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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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