHD.
`In this study, patients undergoing bone marrow transplantation (BMT) who consequently developed
`chronic GVHD were analyzed as to their cytokine production during cGVHD and this was correlated
`with their clinical manifestations. A positive correlation was found between the severity as well as
`the number of major clinical complications and high levels of inflammatory cytokines (IL-IPIL-6
`andTNFa) compared to control patients or to normal donors. Patients undergoing BMT who did not
`develop GVHD, did not produce high levels of IL- I PIL-6 or TNF. High levels of cytokines may be
`used as a tool for assessing novel therapeutic modalities and response to GVHD treatment.
`
`KEY WORDS:
`cytokines
`transplantation (BMT)
`
`chronic graft-versus-host disease (cGVHD)
`
`bone marrow
`
`INTRODUCTION
`
`Chronic Graft versus Host Disease is a major obstacle to
`successful allogeneic bone marrow
`transplantation
`(BMT). GVHD following allogeneic BMT is caused by
`donor graft T lymphocytes that recognize antigenic dis-
`parities between donor and
`Tissue damage
`associated with GVHD is thought to be caused by T cell
`mediated cytotoxicity4,' and it is possible that additional
`networks of inflammatory cytokines act as mediators of
`acute GVHD.6.7 GVHD is viewed today as a three step
`process, consisting of upregulation of HLA and leukocyte
`adhesion molecules on host target cells; followed by ac-
`tivation of donor immunocompetent T cells by host his-
`tocompatibility antigens, which then proliferate. Finally,
`
`Address lor correspondence: Dr. Arnon Nagler. Bone Marrow
`Transplantation Dept., Hadassah University Hospital. P.O. Box 12000,
`Jerusalem, Israel 9 I 120.
`
`I69
`
`these activated T cells, then secrete cytokines, recruit ad-
`ditional cells, induce the expression of histocompatibility
`antigens, and focus their attack on recipient targets.
`Cytokines play a key role in each of the above
`steps. *-I1 Many cytokines are produced in an aberrant
`in patients with cGVHD12-14
`fashion
`including
`Interleukin-2 (IL-2), soluble IL-2 Receptor (sIL-~R),
`Tumor Necrosis Factor-alpha
`(TNF-a) and beta (TNF-
`p), Interferon y (IFNy), Interleukin- 1 (IL-la and IL- I p),
`Interleukin-6 (IL-6) and Interleukin-4 (IL-4).
`TNF-alpha has a major function in the first step of
`GVHD. In various studies TNF-a has been shown to be
`released from host tissues and to be elevated both in the
`tissues and the serum of mice and humans with chronic
`GVHD.15 Moreover, Holler et al. 16 found a direct corre-
`lation between high TNF levels and transplant related
`complications or survival. Furthermore, the rise in TNF
`levels were shown to precede the development of
`GVHD.I6 The results of bioassays and immunoassays of
`
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`I70
`
`V. BARAK ETAL.
`
`TNF levels in patients do not always correlate with the
`clinical picture, suggesting the presence of cytokine in-
`hibitors. An example for this would be IL-1 receptor an-
`tagonist (IL-IRA), which inhibits IL-1, and also prevents
`GVHD.I7
`In addition, high levels of IL-6 have been correlated
`with the onset of acute GVHD as well as the development
`of hepatorenal dysfunction (HRD), and bilateral lung in-
`filtration
`(BLI).Ig High amounts of granulocyte
`macrophage-colony stimulating factor (GM-CSF) have
`also been detected in sera from patients with GVHD19 and
`it has been suggested that IFN is an additional mediator
`of GVHD.20
`This study was undertaken in order to assess whether
`the increase in the production of inflammatory cytokines
`could explain the consequent development of cGVHD in
`many patients post-BMT.
`
`MATERIALS AND METHODS
`
`Cytokine levels were examined in the sera of BMT pa-
`tients who were diagnosed as having cGVHD according
`to the Seattle clinical and histopathological criteria.21
`In most of the patients more than one evaluation was
`performed during the cGVHD period. Thirty normal in-
`dividuals of compatible age and sex served as controls. In
`addition, we evaluatedcytokine profiles 5-12 months post
`transplant in 11 acute lymphoblastic leukemia (ALL) pa-
`tients, 8 acute myeloid leukemia (AML) patients and 6
`chronic myeloid leukemia (CML) patients who underwent
`allogenlic BMT and did not develop cGVHD. Transplant
`conditioning regimens, allograft cell numbers and treat-
`ment were similar between the study groups and the con-
`trols.
`
`Patients
`
`Thirteen consecutive patients (10 males and 3 females)
`who developed cGVHD were enrolled in this study; mean
`age 16 (4-3 1) years. Six patients had ALL, four AML, one
`CML and two severe aplastic anemia (SAA). All patients
`underwent HLA matched bone marrow transplantation
`(BMT). Of the leukemic patients, four were in first com-
`plete remission (CR), four in second CR, one in third CR
`and one in relapse. The CML patient was transplanted in
`the first chronic phase.
`Eight patients were conditioned with fractionated total
`body irradiation (TBI), ( 1200 cGy in 6 fractions) followed
`by administration of etoposide ( I500 mg/m*), cyclophos-
`phamide (60 mgkg) and melphalan (60 mg/m2) and frac-
`
`tionated total lymphoid irradiation (TLI) (600 cGy in 4
`fractions). Two patients were conditioned with Busulfan
`(16 mgkg in 4 days), cyclophosphamide (200 mgkg in 4
`days) and TLI. The patient with CML was conditioned
`with fractionated TBI (1200 cGy) and cyclophosphamide
`(120 mgkg in 2 days). The patients with SAA were con-
`ditioned with fractionated TLI (1600 cGy in 8 fractions)
`and cyclophosphamide (120 mgkg in 2 days).
`Bone marrow cells were purged with CAMPATH-1
`(IgG2b), a monoclonal rat anti human lymphocyte anti-
`body (anti-CDW52) (1 ug/106 nucleated cells) kindly sup-
`plied by Drs. G. Hale and H. Waldmann, Cambridge
`University (Cambridge, U.K.) in order to deplete T lym-
`phocytes.* No other anti GVHD prophylaxis was given.
`The patients with SAA were treated with cyclosporine A
`in order to prevent graft rejection. The patient with CML
`was transplanted with non T depleted marrow and received
`a combination of cyclosporine A and methotrexate as
`GVHD prophylaxis.
`All patients had cGVHD according to biopsy results
`which included a typical histopathologic picture.1 Three
`patients had mild cGVHD, 5 moderate and 5 severe ex-
`tensive cGVHD according to the Seattle grading criteria.21
`All cases showed cutaneous involvement, either general-
`ized or localized, most had other organ involvement (oral
`mucosa, ocular, liver, gastrointestinal and musculoskele-
`tal). Eight of the cGVHD patients developed acute GVHD
`in the early post BMT period. Three patients developed
`grade I, 2 grade I1 and 1 grade 111. All were treated with
`cyclosporine A (6 mgkg) and methylprednisolone (1-5
`mgkg) and 7 of them had no signs of active GVHD by
`day 100. Five patients had de novo cGVHD. Median onset
`of cGVHD was 120 days, range 100-166 days. They were
`treated with prednisone, cyclosporine A, azathioprine with
`modulation depending on clinical parameters. In addition,
`thalidomide (2 patients), colchicine (2 patients) and low
`dose TLI (2 patients) were also given without substantial
`clinical benefit.
`The following cytokine levels were determined in pa-
`tients' sera by means of radioimmunoassay (RIA) or enzyme
`immunoassay (EIA): IL-lp, IL-2, IL-6, TNFa, GM-CSF,
`yIFN. Cytokine determinations were performed within 5-27
`months (mean 11) following the onset of cGVHD. Some of
`the patients sera was tested more than once.
`
`Statistical analysis
`
`For comparison of mean levels of cytokines in test
`patients, controls and normal donors, we used the Student
`T-test. The correlation between disease severity and mean
`cytokine levels was determined by Spearman correlation
`coefficients.
`
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`RESULTS
`
`IL-6 in GVHD
`
`CYTOKINE DYSREGULATION IN cGVHD
`
`171
`
`Cytokine production was evaluated in allogeneic BMTpa-
`tients with cGVHD compared to BMT patients who did
`not develop cGVHD as well as in normal individuals.
`The following cytokines were evaluated: IL- 1 p, IL-2,
`IL-6, TNFa, INFy and GM-CSF. The level of TNF was
`found to be high in 10/13 patients or 19/24 evaluations.
`Only 3 patients (5 evaluations) had undetectable levels of
`TNF (Figure 1). The level of TNFa in most of the patients
`was very high (10-1000 pg/ml compared to normal lev-
`els <I0 pg/ml) (p < 0.001). We observed that the three pa-
`tients who had undetectable levels of TNF developed very
`mild cGVHD (n = 3) while patients who had high or ex-
`tremely high levels of TNF had moderate to severe
`cGVHD (n = 10) (c.c = -0.496). There was no difference
`in TNFa levels between patients with moderate and se-
`vere cGVHD (3 of the patients with very high TNF had
`severe cGVHD while 2 had moderate cGVHD). The level
`of IL-6 was found to be very high (in comparison to nor-
`mal levels p < 0.001) in 11/12 pts or 15/17 evaluations
`ranging between 100&6000pg/ml. Only 1 patient’s serum
`(or 2 evaluations) contained relatively low amounts of IL-
`&although much higher than the level found in normal
`human sera (< 100 pg/ml) (Figure 2). This particular pa-
`tient had very mild cGVHD. All of the patients with mod-
`erate or severe cGVHD, had very high levels of IL-6. The
`remaining 2 patients who developed mild cGVHD, had
`high and very high levels of IL-6, respectively. IL- 1 p was
`found also in high levels in 11/13 pts or 25/31 evaluations
`(600-10000 pg/ml) as compared to normal donors (p <
`0.001). 2/13 patients (or 6 evaluations) were in the nor-
`mal range. The 2 patients with low levels of IL- 1 p had
`mild cGVHD. The third patient with mild cGVHD, had
`elevated levels of IL-1p. All of the patients with moder-
`
`TNFa in GVHD
`
`Ir z c
`
`Patients
`
`Figure 1 TNFa levels in GVHD patients and control normal donors.
`
`Patients
`
`Figure 2
`
`IL-6 levels in GVHD patients and control normal donors.
`
`IL-IP in GVHD
`
`-
`i
`
`Figure 3
`
`Patients
`IL- I p levels in GVHD patients and control normal donors.
`
`ate and severe cGVHD had high or very high levels of
`IL-1 p (Figure 3).
`GM-CSF was found to be elevated in 10/12 patients (or
`13/17 evaluations)-although
`it was considered to be high
`only in 5/12 patients. Two patients had undetectable lev-
`els and the 2 patients with low GM-CSF had mild cGVHD
`(Figure 4).
`Most (8/12) of the patients or 10114 evaluations had un-
`detectable or low levels of IFN y. Only 4/12 patients (or
`4/14 evaluations) had high levels (Figure 5). Out of the 4
`pts with high levels of IFN y, 2 had moderate and 2 had
`severe cGVHD.
`IL-2 levels were undetectable in all of the patients (Data
`not shown). As an additional control, we analyzed sera
`from patients diagnosed with ALL, AML and CML who
`underwent allogeneic BMT and were matched in age and
`sex to the test group. These patients did not produce high
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`V. BARAK ET AL.
`
`GM-CSF in GVHD
`
`172
`
`t -
`3
`r m Y
`E
`c1
`
`In this study we have shown that patients who under-
`went BMT and subsequently developed cGVHD produce
`high or even extremely high levels of inflammatory cy-
`tokines as IL- 1 p, IL-6, TNFa. Moreover, we found a good
`correlation between the severity of GVHD and the ele-
`vated cytokine levels.
`In contrast, patients who underwent BMT and did not
`develop cGVHD, produced these cytokines within a nor-
`mal range. The level of other cytokines such as GM-CSF
`or IFNy was relatively low in most of these patients.
`The level of TNF observed is in accordance with results
`obtained by Holler et al. 16 and in discrepancy with other
`studies which did not find any correlation between cy-
`tokine levels and the severity of GVHD.13.18.20 The cause
`for these discrepancies may be dependent on the exact time
`of cytokine evaluation or on the different evaluation meth-
`ods with different sensitivities.
`Holler et a1.,16 however, showed that high levels of TNF
`generally precede GVHD. As we did not measure cytokine
`levels of these patients before the onset of GVHD, we can-
`not verify this observation. In a recent study by Tong er
`d . 1 3 although TNF levels were shown to precede major
`complications and the development of cGVHD in trans-
`plant patients, this cytokine increased significantly within
`seven days post GVHD diagnosis, but did not correlate
`with disease activity. Additional studies did not find a cor-
`relation between IFNy, IL-2 and GM-CSF, and the devel-
`opment of cGVHD.
`Monitoring the level of serum TNF should help the eval-
`uation of severity of chronic GVHD and allow a greater
`degree of differential treatment in the future.
`Future anti-GVHD therapy could perhaps be directed
`towards the blocking of the production of andor the ef-
`fects of cytokines, whereas most of the established thera-
`pies today are directed towards immunosuppression of the
`
`Cytoklnci in GVHD
`
`T
`
`Figure 6
`Cytokines (IL-1,IL-6. TNF) production in controls, GVHD
`and control ALL, CML and AML patients.
`
`Patients
`
`Figure 4 GM-CSF levels in GVHD patients and control normal
`donors.
`
`INF in CVHD
`
`c
`t -
`r 6
`
`Patienls
`Interferon IFN levels in GVHD patients and control normal
`
`Figure 5
`donors.
`
`levels of IL-Ip, IL-6 or TNFa in contrast to the patients
`who developed GVHD (Figure 6).
`In conclusion, a majority of the patients who developed
`cGVHD had high levels of inflammatory cytokines (IL-
`I j3, TNFa and IL-6) in their sera (Figure 6) compared to
`sera from normal healthy donors or controls.
`
`DISCUSSION
`
`Measurement of cytokines has become a valuable tool not
`only in elucidating pathophysiologic mechanisms but
`more importantly in diagnosis and prognosis of a variety
`of diseases. Previous studies carried out in patients with
`acute or chronic GVHD revealed a cytokine cascade that
`IL-lj3, IL-6, TNFa and
`includes production of
`I ~ ~ y . 6 9 .
`I I- I4
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`CYTOKINE DYSREGULATION IN cGVHD
`
`I73
`
`cytotoxic T lymphocyte (CTL) using cyclosporin A,
`FK506, Rifamicin, steroids, methotrexate, or monoclonal
`antibodies. One method attempted to avoid GVHD was T
`cell depletion' while cytokine antagonists (IL-4, IL- 10) or
`combinations of antagonists such as IL-IRA and sTNF-R
`could be used as an alternative approach. Recently, anti
`TNF compounds such as pentoxifylline and ciprofloxacin
`and anti TNF monoclonal antibodies, have been intro-
`duced as a means of prevention and treatment of trans-
`plant related toxicity and GVHD.**-*4 It is important to
`note that cytokine release (TNF-a, IL- 1 p) from host
`macrophages and endothelial tissues in response to injury
`due to conditioning regimens and/or endotoxins, is inde-
`pendent of HLA-restriction. This may explain the occur-
`rence of GVHD despite genotypic HLA identity between
`the donor and the recipient.
`IL-IRA has been shown to prevent GVHD in a murine
`model without impeding the immunologic and hematopoi-
`etic reconstitution,l7 and recently, clinical phase I/II trials
`using IL-IRA to treat steroid resistant GVHD have begun.
`In summary, we have shown increased levels of in-
`flammatory cytokines (IL-I p, IL-6, TNFa) in cGVHD pa-
`tients. Moreover, a statistically significant correlation was
`found between the severity of cGVHD and the levels of
`these cytokines. We believe that this correlation may be
`due to the role of inflammatory cytokines in the patho-
`physiology of cCVHD. Furthermore, levels of these cy-
`tokines may be used as a tool for assessing new therapeutic
`modalities as well as efficacy and response in GVHD.
`
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