Interactions of keratinocyte growth factor with a nitrating species after marrow transplantation in mice

Imad Y. Haddad1, Angela Panoskaltsis-Mortari2, David H. Ingbar3, Ernesto R. Resnik1, Shuxia Yang1, Catherine L. Farrell4, David L. Lacey4, David N. Cornfield1, and Bruce R. Blazar2

Divisions of 1 Pulmonary and Critical Care Medicine, and 2 Bone Marrow Transplantation, Department of Pediatrics, and 3 Department of Pulmonary Medicine, University of Minnesota, Minneapolis, Minnesota 55455; and 4 Amgen Incorporated, Thousand Oaks, California 91320


    ABSTRACT
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ABSTRACT
INTRODUCTION
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We reported that allogeneic T cells given to irradiated mice at the time of marrow transplantation stimulated tumor necrosis factor (TNF)-alpha , interferon (IFN)-gamma , and nitric oxide (· NO) production in the lung, and the addition of cyclophosphamide (known to stimulate superoxide production) favored the generation of a nitrating species. Although keratinocyte growth factor (KGF) prevents experimental lung injury by promoting epithelial repair, its effects on the production of inflammatory mediators has not been studied. KGF given before transplantation inhibited the T cell-induced increase in bronchoalveolar lavage fluid protein, TNF-alpha , IFN-gamma , and nitrite levels measured on day 7 after transplantation without modifying cellular infiltration or proinflammatory cytokines and inducible · NO synthase mRNA. KGF also suppressed · NO production by alveolar macrophages obtained from mice injected with T cells. In contrast, the same schedule of KGF failed to prevent permeability edema or suppress TNF-alpha , IFN-gamma , and · NO production in mice injected with both T cells and cyclophosphamide. Because only epithelial cells respond to KGF, these data are consistent with the production of an epithelial cell-derived mediator capable of downregulating macrophage function. However, the presence of a nitrating agent impairs KGF-derived responses.

nitric oxide; peroxynitrite; idiopathic pneumonia syndrome; polymerase chain reaction; proinflammatory cytokines


    INTRODUCTION
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INTRODUCTION
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IDIOPATHIC PNEUMONIA SYNDROME (IPS) remains a significant cause of morbidity and mortality and a major factor limiting the success of bone marrow (BM) transplantation (BMT). Allogeneity is an established risk factor for the development of IPS (5, 19, 39). Once infiltrating T cells, alloactivated by antigen-presenting cells, encounter pulmonary antigens, immune-mediated damage begins. Major mediators responsible for killing by cytolytic T cells are via release of perforin (cytolysin, a pore-forming protein) or granzymes (10) and a Fas-dependent lytic pathway (23). In addition, T cells can activate alveolar macrophages to release tissue-damaging mediators such as tumor necrosis factor (TNF)-alpha (3).

Our group (14) recently reported that alveolar macrophage-derived reactive oxygen and nitrogen species such as nitric oxide (· NO), superoxide (O-2·), and · NO-derived species play a central role in the development of lung injury in a murine IPS model after allogeneic BMT. The specific nature of the reactive species was dependent on the transplantation conditions. Total body-irradiated (TBI) mice given alloreactive donor spleen T cells at the time of BMT (BMS) developed lung dysfunction associated with the induction of TNF-alpha , interferon (IFN)-gamma , and · NO. However, the combined injection of T cells and a conditioning regimen of cyclophosphamide (Cy), known to enhance O-2· production (4), favored the generation of a nitrating agent, most likely peroxynitrite (ONOO-), formed by the rapid reaction between · NO and O-2· (18a). The presence of a nitrating agent was associated with the greatest severity of lung dysfunction. Because Cy in the absence of donor T cells did not result in significant lung dysfunction (35), ONOO- formation clarifies the dependence of Cy-facilitated injury and lethality on the presence of allogeneic T cells.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is protective in lethal experimental lung injury models of hyperoxia, acid instillation, radiation, and bleomycin (32, 47, 48). The mechanisms of the protective effects of KGF involve the selective stimulation of alveolar type II pneumocyte proliferation and restoration of normal alveolar structure and function in vivo (42). However, the observation that the preinsult intravenous injection of KGF was as effective as an intratracheal administration despite a significantly less alveolar type II cell proliferative response suggested that the mechanism of protective effects are incompletely understood (13). The effects and mechanisms of KGF on proinflammatory cytokines and · NO production in the injured lung have not been elucidated.

Nitrating agents such as ONOO- can disable tyrosine phosphorylation (9, 21, 22). KGF binds to epithelial cell surface receptors, which results in receptor autophosphorylation (on tyrosine) and activation of a phosphorylation cascade (18). We therefore hypothesized that conditions favoring the generation of a nitrating species interferes with the biological effects of KGF by nitration of proteins critical for KGF-derived signaling. Our results indicate that the pre-BMT injection of KGF suppressed T cell-dependent alveolar macrophage activation and the production of inflammatory mediators in the lungs. However, these inhibitory effects of KGF were impaired during conditions associated with the generation of a nitrating species.


    METHODS
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Mice. Female B10.BR (H2K) and C57BL/6 (H2b) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The mice were housed in microisolator cages in the specific pathogen-free facility of the University of Minnesota (Minneapolis) and cared for according to the Research Animal Resources guidelines of our institution. For BMT, donors were 4-6 wk of age and recipients were used at 8-10 wk of age. Sentinel mice were found to be negative for 15 known murine viruses including those that contribute to pneumonitis (e.g., cytomegalovirus, pneumonia virus of mice, K-virus) by our animal facility during repeated extensive evaluations over the study period.

BMT. BMT was performed as previously described (34, 35). B10.BR mice received phosphate-buffered saline (PBS) or recombinant human KGF (5 mg · kg-1 · day-1 subcutaneously; Amgen, Thousand Oaks, CA) on days -6, -5, and -4 pre-BMT. Mice were then segregated into those receiving either PBS or Cy (120 mg · kg-1 · day-1; Cytoxan, Bristol-Myers Squibb, Seattle, WA) as a conditioning regimen on days -3 and -2. All mice were lethally irradiated (7.5-Gy total body irradiation by X ray at a dose rate of 0.41 cGy/min) on the day before BMT (1). Donor C57BL/6 BM was T cell depleted with a monoclonal anti-Thy-1.2 antibody (clone 30-H-12, rat IgG2b; kindly provided by Dr. David Sachs, Massachusetts General Hospital, Boston, MA) plus complement (Neiffenegger, Woodland, CA). For each experiment, a total of 30-40 recipient mice/treatment group were transplanted via the caudal vein with 20 × 106 C57BL/6 marrow cells with and without 15 × 106 natural killer (NK) cell-depleted (PK136, anti-NK1.1 plus complement) spleen cells (BMS) as a source of IPS-causing T cells.

Bronchoalveolar lavage. On day 7 post-BMT, the mice were killed after an intraperitoneal injection of pentobarbital sodium, and the thoracic cavity was partially dissected. The trachea was cannulated with a 19-gauge needle and infused with 1 ml of ice-cold sterile PBS, and the fluid was withdrawn. This was repeated three times, and the return fluids were combined. The bronchoalveolar lavage fluid (BALF) was immediately centrifuged at 500 g for 10 min at 4°C to pellet the cells. BALF total protein was determined with the bicinchoninic acid (Sigma, St. Louis, MO) method, with bovine serum albumin (BSA) as the standard. Nitrite in the BALF was determined with the Greiss method after the conversion of nitrate to nitrite by the NADH-dependent enzyme nitrate reductase (Calbiochem, La Jolla, CA). IFN-gamma and TNF-alpha levels in the cell-free BALF were determined by enzyme-linked immunosorbent assay (ELISA) with commercial kits (R&D Systems, Minneapolis, MN).

PAGE. To characterize the proteins in the BALF, 10-µl samples of cell-free BALF and mouse serum albumin (5 µg; Sigma) were reduced in 0.1 M tris(hydroxymethyl)aminomethane buffer containing 50 µM dithiothreitol, 0.01% bromphenol blue, 1% sodium dodecyl sulfate (SDS; Sigma), and 10% glycerol and boiled for 5 min. Protein separation was carried out with a Mini-PROTEAN slab gel apparatus (Bio-Rad, Hercules, CA) on 7.5% SDS-PAGE gels (Sigma). Separated proteins were visualized with Coomassie staining solution (Bio-Rad).

Macrophage cell culture. The BALF cell pellets from each treatment group were combined, washed twice in cold PBS, and resuspended in RPMI 1640 medium (Celox Laboratories, St. Paul, MN) containing 5% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 µg/ml). Total cell number was determined with a hemocytometer, and cell viability was assessed by trypan blue exclusion. Cells (2 × 105/well) were added to mouse IgG-coated flat-bottom 96-well microtiter plates (Costar, Cambridge, MA), and macrophages were allowed to adhere for 1 h at 37°C in 5% CO2 in air, followed by removal of unbound cells. The cells were maintained in culture at 37°C for 48 h in 5% CO2 in air. At the termination of cell culture, the supernatants were aspirated from the individual culture wells for measurement of nitrite with the Greiss method and lactic dehydrogenase (LDH) with the colorimetric CytoTox 96 assay with bovine heart LDH as the standard (Promega, Madison, WI). The cells were washed twice with PBS and lysed with lysis solution (10× Triton X-100; Promega), and cellular LDH release was measured. The percent cytotoxicity during the culture of macrophages obtained from transplanted mice was calculated by dividing the cell-free supernatant LDH by the total cellular plus supernatant LDH of each well and multiplying by 100%. Total (supernatant plus cellular) LDH values also were used to correct for possible differences in adherent cell number between groups. Nitrite readings were adjusted accordingly with the BM group at an assigned reference value for 2 × 105 cells (the number of cells originally plated per well).

Immunohistochemistry. In one to two mice per group per experiment, a mixture of 1 ml of optimal cutting temperature medium (Miles Laboratories, Elkhart, IN) and PBS (3:1) was infused into the trachea. The lung was snap-frozen in liquid nitrogen and stored at -80°C. Frozen sections were cut 4 µm thick, mounted onto glass slides, and fixed for 10 min in 3% paraformaldehyde at 4°C. Nonantigenic sites were blocked with 10% normal goat serum (Sigma) for 30 min at 23°C followed by incubation overnight at 4°C with 1) rabbit polyclonal anti-nitrotyrosine antibody (NTAb; 1:50 dilution; Upstate Biotechnology, Lake Placid, NY) or 2) rabbit polyclonal anti-mouse macrophage inducible · NO synthase (iNOS) antibody (1:100 dilution; Transduction Laboratories, Lexington, KY). In control measurements, tissues were incubated with NTAb in the presence of 10 mM nitrotyrosine or with nonspecific rabbit IgG (20 µg/ml). All sections were incubated with a secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase (1:500 dilution), for 45 min at 23°C, followed by the addition of 3,3'-diaminobenzidine (Vector Laboratories, Burlingame, CA) chromogenic substrate. The sections were counterstained with hematoxylin, dehydrated, overlaid with Permount (Sigma), and sealed with coverslips.

Semiquantitative RT-PCR. In some animals, day 7 post-BMT lungs were extracted without lavage and immediately frozen in liquid nitrogen. Total RNA was extracted with the guanidium thiocyanate-phenol-chloroform method (TriReagent, Sigma). RT was performed with a cDNA synthesis kit (First-Strand cDNA Synthesis Kit, Amersham Pharmacia Biotech, Uppsala, Sweden). The RT mixture consisted of 2 µg of RNA; 5 µl of Moloney murine leukemia virus RT containing RNase/DNase-free BSA, dATP, dCTP, dGTP, and dTTP; 1 µl of random hexadeoxynucleotide primer [pd(N)6; 0.2 µg/µl]; and 1 µl of dithiothreitol (200 mM). The RT mixture was incubated for 1 h at 37°C. The products were further amplified by PCR with KlenTaq DNA polymerase (Clontech, Palo Alto, CA). The oligonucleotide upstream and downstream primer sequences, annealing temperatures, and cycle numbers were as follows: 1) mouse beta -actin, 5'-AAGTGTGACGTTGACATCCGT-3' and 5'-CTCATCGTACTCCTGCTTGC-3', 60°C for 30 cycles; 2) mouse iNOS, 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' and 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3', 65°C for 35 cycles; 3) mouse IFN-gamma , 5'-TGCATCTTGGCTTTGCAGCTCTTCCTCATGGC-3' and 5'-TGGACCTGTGGGTTGTTGACCTCAAACTTGGC-3', 60°C for 30 cycles; and 4) mouse TNF-alpha , 5'-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3' and 5'-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3', 60°C for 30 cycles. The PCR products were electrophoresed through a 1% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining. The PCR product of the cDNA of interest was included as a positive control (Clontech). To achieve semiquantitative conditions, RT-PCRs were terminated, and the products were quantified when all the samples were in the linear range of amplification. To ensure that the experiments were in the linear range of the amplification cycle, experiments were performed on a representative sample from each group for all PCR products measured. Aliquots were removed during RT-PCR amplification after every five cycles starting with cycle 15 and ending at cycle 40. In addition, in some samples, iNOS or TNF-alpha cDNA was amplified with 18S rRNA as an internal control (Quantu<UNL>mRNA</UNL>, Ambion, Austin, TX). Densitometry was used in the relative semiquantitative assessment of RT-PCR product (NIH Image, Scion, Frederick, MD).

Statistical analysis. Results are expressed as means ± SE. Data were analyzed by ANOVA or Student's t-test. Statistical differences among group means were determined by Tukey's studentized test. P values <=  0.05 were considered significant.


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INTRODUCTION
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KGF does not modify total BALF cellularity or composition. Allogeneic T cells with or without Cy conditioning given to TBI mice at the time of BMT increased the total number of inflammatory cells recovered by bronchoalveolar lavage on day 7 post-BMT and shifted the cellular differential toward more lymphocytes. KGF administered subcutaneously on days -6, -5, and -4 pre-BMT did not modify the effects of allogeneic T cells and Cy-conditioning regimen on the total number or differential of cells in the BALF (Table 1).

                              
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Table 1.   Effects of KGF on lavage fluid cellular number and profile on day 7 after BMT

KGF prevents the increased BALF total protein mediated by T cells (BMS) but not by T cells and Cy (BMS+Cy). The percent recovery of BALF was similar in all groups of mice (>90% of instilled volume). Pre-BMT KGF treatment suppressed the T cell-mediated increase of BALF total protein on day 7 after BMT, suggesting that KGF prevented permeability edema (Fig. 1). In contrast, BALF obtained from mice treated with the same schedule of KGF and injected with both T cells and Cy (BMS+Cy) contained the highest level of protein. To characterize the proteins in the BALF, 10 µl of fluid were subjected to SDS-PAGE and visualized with Coomassie stain. Figure 1 and Western blots probed with anti-mouse serum albumin (data not shown) revealed that the protein most increased in the BALF of mice injected with T cells with and without Cy was of the same molecular weight as serum albumin, consistent with increased epithelial and endothelial permeability.


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Fig. 1.   A: total protein levels in cell-free bronchoalveolar fluid (BALF) of irradiated [7.5-Gy total body-irradiated (TBI)] B10.BR mice 7 days after bone marrow (BM) transplantation (BMT). Values are means ± SE; n = 12-20 mice/group. Allogeneic splenic T cells with (+) and without (-) cyclophosphamide (Cy; 120 mg/kg on days -3 and -2) increased BALF protein levels. Keratinocyte growth factor (KGF) pretreatment restored total protein levels in mice given T cells (BMS) but not in mice given T cells and Cy (BMS+Cy). * P < 0.05 compared with control (BM). + P < 0.05 compared with effect of KGF in each group. B: to characterize proteins in BALF, 10 µl of fluid were subjected to SDS-PAGE and visualized with Coomassie stain. MSA, mouse serum albumin. No. on left, molecular mass.

KGF suppresses the increased BALF nitrite, TNF-alpha , and IFN-gamma levels mediated by T cells (BMS) but not by T cells and Cy (BMS+Cy). Allogeneic T cells increased the BALF levels of the stable by-products of · NO, nitrite and nitrate, and the proinflammatory cytokines TNF-alpha and IFN-gamma measured on day 7 after transplantation. KGF administered before conditioning suppressed the T cell-mediated generation of · NO (Fig. 2), TNF-alpha , and IFN-gamma levels (Fig. 3). In contrast, pre-BMT KGF failed to suppress the levels of these inflammatory mediators in the BALF obtained from mice injected with both T cells and Cy (BMS+Cy; Figs. 2 and 3). Although the reason for the enhanced nitrite levels in KGF-treated BMS+Cy recipient mice compared with the BMS+Cy recipients (P < 0.05; Fig. 2) is unclear at this time, it may be the result of KGF-mediated abnormal epithelial cell proliferation in a milieu containing high levels of nitrated proteins.


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Fig. 2.   Nitrite levels in cell-free BALF of irradiated B10.BR mice 7 days post-BMT. BMS or BMS+Cy mice exhibited increased nitrite levels. Values are means ± SE; n = 12-20 mice/group. KGF pretreatment suppressed nitrite in BMS but not in BMS+Cy recipient mice. Nitrate was reduced with nitrate reductase before nitrite measurement with Greiss reaction. * P < 0.05 compared with control (BM). + P < 0.05 compared with effect of KGF in each group.



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Fig. 3.   Levels of proinflammatory cytokines in cell-free BALF of irradiated B10.BR mice collected 7 days after transplantation. A: interferon (IFN)-gamma measured by ELISA. B: tumor necrosis factor (TNF)-alpha measured by ELISA. Values are means ± SE; n = 7-14 mice/group. BMS and BMS+Cy mice exhibited increased TNF-alpha and IFN-gamma . KGF pretreatment suppressed TNF-alpha and IFN-gamma levels in BMS but not in BMS+Cy recipient mice. * P < 0.05 compared with control (BM). + P < 0.05 compared with effect of KGF in each group.

To begin to understand the mechanisms responsible for the KGF-induced suppression of T cell-mediated induction of · NO, TNF-alpha , and IFN-gamma , we determined the effects of KGF on iNOS, TNF-alpha , and IFN-gamma gene expression. As assessed by relative semiquantitative RT-PCR, day 7 post-BMT iNOS, TNF-alpha , and IFN-gamma messages were unaffected by KGF treatment in either BMS or BMS+Cy recipients (Fig. 4). Two approaches ensured semiquantitative conditions. First, the cycle-titration experiments described in METHODS showed that the RT-PCRs were terminated during the linear range below the saturation of the PCR products. Second, inclusion of an internal standard (18S rRNA) during the RT-PCRs confirmed our semiquantitative conditions (Fig. 5).


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Fig. 4.   Expression of mRNA for IFN-gamma , TNF-alpha , inducible nitric oxide (· NO) synthase (iNOS), and beta -actin in lung tissues of irradiated BMT mice as determined with RT-PCR. A: indicated amplified cDNAs were run on agarose gel and stained with ethidium bromide. Lane 1, positive control PCR product generated with primer of interest (C); lane 2, amplified cDNA from lung tissue of BM mice; lanes 3 and 4, amplified cDNA from BMS mice without and with KGF, respectively, on days -6, -5, and -4; lanes 5 and 6, amplified cDNA from BMS+Cy mice without and with KGF, respectively. B: semiquantitative assessment of amplified cDNA with densitometry. Results, expressed relative to beta -actin band intensity, are means ± SE of duplicate samples obtained from 2 independent experiments. ND, not determined. * P < 0.05 compared with control (BM).



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Fig. 5.   Assessment of RT-PCR semiquantitative conditions with TNF-alpha mRNA expression. A: linear range for PCR products was determined by preparing a PCR master mix from RNA obtained from BMS mice. Aliquots were removed during RT-PCR amplification after every 5 cycles starting with cycle 15. C, positive control TNF-alpha gene expression (30 cycles). B: multiplex quantitative RT-PCR for TNF-alpha mRNA expression (after 30 cycles of amplification) from indicated groups of mice, with 18S rRNA as an internal standard. Shown is a representative figure, which was repeated twice.

Pre-BMT KGF suppresses cytotoxicity and spontaneous · NO production by cultured alveolar macrophages obtained from mice injected with allogeneic T cells (BMS) but not by T cells and Cy (BMS+Cy). Alveolar macrophages obtained on day 7 after BMT from TBI mice injected with allogeneic T cells (BMS group) spontaneously released large amounts of · NO and LDH into the cell supernatant, indicating that the cells were injured during in vivo exposure to inflammatory mediators. Although cultured alveolar macrophages obtained from mice injected with both T cells and Cy (BMS+Cy) did not release significantly higher · NO (compared with that in the BMS group), they released very high levels of LDH (Fig. 6). This increased cytotoxicity in macrophages obtained from BMS+Cy recipient mice were most likely due to enhanced TNF-alpha and IFN-gamma production and the generation of ONOO-. Pre-BMT KGF administration suppressed cytotoxicity and nitrite production in macrophages obtained from mice injected with T cells (BMS; Fig. 6). However, the same dose of KGF failed to prevent injury and · NO production by macrophages obtained from mice injected with allogeneic T cells and Cy (BMS+Cy).


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Fig. 6.   Spontaneous nitrite (NO-2) production in culture supernatant (hatched bars) and percent cytotoxicity (solid bars) of day 7 post-BMT alveolar macrophages obtained from BM mice, BMS mice, and BMT+Cy mice. Macrophages were cultured for 48 h as described in METHODS. Macrophages obtained from KGF-treated BMS mice produced significantly less · NO and were less injured compared with macrophages taken from non-KGF-treated BMS recipients. KGF failed to suppress · NO production and cytotoxicity in macrophages obtained from BMS+Cy mice. Percent cytotoxicity was calculated from supernatant and cellular lactate dehydrogenase (LDH) values as described in METHODS. Values are means ± SE obtained from at least 3 wells of pooled macrophages obtained from 4-6 mice · group-1 · experiment-1, which was repeated once. * P < 0.05 compared with control (BM). + P < 0.05 compared with effect of KGF in each group.

KGF does not modify iNOS protein and nitrotyrosine expression in lungs of mice injected with T cells and Cy (BMS+Cy). Day 7 post-BMT lung sections taken from TBI mice were incubated with polyclonal antibodies to iNOS (Fig. 7). Increased iNOS binding was observed in lung sections obtained from mice given T cells (BMS) and T cells plus Cy (BMS+Cy). Staining was reduced to control levels in lung sections from mice not given T cells. Moreover, background staining with nonimmune IgG was similar in all groups. KGF treatment inhibited iNOS protein expression in BMS but not in BMS+Cy recipients (Fig. 7).


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Fig. 7.   Immunoperoxidase staining for iNOS protein expression in lungs of irradiated BMT mice taken from indicated treatment groups. Staining of epithelial and inflammatory cells was observed in lung sections of BMS and BMS+Cy mice. KGF treatment suppressed iNOS protein expression in BMS but not in BMS+Cy recipients. Shown is a representative experiment, which was repeated once. Sections from BMS+Cy group incubated with nonspecific rabbit IgG did not exhibit staining above background.

Nitrotyrosine formation is considered a "footprint" for the in vivo generation of a nitrating species (20, 30). High levels of nitrated proteins in the concentrated BALF of recipient mice treated with T cells and Cy were reported (14). To determine whether KGF treatment modified nitrotyrosine formation, day 7 post-BMT lung sections were incubated with NTAbs. Only lung sections from BMS+Cy recipients, but not from BMS or BM recipients, exhibited increased immunostaining with the NTAbs. Antibody binding was specific because it was completely blocked in the presence of excess antigen (10 mM nitrotyrosine). Nitrotyrosine in sections of mice injected with Cy without allogeneic T cells was at background levels (data not shown). Pre-BMT KGF failed to prevent nitrotyrosine formation in the lungs of BMS+Cy recipients (Fig. 8). In addition, the high levels of nitrated proteins in the BALF of recipient mice treated with T cells and Cy were not modified by KGF treatment (data not shown).


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Fig. 8.   Immunoperoxidase staining of lung sections taken from indicated treatment groups of irradiated BMT mice and incubated with nitrotyrosine antibody (NTAb) or NTAb in presence of 10 mM nitrotyrosine (Block). Increased staining of epithelium and inflammatory cells was observed only in sections obtained from BMS+Cy mice. Shown is a representative experiment, which was repeated once. NTAb binding was specific because it was completely blocked in presence of excess antigen.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

The two major findings of this study were that 1) KGF given to TBI mice before BMT suppressed T cell-dependent inflammatory events occurring in the early peri-BMT period and 2) the combined administration of donor spleen T cells and Cy (BMS+Cy) was associated with an impaired response to KGF treatment. Because Cy-facilitated lung dysfunction was dependent on allogeneic T cells (BMS+Cy recipients) and associated with the presence of nitrated proteins (14, 35), we hypothesized that the generation of a nitrating agent was the most likely reason for the impaired KGF effects observed in BMS+Cy recipient mice. Although we cannot rule out other pathways capable of nitration reactions in vivo (8, 29), the most likely nitrating species under our conditions is ONOO-, formed by the simultaneous generation of · NO (by T cell-activated epithelial and inflammatory cells) and O-2· [by macrophages after exposure to Cy (4)]. In a cell culture system, Shin et al. (37) reported that growth factors enhanced ONOO--induced apoptosis and suggested a unique interaction between growth factors and ONOO- during the resolution of inflammation and repair processes in vivo.

KGF can prevent lung dysfunction by enhancing proliferation and growth of alveolar type II cells (42, 48), increasing surfactant protein (SP) A mRNA and secretion (45), and facilitating alveolar epithelial cell DNA repair (41, 44). Because KGF did not affect the composition of cells infiltrating the lungs or prevent induction of TNF-alpha , IFN-gamma , or iNOS mRNA expression, the suppression of inflammatory mediators observed in KGF-treated allogeneic T cells recipient mice (BMS) was not only due to a dampened inflammatory response associated with KGF-induced enhanced epithelial repair. Therefore, we hypothesized that KGF prevented epithelial permeability by an additional mechanism: the posttranscriptional suppression of TNF-alpha , IFN-gamma , and iNOS protein expression. This hypothesis is strengthened by the finding that macrophages obtained from KGF-treated mice given T cells spontaneously produced significantly less · NO (Fig. 6) and TNF-alpha (data not shown).

How does KGF suppress the production of inflammatory mediators? The inhibitory effects of KGF on the T cell-mediated production of inflammatory mediators were observed 10 days after cessation of KGF treatment, suggesting the involvement of a KGF-derived mediator. Work in progress in our laboratories suggests that the KGF-derived mediator is SP-A. SP-A, a calcium-dependent lectin produced by alveolar type II cells, possesses immunoregulatory functions including inhibition of TNF-alpha production from lipopolysaccharide-stimulated alveolar macrophages (25). In addition, our group observed the induction of the T helper 2 (Th2)-type anti-inflammatory cytokine interleukin (IL)-13 in KGF-treated mice starting 4 days after the cessation of KGF administration even in the absence of irradiation or BMT (33). IL-13, normally produced by activated T cells and alveolar macrophages (17, 26), is a potent inhibitor of human and rodent macrophage-derived proinflammatory cytokines and · NO production (6, 27). Consistent with our data, IL-13 downregulates inflammatory mediators at the posttranscriptional level (2) and has no effect on the chemotactic properties of monocytes (46). Therefore, we speculate that the KGF-derived increase in SP-A levels is responsible for the induction of IL-13 and the subsequent downregulation of macrophage function.

How does nitration of critical proteins impair the protective effects of KGF? There are two pathways by which nitrating agents may interfere with the ability of KGF to suppress the production of inflammatory mediators. First, the nitration of tyrosine residues of critical proteins is known to prevent phosphorylation by tyrosine kinases (21, 22, 24). KGF mediates biological functions by autophosphorylation of tyrosine residues present on the KGF receptor (18). Therefore, one possibility is that nitration of the KGF receptor may interfere with the ability of KGF to suppress · NO, TNF-alpha , and IFN-gamma production. However, KGF was injected several days before BMT and should have manifested its effects before the generation of a nitrating agent. In addition, IL-13 shares with IL-4 a common receptor subunit, the IL-4 receptor-alpha , essential for intracellular signaling (38, 50). After binding, IL-4 and IL-13 induce receptor association, receptor tyrosine phosphorylation, and coupling with kinases, which activate each other by tyrosine phosphorylation (43). Therefore, the presence of a nitrating species in mice injected with both T cells and Cy may disable IL-13-mediated signaling. The second pathway by which nitrating agents may impair KGF-derived responses is via the nitration of SP-A. Nitration of tyrosine residues of SP-A may abolish its capability to downregulate macrophage and T-cell activation, as was shown for several other properties of SP-A (16, 49). Elimination of the inhibitory effects of IL-13 on proinflammatory cytokines and · NO production will lead to a resumption of iNOS, TNF-alpha , and IFN-gamma mRNA translation and production of high levels of · NO, TNF-alpha , and IFN-gamma protein. The high levels of proinflammatory cytokines and the generation of ONOO- may explain the tissue injury and enhanced permeability edema observed in KGF-treated mice given both T cells and Cy (BMS+Cy).

However, the presence of a nitrating agent impairs some, but not all, of the biological effects of KGF. For example, KGF pretreatment of mice given allogeneic T cells and Cy (BMS+Cy) 1) attenuated BALF LDH levels, although KGF-mediated suppression of LDH levels did not reach significance (P = 0.08; data not shown); 2) suppressed levels of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86); and 3) decreased the number of cells expressing granzyme B mRNA as assessed by in situ hybridization (33). Suppression of granzyme B may explain why KGF pretreatment favorably shifted the survival curves of BMS+Cy recipient mice, although all mice still died, possibly because of increased epithelial permeability and persistent release of inflammatory mediators in the lungs (34). Taken together, these data suggest that KGF prevents lung dysfunction and prolongs survival by tyrosine phosphorylation-dependent and phosphorylation-independent mechanisms. The presence of a nitrating agent will impair only a KGF-mediated tyrosine phosphorylation-dependent signaling pathway.

There is no longer doubt that human monocytes and alveolar macrophages can express functional iNOS, albeit under different and tighter regulatory control compared with rodent cells (11, 28, 36, 40). In humans, other sources of · NO and ONOO- include airway and alveolar epithelial cells (12, 15) and inhaled · NO gas commonly administered to IPS patients to improve oxygenation without consideration to the conditioning regimens received. In addition, other nitrite-dependent reaction pathways may contribute to the in vivo nitration of proteins through the formation of nitryl chloride (NO2Cl) by reaction of nitrite with hypochlorous acid or myeloperoxidase (7).

Because transplantation is always scheduled, the pre-BMT administration of KGF has great potential to prevent the development of IPS. KGF has been shown to prevent lung injury by mediating alveolar type II cell proliferation. We report herein an additional mechanism by which KGF can prevent allogeneic T cell-mediated enhanced epithelial permeability: downregulation of macrophage activation. This mechanism is disabled in the presence of nitrating agents. Further studies are required to determine the signaling proteins responsible for KGF-derived inhibition of proinflammatory cytokines and · NO and establish a cause-and-effect relationship between the generation of a nitrating species and impaired response to KGF treatment. Identification of specific nitrotyrosyl polypeptides may provide insight into mechanisms regulating not only ONOO--induced injury but also signal transduction pathways associated with peri-BMT lung repair. Efforts should be directed toward preventing the formation of a nitrating species without extreme inhibition of · NO production, an essential molecule in the modulation of immune responses.


    ACKNOWLEDGEMENTS

The expert technical assistance of John Hermanson, Chris Lees, Pat Jung, Naomi Fujioka, and Staci Hermanson is greatly appreciated.


    FOOTNOTES

This work was supported by grants from the Minnesota Medical Foundation and the American Heart Association (Minnesota Affiliate); National Heart, Lung, and Blood Institute (NHLBI) Grants HL-56067 and HL55209; National Institute on Aging Grants RO1-AI-34495 and PO1-AI-35296; and NHLBI Acute Lung Injury Grant P50-HL-50152.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: I. Y. Haddad, Univ. of Minnesota, Dept. of Pediatrics, 420 Delaware St. S.E., Minneapolis, MN 55455 (E-mail: hadda003{at}tc.umn.edu).

Received 2 February 1999; accepted in final form 31 March 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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