Fibrocytes develop outside the kidney but contribute to renal fibrosis in a mouse model
Authors: Barbara Reich, Kathrin Schmidbauer, Manuel Rodriguez Gomez, Fabian Johannes Hermann, Nicole Göbel, Hilke Brühl, Isabel Ketelsen, Yvonne Talke, Matthias Mack
Journal: Kidney International (2013)

Collagen-producing bone marrow–derived cells (fibrocytes) have been detected in animal models and patients with fibrotic diseases. In vitro data suggest that they develop from monocytes with the help of accessory cells and profibrotic soluble factors. Using a mouse model of renal fibrosis, unilateral ureteral obstruction, we found the number of circulating fibrocytes was not reduced when monocytes were depleted with a monoclonal antibody against CCR2 or when CCR2-/- mice with very low numbers of circulating or splenic monocytes were analyzed. The absence of CCR2, however, interfered with migration of fibrocytes into the kidney. The phenotype of splenic and renal fibrocytes was very similar and distinct from classical monocytes as fibrocytes expressed no CD115, medium levels of CCR2, and high levels of CD11b and Ly-6G. Using a depleting monoclonal antibody against Ly-6G or bone marrow chimeric mice expressing the diphtheria toxin receptor under the control of CD11b, we could efficiently deplete fibrocytes from the kidney. Depletion of fibrocytes or reduced migration of fibrocytes into the kidney resulted in lower renal expression of collagen-I. Thus, fibrocytes develop outside the kidney independent of infiltrating monocytes and rely on CCR2 for migration into target organs. Collagen-producing bone marrow–derived cells (fibrocytes) have been detected in animal models and patients with fibrotic diseases. In vitro data suggest that they develop from monocytes with the help of accessory cells and profibrotic soluble factors. Using a mouse model of renal fibrosis, unilateral ureteral obstruction, we found the number of circulating fibrocytes was not reduced when monocytes were depleted with a monoclonal antibody against CCR2 or when CCR2-/- mice with very low numbers of circulating or splenic monocytes were analyzed. The absence of CCR2, however, interfered with migration of fibrocytes into the kidney. The phenotype of splenic and renal fibrocytes was very similar and distinct from classical monocytes as fibrocytes expressed no CD115, medium levels of CCR2, and high levels of CD11b and Ly-6G. Using a depleting monoclonal antibody against Ly-6G or bone marrow chimeric mice expressing the diphtheria toxin receptor under the control of CD11b, we could efficiently deplete fibrocytes from the kidney. Depletion of fibrocytes or reduced migration of fibrocytes into the kidney resulted in lower renal expression of collagen-I. Thus, fibrocytes develop outside the kidney independent of infiltrating monocytes and rely on CCR2 for migration into target organs. Fibrocytes are defined as bone marrow–derived cells that produce collagen type I and to a lower degree other connective tissue proteins.1.Bucala R. Spiegel L.A. Chesney J. et al.Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar,2.Chesney J. Metz C. Stavitsky A.B. et al.Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.J Immunol. 1998; 160: 419-425PubMed Google Scholar In addition, they express markers of hematopoietic cells like CD45, CD11b, and CD34.3.Pilling D. Fan T. 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Peng X. et al.Circulating monocytes from systemic sclerosis patients with interstitial lung disease show an enhanced profibrotic phenotype.Lab Invest. 2010; 90: 812-823Crossref PubMed Scopus (179) Google Scholar The differentiation of monocytes into fibrocytes in vitro suggests that in vivo fibrocytes also develop from classical monocytes that have entered injured tissues.1.Bucala R. Spiegel L.A. Chesney J. et al.Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.Mol Med. 1994; 1: 71-81Crossref PubMed Google Scholar, 2.Chesney J. Metz C. Stavitsky A.B. et al.Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.J Immunol. 1998; 160: 419-425PubMed Google Scholar, 4.Curnow S.J. Fairclough M. Schmutz C. et al.Distinct types of fibrocyte can differentiate from mononuclear cells in the presence and absence of serum.PLoS One. 2010; 5: e9730Crossref PubMed Scopus (49) Google Scholar, 5.Niedermeier M. Reich B. Rodriguez Gomez M. et al.CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.Proc Natl Acad Sci USA. 2009; 106: 17892-17897Crossref PubMed Scopus (182) Google Scholar However, fibrocytes are also found in the peripheral blood and the spleen of healthy humans and mice, opening up the possibility that fibrocytes do not develop locally from monocytes but migrate as already differentiated or partially differentiated collagen-producing cells from the peripheral blood or the spleen into injured tissues. To analyze this question and to investigate the contribution of fibrocytes to renal collagen type I expression, we differentially depleted monocytes and fibrocytes with monoclonal antibodies and diphtheria toxin (DT) in bone marrow chimeric mice and analyzed the number of fibrocytes, the time course of their appearance, and the outcome of fibrosis. We used the model of unilateral ureteral ligation that results in rapid infiltration of the obstructed kidney with monocytes, T cells, and neutrophils. Fibrocytes can readily be detected in the obstructed kidney and fibrosis develops within 5–7 days after ureteral ligation.5.Niedermeier M. Reich B. Rodriguez Gomez M. et al.CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.Proc Natl Acad Sci USA. 2009; 106: 17892-17897Crossref PubMed Scopus (182) Google Scholar,23.Sakai N. Wada T. Yokoyama H. et al.Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis.Proc Natl Acad Sci USA. 2006; 103: 14098-14103Crossref PubMed Scopus (220) Google Scholar We have shown previously that fibrocytes develop under appropriate culture conditions from splenic monocytes expressing the surface markers CD11b, CD115, and Gr-1.5.Niedermeier M. Reich B. Rodriguez Gomez M. et al.CD4+ T cells control the differentiation of Gr1+ monocytes into fibrocytes.Proc Natl Acad Sci USA. 2009; 106: 17892-17897Crossref PubMed Scopus (182) Google Scholar In vitro differentiated fibrocytes still express high levels of CD11b and CD45 but only low levels of CD115. In order to characterize the expression of surface markers on fibrocytes in vivo, we performed unilateral ureteral ligation that leads to a rapid and pronounced infiltration of the obstructed kidney with fibrocytes. The gating strategy to identify fibrocytes is shown in Figure 1a. Single cells (Gate A, exclusion of cell doublets) and peripheral blood mononuclear cells (Gate B, exclusion of neutrophils) were identified by light scatter properties. After gating of CD45+ cells (Gate C) expression of CD11b and intracellular collagen-I was displayed. Staining with an isotype control antibody (rabbit IgG) was performed to exclude unspecific staining. Before ureteral ligation (day 0), we detected only very low numbers of fibrocytes in the obstructed kidney, whereas there was a strong increase after ureteral ligation (Figure 1b). In the spleen, fibrocytes were clearly detectable on day 0 and increased on days 3–7 (Figure 1b). Fibrocytes in the spleen and the obstructed kidney were very similar regarding expression of surface markers and, interestingly, displayed a rather unusual combination of surface molecules (Figure 1c). Apart from CD11b and CD45, collagen-I+ cells expressed medium to low levels of CCR2, medium levels of Gr-1 and Ly-6C, high levels of Ly-6G, and were negative for CD115. These markers distinguish fibrocytes clearly from monocytes (Table 1) that typically express CD115 and consist of two main subpopulations, one expressing high levels of CCR2, Gr-1, and Ly-6C, and the other expressing low to negligible levels of CCR2, low levels of Gr-1, and no Ly-6C.36.Auffray C. Sieweke M.H. Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells.Annu Rev Immunol. 2009; 27: 669-692Crossref PubMed Scopus (1161) Google Scholar, 37.Geissmann F. Jung S. Littman D.R. Blood monocytes consist of two principal subsets with distinct migratory properties.Immunity. 2003; 19: 71-82Abstract Full Text Full Text PDF PubMed Scopus (2582) Google Scholar, 38.Sunderkotter C. Nikolic T. Dillon M.J. et al.Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.J Immunol. 2004; 172: 4410-4417Crossref PubMed Scopus (871) Google Scholar We did not detect intracellular expression of collagen-I in CD115+ monocytes (Figure 1c). These data show that fibrocytes have either changed their repertoire of surface markers after development from monocytes or constitute a distinctive subpopulation of cells in the spleen. In order to investigate the contribution of various cell populations for development of fibrocytes, we depleted these cell populations with monoclonal antibodies or DT.Table 1Expression of markers on monocyte subpopulations and fibrocytesMarkerMonocyteMonocyteFibrocytesSubpopulation 1Subpopulation 2Collagen I--+CD45+++CD11b++++++CD115++++-Ly-6C++-+Ly-6G--+Gr-1+-+CCR2++-(+)-, No expression; +, weak expression; ++, strong expression. Open table in a new tab -, No expression; +, weak expression; ++, strong expression. CCR2+ cells were depleted by daily application of the CCR2 antibody MC-21 (50μg intraperitoneal (i.p.)) from day -1 to day 6 (Figure 2).39.Bruhl H. Cihak J. Plachy J. et al.Targeting of Gr-1+,CCR2+ monocytes in collagen-induced arthritis.Arthritis Rheum. 2007; 56: 2975-2985Crossref PubMed Scopus (95) Google Scholar,40.Mack M. Cihak J. Simonis C. et al.Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice.J Immunol. 2001; 166: 4697-4704Crossref PubMed Scopus (347) Google Scholar Unilateral ureteral obstruction (UUO) was performed on day 0 and mice analyzed on day 7. After the first injection of MC-21, Gr-1+ monocytes were almost completely depleted from the peripheral blood as measured by flow cytometry on day 0 (Figure 2a). Further daily injection of MC-21 maintained the depletion of Gr-1+ monocytes until day 3 and also resulted in a considerable reduction of Gr-1− monocytes that are known to develop from Gr-1+ monocytes (Figure 2a).41.Arnold L. Henry A. Poron F. et al.Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.J Exp Med. 2007; 204: 1057-1069Crossref PubMed Scopus (1397) Google Scholar,42.Yrlid U. Jenkins C.D. MacPherson G.G. Relationships between distinct blood monocyte subsets and migrating intestinal lymph dendritic cells in vivo under steady-state conditions.J Immunol. 2006; 176: 4155-4162Crossref PubMed Scopus (119) Google Scholar However, on day 7, Gr-1+ monocytes reappeared in the peripheral blood because the activity of MC-21 was neutralized by a humoral immune response against the antibody (Supplementary Figure S1 online). On day 7, there was also no significant depletion of monocytes in the spleen and obstructed kidney (Figure 2b). Fibrocytes, identified by expression of CD45, CD11b, and collagen-I, were not significantly diminished by treatment with MC-21 in the obstructed kidney or the spleen on day 7 (Figure 2c and d). In addition, there was no change in renal collagen-I expression as measured by reverse transcription–PCR for collagen type I (Figure 2e). Download .pdf (.22 MB) Help with pdf files Supplementary Figures To exclude that neutralization of MC-21 is responsible for the failure of MC-21 to deplete the fibrocytes in the kidney on day 7, we treated mice with MC-21 for only a shorter period of time from day -1 to day 2 and analyzed peripheral blood, spleen, and kidneys on day 3 (Figure 3). There was a marked depletion of Gr-1+ monocytes in the peripheral blood, spleen, and kidney, but no depletion of fibrocytes (Figure 3a and b). The CCR2 antibody MC-21 very efficiently depleted Gr-1+ monocytes and interfered with the development of Gr-1− monocytes, but was unable to deplete fibrocytes from either the spleen or kidney, most likely because of their low expression of CCR2 (the mean fluorescence intensity for CCR2 was 17±2.3 s.d. on fibrocytes and 115±2.6 s.d. on Gr-1+ monocytes.) Similarly, treatment of mice with MC-21 from days 4 to 6 after UUO with analysis at day 7 efficiently depleted the monocytes, but did not reduce fibrocyte numbers or renal collagen-I expression (Supplementary Figure S2 online). The persistence of fibrocytes in the spleen and the accumulation of fibrocytes in the obstructed kidney despite considerable depletion of monocytes indicate that fibrocytes do not develop from monocytes within the kidney. Fibrocytes could either develop from other cell populations within the kidney or more likely migrate from the periphery (for example, spleen) via the peripheral blood into the kidney. To analyze whether monocytes play a role in the development of fibrocytes in the spleen and to investigate whether CCR2 plays a role in the migration of fibrocytes into the kidney, we analyzed CCR2−/− mice in comparison with wild-type mice (Figure 4). Because of the requirement of CCR2 for emigration of monocytes out of the bone marrow, CCR2−/− mice have low numbers of monocytes in the peripheral blood and spleen but increased numbers of monocytes in the bone marrow.25.Serbina N.V. Pamer E.G. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2.Nat Immunol. 2006; 7: 311-317Crossref PubMed Scopus (1192) Google Scholar,26.Tsou C.L. Peters W. Si Y. et al.Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites.J Clin Invest. 2007; 117: 902-909Crossref PubMed Scopus (797) Google Scholar The numbers of monocytes were analyzed before (day 0) and on day 7 after UUO. As expected, CCR2−/− mice contained much lower numbers of monocytes in the peripheral blood, spleen, and kidney (Figure 4a–c). Very strikingly, the number of fibrocytes in the peripheral blood on day 0 and in the spleen and peripheral blood on day 7 was not reduced in CCR2−/− mice compared with wild-type mice, indicating that a reduced frequency of Gr-1+ or Gr-1− monocytes does not translate into reduced numbers of fibrocytes in the spleen (Figure 4d and Supplementary Figure S3 online). However, in the obstructed kidney of CCR2−/− mice, the number of fibrocytes was significantly reduced, indicating that migration of fibrocytes from the spleen into the kidney is dependent on CCR2 (Figure 4e). Reduced numbers of renal fibrocytes correlate with a significantly lower renal collagen-I expression as shown by reduced deposition of collagen type I and reduced levels of collagen-I mRNA in CCR2−/− mice (Figure 4f and g). The depletion of fibrocytes with MC-21 failed because of the low expression of CCR2 on fibrocytes (see Figure 1c). In addition, prolonged treatment with MC-21 resulted in the development of a neutralizing immune response against MC-21. In order to circumvent both limitations, we used transgenic mice expressing the DT receptor under the control of the CD11b promoter (CD11b-DTR mice) allowing depletion of CD11b+ cells with DT (Figure 5). Bone marrow chimeras were generated whereby bone marrow from CD11b-DTR C57BL/6 mice was transplanted into lethally irradiated wild-type C57BL/6 mice. Thereby, the toxic effects of DT on non-bone marrow–derived cells could be excluded and depletion could be restricted to bone marrow–derived CD11b+ cells. Because of the high expression of CD11b on fibrocytes in the spleen and kidney, we expected fibrocytes to be excellent targets for DT in CD11b-DTR mice. DT was administered i.p. from day -1 to day 6 and UUO was performed on day 0. After the first injection of DT, monocytes were reduced to ∼50% in the peripheral blood, whereas subsequent daily injections resulted in an almost complete depletion of monocytes (Figure 5a). In addition, in the obstructed kidney, treatment with DT markedly reduced the number of Gr-1+ and Gr-1− monocytes (Figure 5b). Treatment with DT markedly reduced the numbers of fibrocytes in the spleen and in the obstructed kidney (Figure 5c and d). Reduced numbers of fibrocytes in the kidney correlated with a significantly reduced deposition of collagen-I and a reduced expression of collagen-I mRNA in the obstructed kidney (Figure 5e and f). These data show that depletion of bone marrow–derived CD11b+ cells reduces the number of fibrocytes in the spleen and the kidney and interferes with development of renal fibrosis. Use of bone marrow chimeric mice also excludes that DT depletes non-bone marrow–derived cells that may also express CD11b. The expression of high levels of Gr-1 on the surface of fibrocytes in the spleen and the kidney may allow depletion of these cells with an antibody against Gr-1 (clone RB6-8C5). This rat IgG2b antibody is known to deplete Gr-1+ monocytes as well as Gr-1+ neutrophils in mice.43.Daley J.M. Thomay A.A. Connolly M.D. et al.Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice.J Leukoc Biol. 2008; 83: 64-70Crossref PubMed Scopus (813) Google Scholar To investigate the ability of the antibody to also deplete fibrocytes, UUO was performed on day 0 and mice were injected daily from day 0 to day 6 with anti-Gr-1 or a isotype control antibody (Figure 6). In the peripheral blood, the Gr-1 antibody almost completely depleted Gr-1+ monocytes and neutrophils on day 3 (Figure 6a and b). However, on day 7 the antibody lost its activity and the number of Gr-1+ monocytes and neutrophils increased. In the kidney there was no significant reduction of monocytes on day 7 (Figure 6c). In contrast to the experiments with the CCR2 antibody MC-21, injection of an antibody against Gr-1 resulted in a profound depletion of fibrocytes from the spleen and the kidney (Figure 6d and e) and a significant reduction in renal deposition of collagen type I (Figure 6f). To analyze whether the Gr-1 antibody is also able to deplete fibrocytes that have already entered the kidney, we performed UUO on day 0 and injected the antibody Gr-1 from days 3 to 6 to allow migration of fibrocytes into the kidney from days 0 to 3 (Supplementary Figure S4 online). Short-term late treatment with anti-Gr-1 not only depleted to a great extent the Gr-1+ monocytes and neutrophils in the peripheral blood, spleen, and kidney as measured on day 7 (Supplementary Figure S4a and b online), but also almost completely eliminated the fibrocytes from the spleen and the UUO kidney (Supplementary Figure S4c and d online), suggesting that the antibody also affects fibrocytes that have entered the kidney during the first 3 days of UUO. Late depletion of fibrocytes with anti-Gr-1 also significantly reduced the degree of renal collagen-I expression (Supplementary Figure S4e online). In addition to a late injection, we also injected the antibody before and during the first days of UUO (from day -1 to day 2) and analyzed the mice on day 3 (Supplementary Figure S5 online). The antibody markedly reduced the number of Gr-1+ monocytes, neutrophils, and fibrocytes in the peripheral blood, spleen, and kidney. In summary, these data indicate that fibrocytes can efficiently be depleted from the spleen and kidney with an anti-Gr-1 antibody and that fibrocytes significantly contribute to the development of renal fibrosis in the UUO model. Our data also indicate that fibrocytes do not develop locally in the kidney from monocytes but exist as collagen-producing cells in the spleen of healthy mice and migrate in a CCR2-dependent manner into the obstructed kidney, where they contribute to deposition of collagen. There are two possibilities of how fibrocytes accumulate in injured tissues and contribute to development of fibrosis. First, tissue injury leads to influx of leukocytes, including monocytes and T cells, whereby fibrocytes then differentiate locally from infiltrating monocytes with the help of accessory cells or soluble factors. Reduced or increased migration of monocytes and factors that regulate the differentiation of monocytes into fibrocytes would determine the number of fibrocytes in the tissue. Reduced numbers of tissue fibrocytes have been observed in mice deficient for certain chemokine receptors such as CCR1, CCR2, CCR5, and CCR7.16.Moore B.B. Kolodsick J.E. Thannickal V.J. et al.CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.Am J Pathol. 2005; 166: 675-684Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 17.Moore B.B. Murray L. Das A. et al.The role of CCL12 in the recruitment of fibrocytes and lung fibrosis.Am J Respir Cell Mol Biol. 2006; 35: 175-181Crossref PubMed Scopus (256) Google Scholar, 18.Scho