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Michigan Oncology Journal Summer 99

The Separation of Graft-Versus Leukemia and Graft-Versus-Host Disease Through Cytokine "Shields"

James Ferrara, M.D.,
Professor of Internal Medicine and Pediatrics
Director, Blood and Marrow Transplantation Program

Allogeneic bone marrow transplantation (BMT) is an effective albeit toxic therapy of many hematologic malignancies. The curative properties of allogeneic BMT derive largely from a graft-versus-leukemia (GVL) effect. Unhappily, the GVL effect is very difficult to dissociate from graft-versus-host disease (GVHD), an often-lethal complication of allogeneic BMT. The separation of GVL from GVHD is therefore an extremely important therapeutic goal of BMT.

Acute GVHD Pathophysiology: Three Phases
Our understanding of the pathophysiology of graft-versus-host disease (GVHD) has improved with insights into the cellular interactions that are intrinsic to all inflammatory processes. Recent findings implicate a cascade of cytokines, proteins that are the central regulatory molecules of the immune system, as a primary cause for the induction and maintenance of GVHD (1, 2). This cascade can be conceptualized in three phases (see Figure 1). The first phase of acute GVHD starts before the donor stem cells are infused. The transplant-conditioning regimen damages and activates host tissues, including the intestinal mucosa, liver, and skin. Activated host cells secrete inflammatory cytokines, including tumor necrosis factor (TNF)-a and interleukin (IL)-1, granulocyte-machrophage colony stimulating factor (GM-CSF), and many others. The presence of inflammatory cytokines during this first phase upregulates adhesion molecules and major histocompatibility complex (MHC) antigens (3), thereby enhancing the recognition of host MHC or minor histocompatibility antigens by mature donor T cells present in the bone marrow harvest. We believe that the enhanced risk of GVHD after clinical BMT associated with intensive conditioning regimens is caused by extensive injury to epithelial and endothelial surfaces with a subsequent release of inflammatory cytokines (4, 5).

The second phase of acute GVHD consists of donor T-cell activation, including presentation of host antigens, activation of individual donor T cells, and proliferation and differentiation of activated T cells. Host antigen presenting cells (APCs) provide co-stimulatory activation signals by B7-CD28 interactions and IL-1. In addition to the T cell receptor (TCR), accessory molecules or T cells such as CD4, CD8, leukocyte function-associated antigen (LFA)-1, LFA-2, and CD44 participate in effector-target cell interactions by intensifying cellular contact and com-munication. The activation of individual donor T cells initiates the transcription of the genes for cytokines, such as IL-2, IL-12, IFNg, and their receptors. Once activated, donor T cells expand into clones and differentiate. Cytokines produced by T cells in response to alloantigens are predominantly secreted by the T-helper 1 (Th1) subset of T cells. Both IL-2 and IFNg play central roles at this juncture by amplifying T cell activation, inducing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cell responses, and priming additional donor and residual host mononuclear phagocytes to produce IL-1 and TNFa.

The complexity of the third phase of acute GVHD has only recently been appreciated. The initial hypothesis that CTL directly causes the majority of tissue damage in GVHD targets is too limited. Large granular lymphocytes (LGLs) or NK cells appear to be prominent in the effector arm of GVHD in several animal models, and they may contribute to the pathological damage (6). Mononuclear phagocytes also play an important role in this phase. Monocytes, which are primed with Th1 cytokines during phase two, vigorously secrete inflammatory cytokines such as TNFa and IL-1 when they receive a second, triggering signal. This signal may be provided by lipopolysaccharide (endotoxin, LPS), which can leak through the intestinal mucosa damaged by the conditioning regimen. TNFa can cause direct tissue damage by inducing necrosis of target cells, or it may induce tissue destruction during GVHD through apoptosis, or programmed cell death. Thus, the induction of inflammatory cytokines may synergize with the cellular damage caused by CTLs and NK cells (7, 8), resulting in the amplification of local tissue injury and further promotion of an inflammatory response, which ultimately leads to the observed target tissue destruction in the BMT host.

The conceptual framework of these three sequential steps helps to explain a number of unique and seemingly unrelated aspects of GVHD. The reduction in GVHD seen in gnotobiotic mice and in patients with aplastic anemia undergoing transplantation in laminar airflow environments with gut decontamination may be explained by the reduction of bacterial LPS on the skin and gut. The LPS may leak through damaged intestinal mucosal surfaces and stimulate the numerous gut-associated lymphocytes and macrophages to produce inflammatory cytokines. The beneficial effect of protective environments may be less apparent in patients receiving transplants for malignancies, because prior therapy and associated infections may have resulted in an environment that facilitates GVHD.

Interleukin IL-11 and Keratinocyte Growth Factor: Cytokine Shields for the GI Tract
Our understanding of the importance of the gastrointestinal (GI) tract to the development of GVHD has provided new opportunities to protect our patients from this disease. One new approach to the prevention of acute GVHD is to disrupt the amplification of inflammatory cytokine effectors by shielding the GI tract from early injury (4, 9). Recently identified biologic response modifiers such as keratinocyte growth factor (KGF) or interleukin-11 (IL-11), which have direct protective effects on the GI tract, offer an attractive new approach to GVHD prophylaxis.

KGF is a fibroblast growth factor family member (FGF-7) with a specificity for epithelial tissues expressing its receptors. We have recently shown that KGF administration for ten days, starting three days before BMT, reduces GVHD mortality and long term morbidity in an experimental mouse BMT model, primarily by protecting the GI tract from GVHD damage and subsequent inhibition of LPS translocation and TNFa generation. KGF (10) did not suppress T cell responses to host tissue and preserved a GVL response, resulting in a significant increase in leukemia-free survival (see Figure 2). The dramatic amelioration of gastrointestinal damage in KGF treated recipients subsequently reduced serum TNFa levels in these animals. As mentioned above, LPS is known to be a potent stimulus for inflammatory cytokine production (11), and it augments donor T cell activation, thereby amplifying both inflammatory and cellular effectors of GVHD. Administration of KGF to animals for ten days inhibited TNFa generation by protecting the GI epithelium from the GVHD injury that was mediated by both TBI and alloreactive T cells (4). Disruption of LPS leakage suppressed TNFa generation that mediated ongoing gut injury (4) and provided dramatic protection from GVHD.

Responses of allospecific donor T cells to host antigens were preserved in animals treated with KGF, and these T cell responses are critical to the maintenance of a graft versus leukemia (GVL) effect. KGF does not protect malignant cell lines from chemoradiotherapy in models studied to date, suggesting reduction in the leukemic burden by BMT conditioning will not be adversely affected by KGF administration. Given the favorable clinical toxicity profile of KGF (12), these compelling preclinical data make KGF an attractive candidate to study in clinical trials as an adjunct to standard GVHD prophylaxis. Such a trial is currently under development at the University of Michigan Cancer Center and should be open to patients later this year.

IL-11 is a second molecule that is of great interest for its ability to protect the GI tract and thereby prevent acute GVHD. Using the same mouse BMT model that showed that KGF could effectively separate GVL from GVHD, we have shown that brief administration (nine days) of IL-11 dramatically prevents lethal GVHD (see Figure 3). IL-11 prevented small bowel damage and reduced serum endotoxin levels by 80%. Treatment with IL-11 also reduced TNFa serum levels and suppressed TNFa secretion by macrophages to LPS stimulation in vitro (13). IL-11 is thus a second molecule that is likely to offer significant protection from GVHD by this new mechanism. A clinical trial of IL-11 in patients at high risk for GVHD should also be available later this year at the University of Michigan Cancer Center. We believe that these novel approaches hold the promise of making BMT a safer and better therapy, and thus available to more patients.

References

1. Ferrara J, Antin J. Pathophysiology of Human GVHD. In: Forman S, Blume K, Thomas E, editors. Hematopoietic Cell Transplantation: Blackwell Science, Inc. p. 305-315, 1999. 2. Antin JH, Ferrara JLM. Cytokine dysregulation and acute graft-versus-host disease. Blood. 80:2964-2968, 1992. 3. Norton J, Sloane JP. ICAM-1 expression on epidermal keratinocytes in cutaneous graft-versus-host disease. Transplant. 51:1203, 1991. 4. Hill GR, Crawford JM, Cooke KJ, Brinson YS, Pan L, Ferrara JLM. Total body irradiation effects on acute graft versus host disease. The role of gastrointestinal damage and inflammatory cytokines. Blood. 90(3204), 1997. 5. Clift RA, Buckner CD, Appelbaum FR, Bearman SI, Petersen FB, Fisher LB, et al. Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens. Blood. 76:1867-1871, 1990. 6. Ferrara JLM, Guillen PF, van Dijken PJ, Marion A, Murphy GF, Burakoff SJ. Evidence that large granular lymphocytes of donor origin mediate acute graft-versus-host disease. Transplant. 47:50-54, 1989. 7. Ghayur T, Seemayer TA, Kongshawn PAL, Gartner JS, Lapp WS. Graft-versus-host (GVH) reactions in the beige mouse: an investigation of the role of host and donor natural killer cells in the pathogenesis of GVH disease. Transplantation. 44:261-267, 1987. 8. Hakim FT, Sharrow SO, Payne S, Shearer GM. Repopulation of host lympohematopoietic systems by donor cells during graft-versus-host reaction in unirradiated adult F1 mice injected with parental lymphocytes. J Immunol. 146:2108-2115, 1991. 9. Cooke K, et al. TNFa production to LPS stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J Clin Invest. In press, 1998. 10. Krijanovski O, Hill G, Cooke K, Teshmia T, Crawford J, YS B, et al. Keratinocyte Growth Factor (KGF) separates graft-versus-leukemia effects from graft-versus-host disease. Blood. In press, 1999. 11. Nestel FP, Price KS, Seemayer TA, Lapp WS. Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease. J Exp Med. 175:405-413, 1992. 12. Serdar C, Heard R, Prathikanti D, Lau D, Danilenko D, Hunt T, et al. Safety, pharmacokinetics and biologic activity of rHuKGF in normal volunteers: results in a placebo-controlled randomized double-blind phase 1 study. Blood. 90 (Suppl. 1):172, 1997. 13. Hill GR, Cooke KR, Teshima T, Crawford JM, Keith JC, Brinson YS, et al. Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation. J Clin Invest. 102:115-123, 1998. Figure 1. The pathophysiology of acute GVHD can be summarized as a process with three phases. Figure 2. Preservation of allogeneic GVL effects in KGF treated mice. B6D2F1 mice were conditioned with 1500 cGy TBI and transplanted with 5 x 106 bone marrow and 0.5 x 106 T cells from B6 donors together with 5000 P815 tumor cells. Recipients of T cell depleted (TCD) bone marrow or bone marrow plus T cells from allogeneic B6 donors were injected with KGF or control diluent from day -3 to +7. Results are represented as Kaplan-Meier cumulative survival estimates from two similar experiments. Control diluent treated TCD recipients (_.._.._.,n=11), KGF treated TCD recipients (______, n = 6), control allogeneic BMT recipients (____ _ _ , n = 28 ), KGF allogeneic BMT recipients ( ______, n = 17). KGF vs control (allogeneic BMT groups), P < 0.0001. Adapted from (10). Figure 3. IL-11 reduces death from acute GVHD. B6D2F1 mice received allogeneic BMT as in Figure 2. IL-11 or control diluent was injected subcutaneously from day -2 to day +7. Control treated animals (dashed line, n=20) had less than 20% survival by day 50 compared to 90% in IL-11 treated animals (solid line, n=20). (Adapted from (13).

 

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Please note: The articles listed in the Cancer Center's News Archive are here for historical purposes. The information and links may no longer be up-to-date.