Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome

Abstract

Bronchiolitis obliterans syndrome (BOS) is the major limitation to survival after lung transplantation. Acute rejection, its main risk factor, is characterized by perivascular/bronchiolar leukocyte infiltration. BOS is characterized by persistent peribronchiolar leukocyte recruitment leading to airway fibrosis and obliteration. The specific mechanism(s) by which these leukocytes are recruited are unknown. Because MCP-1, acting through its receptor CCR2, is a potent mononuclear cell chemoattractant, we hypothesized that expression of this chemokine during an allogeneic-response promotes persistent recruitment of leukocytes and, ultimately, rejection. We found that elevated levels of biologically active MCP-1 in human bronchial lavage fluid (BALF) were associated with the continuum from acute to chronic allograft rejection. Translational studies in a murine model of BOS demonstrated increased MCP-1 expression paralleling mononuclear cell recruitment and CCR2 expression. Loss of MCP-1/CCR2 signaling, as seen in CCR2(-/-) mice or in WT mice treated with neutralizing antibodies to MCP-1, significantly reduced recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS. Lymphocyte infiltration was not reduced under these conditions. We suggest that MCP-1/CCR2 signaling plays an important role in recruitment of mononuclear phagocytes, a pivotal event in the pathogenesis of BOS.

Figure 1

(a) MCP-1 protein levels in unconcentrated BALF from healthy lung transplant recipients compared with those of lung transplant recipients with BOS and lung transplant recipients with acute lung allograft rejection, displayed using a box plot summary. Horizontal line represents the median, the box encompasses the 25th to 75th percentiles, and the error bars encompass the 10th to 90th percentiles for MCP-1 protein levels. (b) The period, in months after lung transplantation, when the BALF was obtained for the three groups displayed, using a box plot summary. (c) BALF MCP-1 from patients with acute rejection and BOS is biologically active as determined by mononuclear cell chemotaxis. There was significantly more chemoattraction to the acute rejection and BOS BALF compared with that of healthy transplant recipients. Neutralizing antibodies to MCP-1 inhibited chemoattraction in the acute rejection and BOS groups, but not the healthy group. *P < 0.05. (d–g) BOS lung section (×400). (d and f) Lack of nonspecific staining with NRS; (e and g) anti-human MCP-1 antibodies demonstrating immunolocalization to columnar epithelium and mononuclear cells. HPF, high power field; AR, acute rejection.

Figure 2

Procollagens type I and III and hydroxyproline levels are increased in allografts undergoing BOS. (a) RT-PCR determination of procollagen type I and III mRNA from allografts and syngeneic controls, compared with β-actin at day 21. (b and c) Semiquantitative results are expressed as a ratio of each PCR product to β-actin band density (n = 4 groups, in which each group represents four pooled tracheas at each time point). *P < 0.05. (d) Hydroxyproline measurements from allografts and syngeneic controls at days 7, 14, and 21 (n = 4 groups, in which each group represents two pooled tracheas at each time point). *P < 0.05.

Figure 3

FACS analysis of leukocyte cell surface markers CD3, CD4, CD8, Ly-6G (PMN), and MOMA-2 (mononuclear phagocytes) from tracheal allografts undergoing BOS compared with syngeneic controls (n = 4 groups, in which each group represents four pooled tracheas at each time point). *P < 0.05.

Figure 4

MCP-1 mRNA and protein levels are markedly elevated in murine allografts undergoing BOS. (a) RT-PCR determination of MCP-1 mRNA from allografts and syngeneic controls, compared with β-actin at days 3, 7, 14, and 21. Semiquantitative results are expressed as a ratio of each PCR product to β-actin band density (n = 4 groups, in which each group represents four pooled tracheas at each time point). *P < 0.05. (b) ELISA measurements of MCP-1 protein levels from allografts and syngeneic controls at days 3–21 (n = 4 groups, in which each group represents four pooled tracheas at each time point). *P < 0.05. (c) MCP-1 mRNA in allografts from CCR2^–/– (BALB/c tracheas to CCR2^–/–) versus CCR2^+/+ (BALB/c tracheas to CCR2^+/+) mice at day 7. (d and e) Day 7 murine BOS section (×400). (d) Lack of nonspecific staining with NRS; (e) anti-murine MCP-1 antibodies demonstrating immunolocalization to injured airway columnar epithelium and mononuclear cells.

Figure 5

CCR2 mRNA expression is markedly elevated in murine allografts undergoing BOS. (a) RT-PCR determination of CCR2 mRNA from allografts and syngeneic controls, compared with β-actin at days 3, 7, 14, and 21. (b) CCR2 mRNA in allografts from CCR2^–/– (BALB/c tracheas to CCR2^–/–) versus CCR2^+/+ (BALB/c tracheas to CCR2^+/+) mice at day 7. Semiquantitative results are expressed as a ratio of each PCR product/β-actin band density (n = 4 groups, in which each group represents four pooled tracheas at each time point). *P < 0.05.

Figure 6

(a–e) FACS analysis of leukocyte cell surface markers CD3, CD4, CD8, Ly-6G (PMN), and MOMA-2 (mononuclear phagocytes) in tracheal allografts from CCR2^–/– versus CCR2^+/+ recipient mice over our 21-day time course (n = 4 groups, in which each group represents four pooled tracheas at each time point). *P < 0.05. (f and g) ELISA measurements of protein level in allografts from CCR2^–/– versus CCR2^+/+ mice at days 7, 14, and 21. (f) MCP-1 and (g) MIP-2 (n = 4 groups, in which each group represents four pooled tracheas at each time point). *P < 0.05.

Figure 7

Representative photomicrographs (×400 and ×40) of the histopathology of tracheal allografts from CCR2^–/– versus CCR2^+/+ recipient mice. (a) Day 14 allografts from CCR2^+/+ recipient mice demonstrate marked transmural leukocyte infiltration, moderate ECM deposition, and severe airway obliteration epithelial injury. In contrast, allografts from CCR2^–/– recipient mice show marked reductions in leukocyte infiltration, matrix deposition, epithelial injury, and airway obliteration. (b) Day 21 allografts from CCR2^+/+ recipient mice demonstrate persistent transmural leukocyte infiltration, very severe ECM deposition, airway obliteration, and epithelial injury. In contrast, allografts from CCR2^–/– recipient mice show marked reductions in leukocyte infiltration, matrix deposition, airway obliteration, and epithelial injury.

Figure 8

Hydroxyproline measurements. (a) Allografts from (BALB/c to CCR2^–/– versus BALB/c to CCR2^+/+) recipient mice at days 7, 14, and 21 (n = 4 groups, in which each group represents two pooled tracheas at each time point). *P < 0.05. (b) Allografts from (CCR2^–/– tracheas to BALB/c versus CCR2^+/+ trachea to BALB/c) mice at day 21. (c) Allografts from (BALB/c trachea to C57BL/6) mice treated with anti–MCP-1 versus NRS at day 21 (n = 4 groups, in which each group represents two pooled tracheas at each time point). *P < 0.05.