Post-BMT lung injury occurs independently of the expression of CCL2 or its receptor, CCR2, on host cells

Angela Panoskaltsis-Mortari,1 John R. Hermanson,1 Elizabeth Taras,1 O. Douglas Wangensteen,2 Israel F. Charo,3 Barrett J. Rollins,4 and Bruce R. Blazar1

1Department of Pediatrics, Heme/Oncology/Bone and Marrow Transplant Division and Cancer Center and 2Department of Physiology, University of Minnesota, Minneapolis, Minnesota 55455; 3Gladstone Institute, San Francisco, California 94141; and 4Dana Farber Cancer Institute, Boston, Massachusetts 02115

Submitted 14 May 2003 ; accepted in final form 30 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Idiopathic pneumonia syndrome (IPS) is a significant cause of mortality post-bone marrow transplant (BMT) in humans. In our murine model, lethal pre-BMT conditioning and allogeneic T cells result in the recruitment of host antigen-presenting cells (APC) and donor T cells into the lung post-BMT concomitant with development of severe lung dysfunction. CCL2 induction is found in bronchoalveolar lavage fluid (BALF) before host monocyte influx. The major receptor for CCL2 is CCR2 present on monocytes; this interaction can play a crucial role in monocyte recruitment in inflammation. To determine whether blockade of the CCL2/CCR2 pathway could hinder host monocyte influx, lethally conditioned wild-type (WT), CCL2-/-, or CCR2-/- mice were transplanted with allogeneic marrow and spleen cells. WT and -/- recipients exhibited equivalent lung dysfunction post-BMT. The frequencies of host macrophages as well as donor CD4+ and CD8+ T cells in lungs post-BMT did not differ between WT and -/- recipients. However, the T cell dependency of the host CD11b+ major histocompatibility complex class II+ cell influx was lost in CCR2-/- recipients. In CCR2-/- mice, this influx was accompanied by elevated levels of CCL20. Post-BMT BALF and sera of -/- mice did not reveal any decrease in cytokines or chemokines compared with WT mice. CCL2-/- mice had a deficiency of CCL2 in their BALF and sera post-BMT, confirming our hypothesis that CCL2 is predominantly host derived. Therefore, IPS can occur independently of host expression of CCL2 or CCR2, and compensatory mechanisms exist for regulating APC recruitment into the lung during the early post-BMT period.

chemokines; mouse models; monocytes; idiopathic pneumonia syndrome; bone marrow transplant


IDIOPATHIC PNEUMONIA SYNDROME (IPS) remains a major complication after bone marrow transplantation (BMT) (11). Risk factors for developing IPS are related to the intensity of the conditioning regimen used and the degree of alloreactivity of the donor graft (15). We have characterized a murine model of IPS caused by the influx of host monocytes and donor T cells into the lungs early postallogeneic BMT of lethally irradiated mice (30). Intensifying the pre-BMT conditioning with cyclophosphamide potentiates the development of alloreactive T cell-dependent IPS. Lung dysfunction in our model presents as reduced specific compliance, decreased total lung capacity, and increased wet and dry lung weights. Histologically, IPS is associated with injured alveolar type II cells and increased frequencies of cells expressing B7 ligands (costimulatory for T cells) and the cytolytic protein granzyme B (28, 30). Bronchoalveolar lavage fluid (BALF) of mice with IPS contain elevated levels of inflammatory cytokines as well as other indexes of lung injury as evidenced by increased levels of nitrite, lactate dehydrogenase, and protein (17).

We reported that monocyte- and T cell-attracting chemokines are produced in the lung during the generation of IPS in our model (29). Induction of CCL2 (macrophage chemotactic protein-1 or MCP-1) was found in BALF of these recipient mice before host monocyte recruitment on day 3 post-BMT. CCL2 is produced by numerous cell types including monocytes, epithelial cells, fibroblasts, and tumor cells (33). Endothelial cells are also major producers of CCL2 in response to inflammatory cytokines (32). In murine IPS, the highest levels of CCL2 are produced after allogeneic BMT in a T cell-dependent manner (29). In vitro studies have shown that high amounts of CCL2 are produced by antigen-presenting cells (APC) that are effective inducers of T cell responses (34). The major receptor for CCL2 is CC chemokine receptor 2 (CCR2), present on monocytes, and, in rodent systems, this interaction has been shown to play a crucial role in monocyte/macrophage recruitment in inflammation, autoimmunity, and resistance to infectious organisms (16, 20, 21, 24, 35, 38, 39). In relationship to the lung, reduced recruitment of monocytes, decreased T helper cell type 1 (Th1) cytokine responses, and early death have been seen in rodent models of bacterial-induced allergic airway inflammation, granulomatous lung disease, alloantigen-induced bronchiolitis obliterans, and endotoxin administration, in the presence of neutralizing antibodies to CCL2 and in CCL2-/- or CCR2-/- mice (1, 8, 10, 25, 27, 31).

Our previous findings in murine IPS demonstrated the association of early CCL2 production in the lung followed by monocyte influx and inflammatory cytokine production (29). We hypothesized that prevention of this initial monocyte recruitment might blunt the subsequent inflammatory cascade. Therefore, we tested whether CCL2 or CCR2 might be critical mediators of IPS and whether blockade of the CCL2/CCR2 recruitment pathway could hinder the influx of host monocytes. Contrary to what we anticipated, CCL2-/- and CCR2-/- mice still developed IPS as severe as wild-type (WT) BMT recipients, and recruitment of host major histocompatibility complex (MHC) class II+ cells was unimpaired.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice. C57BL/6 (H2b) mice were purchased from the National Institutes of Health (Bethesda, MD). B10.BR (H2k) mice were purchased from Jackson Laboratories (Bar Harbor, ME). CCL2-/- and CCR2-/- mice backcrossed onto the C57BL/6 background (>10 generations) have been described previously (10, 24). Mice were housed in microisolator cages in the specific pathogen-free facility of the University of Minnesota and cared for according to the Research Animal Resources guidelines of our institution. Experiments were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. Sentinel mice were found to be negative for infectious microorganisms known to cause pulmonary pathology such as pneumonia virus, K virus, Sendai, etc. For BMT, donors were 8-12 wk of age, and recipients were used at 8-10 wk of age.

Pre-BMT treatment and conditioning. C57BL/6 WT or CCL2-/- or CCR2-/- mice received PBS or cyclophosphamide (Cytoxan; Bristol Myers Squibb, Seattle, WA), 120 mg·kg-1·day-1 ip, as a conditioning regimen pre-BMT on days -3 and -2. All mice were lethally irradiated on the day before BMT (7.5 Gy TBI) by X-ray at a dose rate of 0.41 Gy/min as described previously (3).

BMT. Our BMT protocol has been described previously (2). Briefly, donor B10.BR bone marrow was T cell depleted (TCD) with anti-Thy 1.2 monoclonal antibody (MAb; clone 30-H-12, rat IgG2b, kindly provided by Dr. David Sachs, Charlestown, MA) plus complement (Nieffenegger, Woodland, CA). C57BL/6 WT or CCL2-/- or CCR2-/- recipient mice were transplanted via caudal vein with 20 x 106 TCD B10.BR (H2k) marrow with or without 15 x 106 natural killer (NK) cell-depleted (PK136, anti-NK1.1 + complement) spleen cells as a source of IPS-causing T cells.

Lung weights. Mice were euthanized with pentobarbital sodium, and the thoracic cavity was partially dissected. Lungs were exsanguinated by perfusion with 1.0 ml of saline via the right ventricle of the heart. To minimize the number of mice needed for the study without compromising the data, the right lung (bilobed) was used for weight determinations while the left lung was processed for histopathology. For each mouse, the wet weight was taken immediately after right lung removal from the thorax. Lungs were dried overnight to a constant weight at 80°C followed by determination of dry weights. The wet-to-dry weight ratio was calculated and taken as a measure of the severity of lung injury (37). No correction for extravascular blood content was used in the calculations.

Pressure-volume curves. After full heart-lung excision, the lungs were suspended via the trachea and kept moist with saline. Pressure-volume (P-V) curves of air-filled lungs were obtained as previously described (30). Air was delivered into the lungs via a tracheal cannula in 0.05-ml increments with a syringe while measuring intratracheal pressure with a transducer until 30 cmH2O pressure was reached. The volume at this pressure was denoted as the total lung capacity (TLC), assuming the volume of air in the collapsed lungs before inflation was negligible (compared with the TLC). Air was then withdrawn in 0.05-ml increments until pressure was atmospheric. This was done three times, and data were procured from the third series. Specific lung compliance was calculated from the slope of the deflation curve from points flanking 5 cmH2O pressure, considered normal breathing range, by the formula ({Delta}volume/{Delta}pressure)/Av volume, where volume is in milliliters, pressure is in cmH2O, and Av volume is average volume over the pressure range used to generate the slope of the P-V curve (i.e., {Delta}volume/{Delta}pressure).

Bronchoalveolar lavage. The trachea was cannulated with a 19-gauge needle, infused with 0.5 ml of PBS, and the fluid was withdrawn. This was repeated twice, and a total of 1.5 ml of BALF were collected per mouse, centrifuged (1,000 g) at 4°C for 10 min to pellet the cells, and stored at -80°C.

Serum collection. At the time of death, blood was collected by cardiac puncture, placed immediately at 4°C, and the serum was separated at 4°C and stored at -80°C.

Chemokine/cytokine level determination. Bronchoalveolar lavage (BAL) and serum levels of predominant monocyte/macrophage attractants CCL2 (MCP-1), CCL7 (MCP-3), and CCL20 (macrophage inflammatory protein-3{alpha} or MIP-3{alpha}); predominant T cell attractants CCL3 (MIP-1{alpha}), CCL4 (MIP-1{beta}), CCL5 (RANTES), CCL11 (eotaxin), CCL17 (TARC), XCL1 (lymphotactin), CCL6 (C10), CCL21 (Exodus-2), and CXCL10 (IP-10); neutrophil attractants CXCL1 (KC) and CXCL2 (MIP-2); proinflammatory Th1-type cytokines IFN-{gamma}, TNF-{alpha}, IL-1{beta}, and IL-6; and anti-inflammatory Th2-type cytokines IL-13 and IL-10 were determined by sandwich ELISA using mouse-specific commercial kits (R&D Systems, Minneapolis, MN; sensitivity 1.5-3.0 pg/ml) or by sandwich ELISA (sensitivity 1 pg/ml) empirically developed using specific MAbs and results interpolated from standard curves of the relevant recombinant proteins (R&D Systems).

Frozen tissue preparation. A mixture of 0.5 ml of optimal cutting temperature compound (OCT; Miles, Elkhart, IN)-PBS (3:1) was infused via the trachea into the lungs. Lung tissue was embedded in OCT, frozen in liquid nitrogen, and stored at -80°C. Histology was scored using a previously documented scoring system (4).

Immunohistochemistry. Cryosections (6 µm) were fixed in acetone and immunoperoxidase stained using biotinylated MAbs as described previously (5) with avidin-biotin blocking reagents, ABC-peroxidase conjugate, and 3,3'-diaminobenzidine chromogen purchased from Vector Laboratories (Burlingame, CA). The biotinylated MAbs used were as follows: anti-CD4 (clone GK1.5), anti-CD8 (clone 2.43), anti-CD11b (Mac-1, clone M1/70), and anti-Gr-1 (clone RB6-8C5), all purchased from BD Pharmingen (San Diego, CA). The number of positive cells in the lung was quantitated as the percent of nucleated cells under x200 magnification (x20 objective lens). Four fields per lung were evaluated. For immunofluorescent costaining, FITC-labeled CD11b was used with Cy3-labeled CD11c (BD Pharmingen), and images were obtained using an Olympus FV500 confocal laser scanning fluorescent microscope with Fluoview software (Olympus America, Melville, NY).

Flow cytometry. Phenotyping of BAL cells was evaluated on days 3 and 7 post-BMT by quantitation of donor cells using biotin-labeled anti-H2k MAb (clone 11-4.1, BD Pharmingen) with SA-PerCp and host cells using phycoerythrin (PE)-labeled anti-H2b (clone EH144, BD Pharmingen). T cells and monocytes/macrophages were quantitated using fluorochrome-labeled MAbs (FITC or PE, Pharmingen) directed to CD3 (clone 1452C11), CD4 (clone GK1.5), CD8 (clone 53-6.72), and CD11b (clone M1/70). Flow cytometry was done on a FACS Calibur (BD Biosciences, Mountain View, CA) with 10,000 events analyzed (determined by forward and side scatter).

Statistical analysis. Survival data were analyzed by life table methods using the Mantel-Peto-Cox summary of chi-square. Other data were analyzed by ANOVA or Student's t-test. P values <= 0.05 were considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Post-BMT lung dysfunction and inflammatory cell recruitment are not decreased in CCL2-/- or CCR2-/- recipients. In our initial description of mouse IPS post-BMT (30), lung injury was highly correlated with decreased total lung capacity and inflammatory cell influx. Therefore, we wanted to know whether these parameters were affected by loss of CCL2 or CCR2. Lethally irradiated C57BL/6, CCL2-/-, or CCR2-/- mice were transplanted with B10.BR bone marrow with or without 15 x 106 splenocytes. CCL2-/- and CCR2-/- mice exhibited similar decreases in total lung capacity and specific compliance on day 7 post-BMT equivalent to WT recipients (total lung capacities shown in Fig. 1). Increases in both wet and dry lung weights, indicative of cellular inflammation and of fluid leakage, were equally elevated among the WT, CCL2-/-, and CCR2-/- recipients (not shown). Histological examination of the lungs procured on day 7 post-BMT showed equivalent degrees of perivascular and peribronchiolar cuffing in WT, CCL2-/-, and CCR2-/- recipients of allogeneic bone marrow and IPS-causing splenocytes (Fig. 2) with equivalent histological scores (4) (not shown). Lungs were examined by immunohistochemistry to determine whether accumulation of T cells or monocytes/macrophages in the interstitial parenchyma of the lungs had been altered. No decreases in donor CD4+ or CD8+ T cell or CD11b+IAb+ (host haplotype) macrophage numbers were seen (CD11b data shown in Fig. 3; T cell data not shown). Flow cytometric analysis of BAL cells on day 7 post-BMT revealed no differences in the percentages of macrophages, MHC class II+ cells (predominantly of host haplotype), or donor CD4+ or CD8+ T cells that had accumulated in the alveolar airways (not shown). These data are consistent with the similar degrees of decreased lung capacity and specific compliance seen at this time point in WT, CCL2-/-, and CCR2-/- recipients. Therefore, contrary to what we expected, the frequencies of monocytes/macrophages, the cells whose migration is presumed to be dictated by the CCL2/CCR2 pathway, did not decrease, indicating that the APC influx had not been affected. However, CCR2-/- recipients had elevated numbers of CD11b+ cells on day 3 post-BMT of bone marrow alone (i.e., without allogeneic splenocytes) in the lung interstitium compared with pretransplant levels (Fig. 3A, P = 0.004, day 3 time point vs. day -3) such that the T cell-dependent increase in macrophages was no longer apparent (see also Fig. 4A). This early, T cell-independent CD11b+ cell influx was not as dramatic in WT recipients and was not seen in CCL2-/- recipients until day 7 post-BMT (Fig. 3A). In nonmanipulated CCR2-/- control mice, there appeared to be increased frequencies of CD11b+IAb+ cells compared with WT B6 mice (Fig. 3A, day -3 levels, P = 0.01). Therefore, CCR2-/- mouse lungs (but not CCL2-/- lungs) differ from WT in the proportion of macrophages present in their lungs before conditioning and BMT. Because donor T cell infiltration into the lung was low in the bone marrow-only group, lung dysfunction did not occur in CCL2-/- recipients of bone marrow alone.



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Fig. 1. The reduction in total lung capacity (TLC) postallogeneic bone marrow transplant (BMT) is not affected by absence of CCL2 (macrophage chemotactic protein-1 or MCP-1) or CC chemokine receptor 2 (CCR2) in the host. After full heart-lung excision, the lungs were suspended via the trachea and kept moist with saline. TLC was determined from the injected volume of air needed to reach 30 cmH2O pressure as measured with a transducer. Baseline TLC of the CCL2-/- and the CCR2-/- mice did not differ significantly from the C57BL/6 mice. *P < 0.05 vs. corresponding non-manipulated strain. BM, allogeneic bone marrow alone; BMS, allogeneic bone marrow with splenocytes.

 


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Fig. 2. Mice deficient in CCL2 or CCR2 develop idiopathic pneumonia syndrome (IPS) postallogeneic BMT. Hematoxylin & eosin stains of 6-µm cryosections of control lungs (left) and lungs taken on day 7 post-BMT (right) show septal thickening and perivascular and peribronchiolar cuffing (arrows) as well as infiltration into the lung parenchyma and alveolar airways in lungs with IPS. Magnification, x200. WT, wild type.

 


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Fig. 3. Influx of host CD11b+ cells into the lungs of allogeneic BMT recipients is not altered by absence of either CCL2 or CCR2 in the host, but T cell dependency is lost in CCR2-/- recipients. Expression of CD11b and host IAb on the indicated days relative to BMT was determined by immunoperoxidase staining with biotinylated monoclonal antibodies. Data are presented as percent of nucleated cells. Mean values ± SE are indicated for 6 mice/group pooled from 2 experiments. A: groups receiving allogeneic BM alone. B: groups receiving allogeneic BM + splenocytes. *P < 0.05 vs. pre-BMT level.

 


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Fig. 4. Increased numbers of host CD11b+ CD11c+ cells and CCL20 (macrophage inflammatory protein-3{alpha}) in CCR2-/- recipient lungs postallogeneic BMT. A: immunoperoxidase staining of CD11b on day 3 post-BMT of representative WT (left) and CCR2-/- (right) mice given allogeneic BM alone; magnification, x200. B: levels of CCL20 in lung protein extracts as determined by ELISA. Mean values ± SD are indicated for 6 mice/group pooled from 2 experiments. *P < 0.05 vs. corresponding WT control. C: immunofluorescence costaining for CD11b (green) and CD11c (red) on day 3 post-BMT of representative WT (left) and CCR2-/- (right) mice given allogeneic BM alone. Cells coexpressing both markers are depicted in yellow; magnification, x400 under oil immersion.

 

Recipients deficient in CCR2 have elevated levels of the myeloid dendritic cell chemokine MIP-3{alpha} (CCL20). Because CCR2-/- mice exhibited an accelerated early, T cell-independent influx of CD11b+ cells (Fig. 4A), we analyzed the lungs for changes in the levels of chemoattractants at different time points in the peri-BMT period. Of the panel of chemokines we were able to measure (listed in MATERIALS AND METHODS), a dramatic difference was seen for CCL20/MIP-3{alpha} (and moderately so for CCL2). Compared with WT, CCR2-/- mice (but not CCL2-/- mice) have inherently higher levels of CCL20 in their lungs before conditioning (day -3), and these levels remain elevated post-BMT (Fig. 4B). Interestingly, the highest levels of CCL20 were seen on day 3 post-BMT in the lungs of CCR2-/- mice receiving allogeneic bone marrow alone (i.e., in the absence of IPS-causing splenocytes). Cells bearing the CD11b+ MHC class II+ APC phenotype are consistent with those of the myeloid dendritic cell lineage that express CCR6, the only known receptor for CCL20 (MIP-3{alpha}). Myeloid dendritic cells also coexpress CD11c along with CD11b, and immunofluorescent costaining for these markers also showed increased numbers of cells coexpressing CD11b and CD11c in CCR2-/- recipient mouse lungs on day 3 post-BMT as shown in Fig. 4C. As expected, there was a lack of CCL2 in the lungs and sera of the CCL2-/- mice post-BMT (Fig. 5), confirming our previous hypothesis that CCL2 is host and not donor derived post-BMT (29). These same CCL2-/- recipients of allogeneic bone marrow and spleen had elevated lung (but not systemic) levels of CCL21 (predominant T cell attractant) compared with WT or CCR2-/- recipients (not shown). However, this was seen only at the later day 7 post-BMT time point and did not lead to increased numbers of donor T cells that were elevated compared with WT or CCR2-/-. Thus elevated levels of CCL20 may be compensatory for the lack of CCL2 in facilitating host APC recruitment into the lung post-BMT.



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Fig. 5. IFN-{gamma}, IL-10, and CCL2 levels in the bronchoalveolar lavage fluid (BALF; A) and serum (B) on day 7 postallogeneic BMT of WT, CCL2-/-, and CCR2-/- recipient mice. Levels of the indicated cytokines/chemokines were determined by ELISA. Mean values ± SD are indicated for 6 mice/group pooled from 2 experiments. *P < 0.05 vs. corresponding WT group.

 

Higher levels of both pro- and anti-inflammatory T cell mediators in CCL2-/- and CCR2-/- recipients. Because differences in some chemokine levels were found in the lungs of CCL2-/- and CCR2-/- recipients, levels of Th1- and Th2-related inflammatory cytokine mediators in BALF and lung protein extracts were measured to ascertain whether changes in cytokines may have been affected. We found an elevated level of IFN-{gamma} in the BALF of CCL2-/- recipients of allogeneic bone marrow and spleen compared with WT B6 recipients (Fig. 5). Therefore, this elevated level of IFN-{gamma} coexisted, in these mice, with an elevated level of CCL21 but was not associated with elevated T cell numbers compared with either WT or CCR2-/- recipients of allogeneic bone marrow and spleen.

Systemic levels of cytokines and chemokines may also influence the pulmonary vasculature, the ensuing inflammatory milieu, chemokine gradients, and the extravasation events post-BMT. Of the panel of cytokines and chemokines measured (listed in MATERIALS AND METHODS) in the sera, most were not affected by the lack of CCL2 or CCR2 in the recipient mice on day 7 post-BMT. Besides the expected paucity of CCL2 in the CCL2-/- mice (as stated above), the exception was IL-10 that was elevated, compared with WT, in the sera of CCL2-/- and CCR2-/- mice given bone marrow and allogeneic spleen cells (Fig. 5, P < 0.05). Other Th2 cytokines, such as IL-4 and IL-13, were present at equivalent levels in knockout (KO) mice compared with WT mice post-BMT, despite the fact that CCL2 is considered to be critical for Th2 responses. The elevated post-BMT levels of IL-10 in the KO mice were T cell dependent, consistent with our previous findings in WT mice (30).

Graft-vs.-host disease-mediated mortality is accelerated in the absence of host CCL2 or its receptor CCR2. To determine whether host expression of CCL2 or CCR2 would have an effect on the generation of graft-vs.-host disease (GVHD; that is associated with IPS), lethally irradiated C57BL/6, CCL2-/-, or CCR2-/- mice were transplanted with B10.BR bone marrow with or without 5 or 25 x 106 splenocytes. Figure 6A shows that CCL2-/- recipients of allogeneic splenocytes had an accelerated lethality compared with WT recipients at both spleen cell doses (P < 0.04). Similar findings were found with CCR2-/- mice (Fig. 6B), and these recipients appeared to be five times more sensitive to the GVHD induced by allogeneic T cells since CCR2-/- recipients of 5 x 106 spleen cells had a mortality rate equivalent to WT B6 mice receiving 25 x 106 splenocytes. GVHD-induced weight loss paralleled the survival data (not shown). Therefore, preclusion of the CCL2/CCR2 pathway does not hinder GVHD. On the contrary, GVHD mortality is accelerated.



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Fig. 6. Acceleration of graft-vs.-host disease-mediated mortality in the absence of host CCL2 or CCR2. TBI-conditioned WT, CCL2-/- (A), or CCR2-/- (B) mice were transplanted with B10.BR TCD bone marrow with or without 5 or 25 x 106 splenocytes. CCL2-/- recipients had an accelerated lethality compared with WT recipients of allogeneic bone marrow and spleen (BMS groups). P < 0.04 for comparisons of both cell doses shown. CCR2-/- recipients had an accelerated lethality compared with WT recipients of allogeneic bone marrow and spleen (BMS groups). P < 0.04 for comparison of 5 x 106 cell dose; P = NS for CCR2-/- recipients of 5 x 106 cells vs. WT recipients of 25 x 106 cells. N = 8/group. NS, not significant.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We demonstrate in this study that the presence of the CCL2/CCR2 pathway is not needed for the induction of early post-BMT-related lung injury. Previous data from our laboratory indicated an induction of CCL2 by resident pulmonary cells post-BMT that preceded the influx of host macrophages. These cells comprise the initial infiltrate into the lung early after BMT and may serve to costimulate alloantigen-reactive donor T cells that enter the lung subsequently. The preferred receptor for CCL2 is CCR2 present on monocytes, and this ligand-receptor interaction has been shown to be critical for monocyte recruitment in various rodent models of inflammation. We had, therefore, reasoned that the use of CCL2 or CCR2 knockout mice as recipients would hinder the influx of these cells. Contrary to what we expected, manifestations of IPS were not ameliorated, nor was the cellular influx hindered, most likely due to a compensatory increase of other chemoattractants for host APC (e.g., CCL20).

The paucity of systemic and pulmonary levels of CCL2 in the CCL2-/- recipients post-BMT confirms our earlier hypothesis that CCL2 is host derived post-BMT (29). Because CCL2 production in the lung preceded the influx of host monocytes, we anticipated that mice deficient in CCL2 would exhibit less lung injury post-BMT. This was not the case. Others have also found a minimal role for CCL2 in immune complex-mediated lung injury in rats (9). We did not find a compensatory increase in other monocyte attractants such as CCL7 (MCP-3) or MCP-5, so it remains unknown what the potential recruitment pathway may be. Furthermore, we found no compensatory increase in chemokine receptors CCR1-5 as assessed by RNase protection assay (data not shown). In another murine system in which a minimal role for CCL2 in recruiting monocytes was found, it was shown that another chemokine, MIG (CXCL9), was responsible for monocyte recruitment (22). Although we did not measure MIG levels in our BMT recipient mice in the current study, in other studies we have used CXCR3-/- mice and anti-CXCR3 MAbs (CXCR3 is the receptor for MIG and IP-10) and found that IPS and GVHD occurred independently of donor CXCR3 expression (B. R. Blazar, P. A. Taylor, and A. Panoskaltsis-Mortari, unpublished data).

We found that mice deficient in CCR2 have elevated levels of the myeloid dendritic cell chemokine CCL20 (MIP-3{alpha}) in their lungs both pre-BMT and post-BMT. This correlated with elevated levels of CD11b+IA+ cells coexpressing CD11c consistent with the myeloid dendritic cell lineage that expresses CCR6 (23). CCR6 is the only known receptor for CCL20 that is produced predominantly in epithelial-rich tissues including the lungs (19). Therefore, it would be interesting to determine whether the CCR6/CCL20 pathway plays a critical role in the recruitment of host APC into the lungs post-BMT.

The elevation in IFN-{gamma} levels in the lungs of CCL2-/- recipients is consistent with in vitro data of Hogaboam et al. (18) showing enhanced production of IFN-{gamma} by, and proliferation of, CD4+ T cells in coculture with stimulated lung fibroblasts and neutralizing CCL2 antibody. This is in contrast to other recent findings demonstrating decreased IFN-{gamma} production in the lungs of CCL2-neutralized mice with pulmonary Cryptococcus neoformans infection (36). Furthermore, these authors found that CCL2-neutralized mice did have Th1 inflammatory cells in lung-associated lymph nodes, but that these T cells failed to traffic to the lungs with resultant Th2 T cell predominance in the lungs. In our study, we found no decrease in T cell influx into the lungs post-BMT and no significant Th1/Th2 skewing. In addition, we found no differences in the percentage of cells expressing mRNA for cytolytic granzymes by in situ hybridization (data not shown). Contradictory findings in the literature reasonably stem from the different forms of inflammatory insult (e.g., exogenous infectious agents or allergens vs. internally derived BMT-related injury), so the roles of chemokine/chemokine receptor pathways will differ.

Mice deficient in CCL2 or CCR2 exhibited elevated levels of IL-10 systemically early post-BMT. Induction of IL-10 by blockade of CCL2 was also found in a mouse model of acute septic peritonitis by in vivo administration of anti-CCL2 antibodies (26). Indeed, we found higher systemic levels of IL-10 in otherwise non-manipulated CCL2-/- mice compared with WT B6 mice (22 ± 13 pg/ml serum vs. 0 ± 0 pg/ml, P = 0.0002). Therefore, CCL2-/- mice have a significantly elevated level of IL-10 even before BMT. IL-10 is normally considered an anti-inflammatory cytokine being produced by Th2-type cells. However, we have previously demonstrated that high levels of exogenously administered IL-10 can exacerbate both CD4 and CD8 T cell-mediated GVHD in a dosedependent fashion (6, 7). In fact, we observed that deficiency of CCL2 or CCR2 did not ameliorate GVHD but accelerated mortality and body weight loss. Therefore, preclusion of the CCL2/CCR2 pathway does not hinder GVHD. However, cells other than monocytes can also express CCR2, and it has been recently described that bronchiolar and alveolar epithelial cells express CCR2 receptors that functionally respond to CCL2, resulting in proliferation (13, 14). Therefore, we suggest that the induction and presence of CCL2 in the lung post-BMT may be an attempt by the host to stimulate epithelial cells to reepithelialize the alveolar basement membrane and not to recruit monocytes per se. Furthermore, the production of CCL2 by these same cell types post-BMT (29) raises the possibility of an autocrine loop for epithelial cell repair via binding to CCR2 or other chemokine receptor/ligands as well.

In conclusion, we report that IPS and GVHD can occur independently of host expression of CCL2 or CCR2. The data suggest redundancies in the chemokine recruitment pathways in the BMT setting and that other chemokine/chemokine receptor pathways suffice to execute post-BMT complications.


    ACKNOWLEDGMENTS
 
The expert technical assistance of Chris Lees, Kalpesh Joshi, Melinda Berthold, Mike Erhardt, and Matt Kramer is greatly appreciated. We are grateful to Drs. Robert Strieter and Patricia Taylor for helpful discussions.

GRANTS

This study was supported by the Children's Cancer Research Fund, the Viking Children's Fund, and National Heart, Lung, and Blood Institute Grants R01-HL-55209 and R01-HL-66308 and NCRR Shared Instrument Grant 1-S10-RR-16851.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Panoskaltsis-Mortari, Univ. of Minnesota, Dept. of Pediatrics, Division of Hematology-Oncology, Blood and Marrow Transplant Program, Mayo Mail Code 366, 420 Delaware St. SE, Minneapolis, MN 55455 (E-mail: panos001{at}umn.edu).

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. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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