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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2581-2589
A Role for Transforming Growth Factor- 1 in the Increased
Pneumonitis in Murine Allogeneic Bone Marrow Transplant Recipients With
Graft-Versus-Host Disease After Pulmonary Herpes Simplex Virus Type 1 Infection
By
Heiko Adler,
Janice L. Beland,
Wende Kozlow,
Nadia C. Del-Pan,
Lester Kobzik, and
Ilonna J. Rimm
From the Division of Pediatric Hematology-Oncology, Dana-Farber
Cancer Institute, Department of Pediatrics, Children's Hospital,
Harvard Medical School; and the Physiology Program, Harvard School of
Public Health and Department of Pathology, Brigham and Women's
Hospital, Boston, MA.
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ABSTRACT |
To gain further insights in the pathogenesis of herpesvirus
pneumonia in allogeneic bone marrow transplant recipients, transplanted mice (B10.BR  CBA) with graft-versus-host
disease (GVHD) and control mice (transplanted mice without GVHD and
normal CBA mice) were infected intranasally with herpes simplex virus
type 1 (HSV-1). When compared with infected control mice, infected
allogeneic transplant recipients with GVHD showed increased periluminal
mononuclear cell infiltrates. However, infected allogeneic transplant
recipients with GVHD showed lower virus content in the lung tissue than
infected control mice. High concentrations of transforming growth
factor-beta 1 (TGF- 1) were detected in the bronchoalveolar lavage
(BAL) fluid of mock-infected allogeneic transplant recipients with
GVHD, which increased slightly after infection. Anti-TGF- treatment
of allogeneic transplant recipients with GVHD significantly decreased
the histological evidence of pneumonitis at day 4 after HSV-1
infection. We conclude that allogeneic transplant recipients with GVHD
have (1) increased pneumonia, (2) highly elevated levels of TGF-1 in
the BAL fluid, and (3) reduced pulmonary virus content after HSV-1
infection. Our data suggest that the newly recognized dysregulation of
cytokine (TGF-1) production may be more important than the viral
load for the increased severity of HSV-1 pneumonia in allogeneic
transplant recipients with GVHD.
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INTRODUCTION |
ALLOGENEIC BONE MARROW transplantation
(BMT) has become an important treatment for a number of hematologic,
malignant, and genetic disorders. Late infectious complications are the
leading cause of nonrelapse death, and they are often associated with the presence of graft-versus-host disease (GVHD).1,2 A
recent study showed that viruses, particularly herpesviruses including cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1), caused
37% of late infections after allogeneic BMT.3 The lung was
one of the most common single organ sites of life-threatening infections, accounting for 22.8% of the localized
infections.3
Our new model of HSV-1 pneumonia4 allows a direct
comparison of viral pneumonia in normal and immunocompromised
(transplanted) animals. The HSV-1 system may be more useful for
clinically related studies than the murine cytomegalovirus (MCMV)
model, because MCMV causes pneumonia only in immunosuppressed, but not
normal mice.5,6 Antigen-specific humoral and cellular
mechanisms and innate immunity may protect against HSV
infection.7,8 Antibody-mediated protection in vivo may
involve neutralization, antibody-dependent cellular
cytoxicity, and Fc receptor-dependent mechanisms.8 Cellular protective mechanisms against HSV
include T-cell-mediated cytokine release by CD4+ T cells
and cytolysis of infected cells by CD8+ T
cells.8,9 Mediators of innate immunity include macrophages and natural killer cells.7,10 For example, macrophages
stimulated by interferon- (IFN-) inhibit the replication of HSV-1
by the generation of nitric oxide (NO).11,12
In contrast to the protective responses described above, immune
responses during viral infections can also be detrimental to the host.
For example, T cells mediate the tissue injury in herpetic stromal
keratitis.13 Furthermore, the inflammatory response
associated with NO production can increase pathology; we showed
recently that inhibition of NO production decreased the cellular influx
and pathology in HSV-1 pneumonia.4
In this study, we wished to model late posttransplant pulmonary
infection in BMT recipients, in particular with regard to the presence
or absence of GVHD. Specifically, we wanted to address the following
questions. First, do allogeneic BMT recipients with GVHD develop more
severe pneumonitis than allogeneic BMT recipients without GVHD after
pulmonary HSV-1 infection? Second, is there a correlation between the
severity of HSV-1-induced pneumonitis and the pulmonary viral titer?
Finally, are there differences in the pulmonary immune response after
HSV-1 infection with regard to inflammatory cell influx and cytokines
between allogeneic BMT recipients with and without GVHD?
Previous murine studies of posttransplant viral infections were
performed in the early period after BMT rather than after engraftment.14 Because the current clinical problem is
infection after engraftment, mice were infected intranasally with HSV-1 at 12 weeks after BMT across minor histocompatibility
differences.15 The number of T cells injected into
recipient mice was chosen to produce a mild GVHD in the experimental
group (Allogeneic GVHD) to determine the effects of mild GVHD on
HSV-1-induced pneumonia. Recipients injected with T-cell-depleted
bone marrow only (Allogeneic No GVHD), as well as normal CBA mice, were
used as controls.
Our data show that, when compared with control mice, allogeneic
transplant recipients with GVHD had more severe pneumonitis, reduced
pulmonary influx of inflammatory cells, and antibody production after
intranasal HSV-1 infection as well as highly elevated levels of
transforming growth factor-beta 1 (TGF-1) in the bronchoalveolar lavage (BAL) fluid. Unexpectedly, they also showed reduced pulmonary virus content. Treatment with anti-TGF- monoclonal antibody (MoAb) decreased the histological evidence of pneumonitis. Therefore, dysregulated cytokine production may be more important than the cytopathic effects of the virus in the development of HSV-1 pneumonia in allogeneic transplant recipients with GVHD.
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MATERIALS AND METHODS |
Mice and BMT.
Female CBA/J (H2k) and B10.BR (H2k) mice were
purchased from The Jackson Laboratories (Bar Harbor, ME), and BMT was
performed between the ages of 10 to 14 weeks. Bone marrow cells were
obtained from the femurs and tibias of donor B10.BR mice, and
transplanted into B10.BR recipients as syngeneic transplant control.
After T-cell depletion by treatment with anti-Thy1.2 and complement (Accurate Chemicals & Scientific Corp, Westbury, NY), 5 × 106 bone marrow cells alone or mixed with 4 × 105 nylon wool nonadherent donor splenic T cells were
resuspended in Leibovitz's L-15 medium (Life Technologies, Grand
Island, NY) and transplanted into CBA recipients via tail vein infusion
(0.5 mL total volume per mouse). Before transplantation, recipient mice
received 11 Gy of total body irradiation (137Cs source)
delivered in two fractions separated by 3 hours to reduce
gastrointestinal toxicity. Mice were subsequently housed in sterilized
micro-isolator cages and received normal chow and autoclaved
hyperchlorinated water for the first 2 weeks after BMT and filtered
water thereafter.
Assessment of GVHD.
The severity of GVHD was assessed by following the weights of mice
after BMT. Failure to gain weight has been found to be a reliable
indicator of systemic GVHD in this murine model.16 Using
the above described transplant parameters, a mild GVHD was induced in
allogeneic recipients transplanted with donor bone marrow and nylon
wool nonadherent donor splenic T cells. Approximately 80% of the
allogeneic transplanted mice with GVHD were available for infection at
12 weeks after BMT. At this time point after BMT, the mice with GVHD
showed a 20% weight loss due to GVHD as compared with the control
mice. The mortality related to the BMT in this model occurred within
the first 6 to 8 weeks after BMT; beyond 8 weeks after BMT, little
further mortality was observed.
Infection of mice.
Mice were infected intranasally with 2.5 × 105
plaque-forming units of HSV-1, strain KOS
1.1,17 kindly provided by Drs R. Finberg and J. Brubaker
(Dana-Farber Cancer Institute, Boston, MA), as described.4
This viral dose caused pneumonitis without mortality. The virus stocks
were prepared as clarified lysates of infected Vero cells. Briefly,
Vero cells were infected at a multiplicity of infection of 0.1. After
72 hours, virus stocks were prepared, after removing the supernatants,
by two rounds of freezing and thawing of the infected cell pellets in
culture medium. The clarified lysates were aliquoted and stored at
 80°C until used. Uninfected control stocks were prepared
from Vero cells in the same manner. The Vero cells and culture media
were screened and shown to be free of mycoplasma and endotoxin
contamination.
Tissue sampling and flow cytometry.
BAL and preparation of lung tissue for virus titration and histological
examination were performed as previously described.4 BAL
lymphocytes were stained for flow cytometry and analyzed using CellQuest on a FACScan (Becton Dickinson, Mountain View,
CA) as described.4
Virus titration and histology.
Viral titers in lung tissue were determined by standard plaque assay on
Vero cells as described.4 Sections for histopathologic examination were prepared as described,4 and analyzed and
scored microscopically in a blind study on coded slides as previously described.16
Determination of HSV-1-specific antibodies.
HSV-1-specific IgG1 and IgG2a antibodies in BAL samples of mice 14 days after infection were determined as described,4 using a
specific enzyme-linked immunosorbent assay (ELISA).18 In
all assays, samples and standards were run at least in duplicate. The
quantitation of HSV-1-specific antibodies was performed by the method
of Zollinger and Boslego.19
Cytokine determinations.
Levels of IFN-, IL-2, IL-4, IL-10, IL-5, tumor necrosis factor-
(TNF-), and TGF- in BAL fluids were determined by ELISA. Except
for TGF-, all measurements were performed using recombinant cytokine
standards, and "pairs" of capture and peroxidase-conjugated secondary antibodies, all purchased from Pharmingen (San Diego, CA),
were used according to the protocol supplied by
Pharmingen. The plates were developed using avidin-peroxidase and
2,2 -Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)
substrate (Sigma Chemical Co, St Louis, MO), and read at
405 nm using an ELISA reader. The lower detection limit for all assays
was 100 pg/mL. In some cases, BAL fluids were tested after
approximately 5-fold concentration by using Centricon-10 concentrators
(Amicon, Beverly, MA). Total TGF-1 in BAL fluids was assayed using a
previously described capture ELISA.20 Briefly, 96-well
microtiterplates were coated overnight at 4°C with polyclonal
chicken-anti-TGF-1 antibody (R & D Systems, Minneapolis, MN), and
then blocked for 2 hours at room temperature with 10% BSA
Diluent/Blocking Solution Concentrate (Kirkegaard & Perry Laboratories
Inc, Gaithersburg, MD). After washing with phosphate-buffered saline
containing 0.05% Tween 20, 50 µL of samples, or recombinant human
TGF-1 (R & D Systems) as standard were added to duplicate wells,
followed by overnight incubation at 4°C. After washing, plates were
incubated with a mouse anti-TGF-1, -2, -3 MoAb (Genzyme,
Cambridge, MA) for 45 minutes at room temperature. After washing,
plates were incubated with peroxidase-labeled goat anti-mouse IgG
(Kirkegaard & Perry) for 30 minutes at room temperature, washed again,
developed using 2,2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS) substrate (Sigma), and read at 405 nm. This ELISA has been
shown to be 10-fold more sensitive in measuring TGF- than the
bioassay using mink lung epithelial cells.20
Treatment of mice with anti-TGF- MoAb.
Mouse anti-TGF-1, -2, -3 MoAb (Genzyme) was administered at a
dose of 100 µg per injection intraperitoneally (IP) in a volume of
0.2 mL at days 1, 1, and 3.21,22 Control mice were treated with the same amount of mouse IgG (Sigma). Mice were infected intranasally at day 0, and lung samples were prepared for histological examination at day 4 after infection.
Statistical analyses.
The Mann-Whitney test and the Student's t-test (unpaired) were
used for data analysis where appropriate. Group differences in the
viral titer per lung over time after infection (see Fig 3B) were
analyzed by the Kruskal-Wallis test  a nonparametric analysis of
variance (ANOVA).
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| Fig 3.
Allogeneic transplant recipients with GVHD showed less
pulmonary virus content. Viral titers in the lungs were determined by
plaque assay at day 4 (A) and at the indicated time points (B) after
intranasal HSV-1 infection. Data represent means ± SE of individual
mice, derived from at least two separate experiments. Numbers of mice
in (A) were: 7 (normal CBA), 5 (allogeneic GVHD), and 3 (allogeneic No
GVHD). Numbers of mice in (B) were: 3, 3, 3, 3, 7, and 6 (normal CBA; 3 hours, Days 1, 2, 3, 4, and 7, respectively); and 4, 5, 3, 5, 5, and 3 (allogeneic GVHD; 3 hours, Days 1, 2, 3, 4, and 7, respectively). The
asterisk in (A) indicates a significant difference from both normal CBA
and allogeneic No GVHD (P < .04; Student's
t-test). The difference between normal CBA and allogeneic GVHD (B)
was significant (P = .0037 using the Kruskal-Wallis test, a
nonparametric ANOVA).
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RESULTS |
Increased periluminal mononuclear cell infiltrates in allogeneic
transplant recipients with GVHD.
Allogeneic transplanted mice (B10.BR CBA) with GVHD and, as
controls, allogeneic transplanted mice without GVHD and syngeneic transplanted mice (B10.BR B10.BR) were infected intranasally with HSV-1 at 12 weeks after transplantation. As an additional control,
age-matched normal CBA mice were infected as well. Histological examination of lungs was performed in mock-infected mice and in mice at
day 7 after infection. Allogeneic transplant recipients with GVHD
showed increased pathology
(Fig 1).
Scores reflecting the periluminal histopathologic changes were
significantly higher (P = .05; Student's t-test) in
allogeneic transplant recipients with GVHD, when compared with control
mice (Fig 2). The periluminal histopathologic scores were also higher in allogeneic transplant recipients with GVHD, when compared with normal CBA mice, at days 4, 10, and 14 after infection (data not shown). No significant differences
were observed in the scores reflecting the parenchymal histopathologic
changes (data not shown). Because the results in syngeneic transplanted
mice were very similar to those in allogeneic transplanted mice without
GVHD, the latter, which are the most appropriate control for procedural
effects and for the effect of GVHD, and normal CBA mice, were used as
controls in further experiments.
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| Fig 1.
Allogeneic transplant recipients with GVHD
showed increased pathology. Photomicrographs of lung sections stained
with hematoxylin and eosin show evidence of increased pathology in
infected allogeneic GVHD mice. (A) Normal CBA, mock-infected, original
magnification × 25; (B) normal CBA, mock-infected, original
magnification × 100; (C) normal CBA, infected, original
magnification × 40; (D) normal CBA, infected, original
magnification × 100; (E) allogeneic GVHD, mock-infected, original
magnification × 10; (F) allogeneic GVHD, mock-infected, original
magnification × 75; (G) allogeneic GVHD, infected, original
magnification × 10; (H) allogeneic GVHD, infected, original
magnification × 100.
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| Fig 2.
Allogeneic transplant recipients with GVHD showed
increase of periluminal mononuclear infiltrates. The pathology scores
of mock-infected mice and mice 7 days after intranasal HSV-1 infection
were determined. Data represent means ± SE of individual mice,
derived from at least two separate experiments. Numbers of
mock-infected mice were: 11 (normal CBA), 9 (allogeneic GVHD), 6 (allogeneic No GVHD), and 2 (syngeneic). Numbers of infected mice were:
17 (normal CBA), 13 (allogeneic GVHD), 8 (allogeneic No GVHD), and 4 (syngeneic). The asterisks indicate a significant difference from both
normal CBA and allogeneic No GVHD (P = .05; Student's
t-test).
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Allogeneic transplant recipients with GVHD showed lower pulmonary
virus burden.
Because infected allogeneic transplant recipients with GVHD displayed
more severe pulmonary pathology when compared with allogeneic transplant recipients without GVHD, higher pulmonary viral titers were
expected in mice with GVHD. However, somewhat surprisingly, allogeneic
transplant recipients with GVHD had significantly (P < .04;
Student's t-test) less pulmonary virus content than both control groups at day 4 after infection
(Fig 3A). Kinetic studies comparing
allogeneic transplant recipients with GVHD and normal CBA mice showed
that allogeneic transplant recipients with GVHD had lower pulmonary
virus content than normal CBA mice at all time points (Fig 3B). The
difference in viral titer per lung over time after infection between
normal CBA mice and allogeneic transplant recipients with GVHD was
significant (P = .0037 using nonparametric ANOVA). The virus
was cleared from the lungs in all groups of mice at day 7 after
infection.
Reduced influx of lymphocytes to the lungs in allogeneic transplant
recipients with GVHD.
Intranasal infection with HSV-1 led to a pronounced increase in the BAL
lymphocyte number at day 7 after infection
(Fig 4). In all groups of both
mock-infected and infected mice, we observed a dominance of
CD3+ T cells. The vast majority of the cells were /
TCR+; only very low numbers of / TCR+
cells were detected (data not shown). In mock-infected mice, we found
higher numbers of CD4 than CD8+ cells. After infection, the
CD4/CD8 ratio in infected control mice changed to 1:3. However, in
allogeneic transplant recipients with GVHD, the corresponding ratio was
only 1:1 (Fig 4).
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| Fig 4.
Characterization of the lymphocyte subsets in the BAL
fluid of mock-infected mice and mice 7 days after intranasal HSV-1
infection. The number of lymphocytes was calculated from the percentage
determined by fluorescence-activated cell sorter (FACS) analysis. Data
represent means ± SE of individual or pooled BAL samples, derived
from at least two separate experiments. Numbers of mock-infected mice
were: 12 (normal CBA), 12 (allogeneic GVHD), and 6 (allogeneic No
GVHD). Numbers of infected mice were: 17 (normal CBA), 13 (allogeneic
GVHD), and 8 (allogeneic No GVHD). The asterisks indicate a significant
difference from both normal CBA and allogeneic No GVHD (P < .02; Student's t-test).
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This result suggested a selective deficit in the influx of
CD8+ T cells to the lungs of allogeneic transplant
recipients with GVHD. When expressed as a multiple of cells in infected
versus mock-infected mice, the increase in the total lymphocyte number in the control mice was between 24- to 36-fold
(Fig 5A), and in the CD8+
T-cell number, between 195- to 355-fold (Fig 5B). However, allogeneic transplant recipients with GVHD showed only a 7-fold increase of total
lymphocytes (Fig 5A), and only a 39-fold increase of CD8+ T
cells (Fig 5B) (P < .04; Student's t-test).
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| Fig 5.
Allogeneic transplant recipients with GVHD showed a
reduced pulmonary influx of lymphocytes. BAL cell numbers of
mock-infected mice and mice 7 days after intranasal HSV-1 infection
were determined. (A) Total lymphocytes recovered by BAL. (B)
CD8+ T cells recovered by BAL. Numbers are expressed as
the multiple of the cells recovered in infected versus mock-infected
mice. The numbers were calculated from the percentages determined by
FACS analysis. Data represent means ± SE of individual or pooled BAL
samples, derived from at least two separate experiments. Numbers of
mice were as shown in Fig 4. The asterisks indicate a significant
difference from both normal CBA and allogeneic No GVHD (P < .04; Student's t-test). The error bar for the allogeneic no
GVHD group in (B) falls within the symbol.
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Reduced production of HSV-1-specific antibodies in allogeneic
transplant recipients with GVHD.
Because a humoral immune response may also be important in HSV-1
infection,8 we determined HSV-1-specific IgG1 and IgG2a, the latter isotype being predominantly induced during viral
infections,23 in BAL fluids of infected mice. Allogeneic
transplant recipients with GVHD produced significantly less antibodies
of the IgG2a subclass, when compared with infected control mice
(P < .04; Student's t-test)
(Fig 6).
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| Fig 6.
Allogeneic transplant recipients with GVHD showed reduced
HSV-1-specific antibody production. HSV-1-specific IgG1 (A) and IgG2a
(B) antibodies in the BAL fluid were determined by ELISA 14 days after
intranasal HSV-1 infection. Data represent means ± SE of individual
or pooled BAL samples, derived from at least two separate experiments.
Numbers of mice were: 5 (normal CBA), 4 (allogeneic GVHD), and 10 (allogeneic no GVHD). The asterisks indicate a significant difference
from both normal CBA and allogeneic no GVHD (P < .04;
Student's t-test) for IgG2a, and a significant difference from
normal CBA (P < .02; Student's t-test) for IgG1.
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Increased levels of TGF-1 in allogeneic transplant recipients with
GVHD.
Because cytokines are involved in the pathogenesis of viral lung
infections24 as well as in the pathophysiology of
GVHD,25 we determined cytokine levels in BAL fluid of
mock-infected mice, and at an early (day 4) as well as late (day 14)
time point after infection. TGF-1 and TNF- were measured, because
these cytokines have been shown to be induced after pulmonary radiation
injury26 as well as during pulmonary GVHD.27
Interestingly, both mock-infected as well as infected allogeneic
transplant recipients with GVHD showed markedly elevated levels of
BAL-fluid TGF-1, when compared with control mice
(Fig 7). Mice transplanted with higher
T-cell numbers, and therefore increased GVHD, showed further elevated levels of TGF-1 in the BAL fluid (data not shown). Elevated levels of TGF-1 were not detected in the sera (data not shown). Additional cytokines assayed were IFN-, IL-2, IL-4, IL-5, TNF-, and IL-10. Only IFN- was readily detectable in BAL fluids of normal CBA mice at
days 4 and 7 after infection. None of the other cytokines could be
detected in BAL fluids using ELISA, even after concentration of the
samples (data not shown). However, cytokine levels in BAL fluid
represent the excess after consumption or degradation,24 and the cytokine levels may be below the detection limit of ELISA.
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| Fig 7.
Allogeneic transplant recipients with GVHD showed
increased levels of TGF-1 in the BAL fluid. Total TGF-1 was
determined by ELISA. Data represent means ± SE of individual or
pooled BAL samples, derived from at least two separate experiments.
Numbers of mice were: Day 0: 8, 9, and 7; Day 4: 6, 4, and 6; Day 14:
6, 4, and 10; for normal CBA, allogeneic GVHD, and allogeneic no GVHD,
respectively. BAL fluids of mock-infected mice were collected at
several time points after mock infection. Because we observed no
differences in the TGF-1 levels at different time points after mock
infection, the data are presented together. The asterisks indicate a
significant difference from both normal CBA and allogeneic no GVHD
(P < .05; Student's t-test).
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Treatment with anti-TGF- MoAb decreased the histological evidence
of pneumonitis in infected allogeneic transplant recipients with GVHD.
Because highly elevated levels of TGF-1 were measured in the BAL
fluid of allogeneic transplant recipients with GVHD, we hypothesized
that TGF-1 might be involved in the pathogenesis of HSV-1-induced
pneumonia in allogeneic transplant recipients with GVHD. To test this
hypothesis, infected allogeneic transplant recipients with GVHD were
treated with anti-TGF- MoAb, or as a control, with mouse IgG.
Treatment with anti-TGF- MoAb significantly decreased the
histological evidence of pneumonitis (Fig
8). We did not observe changes in the pulmonary viral titer or in the number and composition of the cells recovered by BAL after treatment with anti-TGF- MoAb (data not shown).
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| Fig 8.
Treatment with anti-TGF- MoAb decreased the
histological evidence of pneumonitis in infected allogeneic transplant
recipients with GVHD. Animals were treated with 100 µg anti-TGF-
or control IgG at days 1, 1, and 3. Mice were infected intranasally
at day 0, and histology was performed 4 days after infection. Data
represent means ± SE of six individual mice per group, derived from
three separate experiments. The difference between
anti-TGF-treated animals and IgG control-treated animals was
significant (P = .0336; Mann-Whitney test).
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DISCUSSION |
In this study, intranasal HSV-1 infection of murine BMT recipients was
used to gain insights in the pathogenesis of HSV-1-induced pneumonitis
in allogeneic transplant recipients. The key findings were that (1)
allogeneic transplant recipients with GVHD showed more severe
pneumonitis, when compared with allogeneic transplant recipients
without GVHD or normal CBA mice; (2) allogeneic transplant recipients
with GVHD showed less pulmonary virus than the control mice; and (3)
allogeneic transplant recipients with GVHD showed highly elevated
levels of TGF-1 in the BAL fluid. Treatment with anti-TGF-
significantly decreased the histological evidence of pneumonitis.
Our studies were designed to model pneumonia in a clinical BMT unit.
Previous studies of viral infection after BMT predominantly used the
Parent into F1 model, combined with virus inoculation in the immediate
peritransplant period.28-30 These Parent into F1 models did
not involve irradiation.28-30 Because patients receive total body irradiation, accompanied by the induction of
cytokines25 capable of affecting the immune response to a
subsequent infection, a murine BMT system involving irradiation is
closer to the clinical setting. In this study, mice were infected at 12 weeks after transplantation, which is more appropriate for the clinical
problem of late infection.
The results clearly showed that the increased pneumonitis was due to
GVHD, because allogeneic transplant recipients without GVHD, which also
received irradiation, showed less pneumonitis than mice with GVHD.
Viral inoculation after allogeneic BMT resulted in an exacerbation of
both viral infection and GVHD.28,31-34 However, mice with
the more severe pneumonitis (allogeneic GVHD) had lower pulmonary virus
content, when compared with control mice (allogeneic no GVHD, normal
CBA). Therefore, the data suggested that the histopathology, rather
than the viral titer, is the more important endpoint. We showed in our
previous study that the histopathology correlated with lung compliance,
a parameter of in vivo global lung function.4
Previously published data suggested that pulmonary pathology and viral
titer may not be correlated. Shanley et al14 have found
higher viral titers in the lungs of allogeneic transplanted mice, when
compared with syngeneic recipients, after infection with murine CMV. In
that study, mice were infected subcutaneously early (14 days) after
BMT, and viral titers in the lung were determined 14 days after
infection. The difference between the two studies might be due to the
different time point of infection after BMT (14 days v 12 weeks), to the different virus and route of infection used, or to
differences in the immune response to MCMV as opposed to HSV-1. The
presence or absence of lung disease was not investigated in that study.
However, Grundy et al30 showed that pulmonary disease did
not correlate with the level of MCMV replication in the lung after IP
infection of mice with GVHD in the Parent into F1 model. The lack of
correlation between the extent of pneumonitis and pulmonary viral
titers in MCMV lung infection is in agreement with our earlier
work4 and with the results of our current study in
HSV-1-induced pneumonitis.
The result that allogeneic transplant recipients with GVHD showed more
severe pneumonitis in the presence of less virus suggested that
alterations in the immune response of allogeneic transplant recipients
with GVHD to HSV-1 infection may be more important for the development
of pneumonitis than direct viral cytocidal effects. In particular, we
hypothesized that an impaired cytokine production may be involved.
Inappropriate production of cytokines may play an important role in the
induction and progression of GVHD.25
Indeed, the second major finding was that allogeneic transplant
recipients with GVHD had highly elevated BAL fluid TGF-1 levels.
TGF- is a pleiotropic cytokine and can exert both immunosuppressive and proinflammatory effects.35 For example, TGF-
inhibits lymphocyte proliferation,35 suppresses human
immunodeficiency virus expression and replication in infected
cells,36 induces apoptosis,37 downregulates NO
production,38 and is involved in fibrotic disorders of the
lung by its ability to upregulate collagen expression.35 Thus, TGF- might be responsible for the decreased BAL lymphocyte number by interfering with lymphocyte proliferation and/or
influx. By its immunosuppressive effects, TGF- may enhance
susceptibility to lung infections.3 TGF- may account for
the lower pulmonary virus content observed in allogeneic transplant
recipients with GVHD by inhibiting proliferation of lung epithelial
cells, a HSV-1 replication site,39,40 or by causing
apoptosis of epithelial cells. TGF- may be involved in the apoptosis
of epithelial cells which is induced during GVHD.25,41
Furthermore, TGF- is involved in the late fibroproliferative phase
of the idiopathic pneumonia syndrome, predominantly in patients with
chronic GVHD.42 We did not observe changes in the pulmonary
viral titer and in the BAL cell number after treatment with
anti-TGF- MoAb, possibly due to the particular dose of antibody and
time point studied in our experiments.
This is the first report of a role of TGF-1 in the pathogenesis of
HSV-1-induced pneumonia in allogeneic transplant recipients with GVHD.
Interestingly, TGF- was shown to be involved in intestinal GVHD.43 The BAL fluid TGF-1 levels increased slightly
after i.n. infection in allogeneic transplant recipients with GVHD. In
contrast, TGF-1 was induced after intranasal infection of normal
mice, in agreement with the report of the generation of TGF- after
IP lymphocytic choriomeningitis virus infection of mice.44,45
If the immune response is more important than the viral load, curative
therapy for posttransplant viral infections may include the modulation
of immune functions. The specific inhibition of mediators of the immune
response, which may be produced in excess in response to a given
infection and during GVHD, might be an effective treatment for
posttransplant infection. Consistent with this hypothesis, we could
show that anti-TGF- treatment decreased the histological evidence
of pneumonitis.
TGF- downregulates NO,38 which is produced during
GVHD46 and HSV-1-induced pneumonitis.4
Consequently, whereas elevated levels of nitrite/nitrate in BAL fluids
of infected normal mice were detected after intranasal HSV-1 infection,
no increase was observed in allogeneic transplant recipients with GVHD.
This absence of nitrite/nitrate elevation may be due to the high
TGF-1 concentrations in the BAL fluid of these animals. Thus,
inhibition of NO production suppressed histological evidence of
HSV-1-induced pneumonia in normal mice, but not in allogeneic
transplant recipients with GVHD.4 Because TGF-
downregulates NO production, inhibition of TGF- may lead to an
enhanced generation of NO. Therefore, in allogeneic transplant
recipients with GVHD, a combined treatment with inhibitors of both
TGF- and NO may be more effective than anti-TGF- alone.
In conclusion, we showed that allogeneic transplant recipients with
GVHD developed more severe pneumonitis after intranasal HSV-1 infection
than control mice without GVHD, and that in allogeneic transplant
recipients with GVHD an enhanced TGF-1 production may be more
important for the development of pneumonitis than the pulmonary viral
load. Consistent with this hypothesis, we could show that anti-TGF-
treatment decreased the histological evidence of pneumonitis.
| |
FOOTNOTES |
Submitted November 20, 1997;
accepted June 2, 1998.
Supported by grants from the National Institutes of Health, the
Mallinckrodt Foundation, and the Aid for Cancer Research. H.A. is
supported by a grant from the Deutsche Forschungsgemeinschaft (DFG).
I.J.R. is a Claudia Adams Barr Investigator.
Address reprint requests to Ilonna J. Rimm, MD, PhD, Genetics
Institute, 87 CambridgePark Dr, Cambridge, MA 02140; e-mail: irimm{at}genetics.com.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Steven Burakoff for helpful discussions, and Linda Callahan
for the expert technical support. We thank Drs V. Kuchroo and M. Prabhu
Das for the help in establishing the TGF-1 ELISA.
| |
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Abstract
Introduction
Abstract