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. 2007 Dec 1;179(11):7883-90.
doi: 10.4049/jimmunol.179.11.7883.

Cysteinyl leukotrienes are autocrine and paracrine regulators of fibrocyte function

Kevin M Vannella  1 , Tracy R McMillan, Ryan P Charbeneau, Carol A Wilke, Peedikayil E Thomas, Galen B Toews, Marc Peters-Golden, Bethany B Moore
Affiliations

Cysteinyl leukotrienes are autocrine and paracrine regulators of fibrocyte function

Kevin M Vannella et al. J Immunol. .

Abstract

Pulmonary fibrosis is characterized by the accumulation of fibroblasts and myofibroblasts. These cells may accumulate from three potential sources: the expansion of resident lung fibroblasts, the process of epithelial-mesenchymal transition, or the recruitment and differentiation of circulating mesenchymal precursors known as fibrocytes. We have previously demonstrated that fibrocytes participate in lung fibrogenesis following administration of FITC to mice. We now demonstrate that leukotriene-deficient 5-LO(-/-) mice are protected from FITC-induced fibrosis. Both murine and human fibrocytes express both cysteinyl leukotriene receptor (CysLT) 1 and CysLT2. In addition, fibrocytes are capable of producing CysLTs and can be regulated via the autocrine or paracrine secretion of these lipid mediators. Exogenous administration of leukotriene (LT) D(4), but not LTC(4) induces proliferation of both murine and human fibrocytes in a dose-dependent manner. Consistent with this result, CysLT1 receptor antagonists are able to block the mitogenic effects of exogenous LTD(4) on fibrocytes. Endogenous production of CysLTs contributes to basal fibrocyte proliferation, but does not alter fibrocyte responses to basic fibroblast growth factor. Although CysLTs can induce the migration of fibrocytes in vitro, they do not appear to be essential for fibrocyte recruitment to the lung in vivo, possibly due to compensatory chemokine-mediated recruitment signals. However, CysLTs do appear to regulate the proliferation of fibrocytes once they are recruited to the lung. These data provide mechanistic insight into the therapeutic benefit of leukotriene synthesis inhibitors and CysLT1 receptor antagonists in animal models of fibrosis.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
FITC deposition results in release of CysLTs. A, WT (C57BL/6) mice were injected with FITC on day 0. Lung homogenates were collected on days 0, 3, and 7 after FITC treatment. Lipids were extracted and levels of CysLTs were determined by specific EIA, n = 4–6/group, p < 0.05 by ANOVA at day 7. B, Mice were injected with FITC on day 0 and plastic-adherent BAL cells were harvested on day 0 or 7. These cells (which consist mostly of AMs but may contain some fibrocytes and neutrophils after FITC treatment) were cultured at 5 × 105/ml for 1 h in the presence of 5 μM A23187 and supernatants were analyzed for CysLTs via specific EIA, n = 4, p = 0.0002 by Student’s t test. C, WT (129SvEv) or 5-LO−/− mice were injected with FITC intratracheally on day 0. On day 21, mice were euthanized, lungs were removed, and collagen content was determined via hydroxyproline assay, n = 10, p = 0.01 by Student’s t test.
FIGURE 2
FIGURE 2
Murine and human fibrocytes express CysLT1 and CysLT2. A, Murine fibrocytes were purified from C57BL/6 lung digest cultures, and total RNA was prepared and analyzed for the expression of CysLT1 and CysLT2 by real-time RT-PCR. The expression level of each gene was first normalized to β-actin and next compared with the expression level of CysLT1 in AMs. This data are representative of two independent experiments. B, Murine fibrocytes and fibroblasts were purified from lung digest cultures. AMs were isolated from BAL. Four micrograms of protein from each lysate was run and analyzed by Western blotting with anti-human CysLT1 or anti-murine CysLT2 receptor Abs. Each lane represents cells from a single animal. Blots were stripped and probed for β-actin as a housekeeping control. C, Human fibrocytes were isolated from buffy coats of two normal volunteers. Western blotting was performed on 4 μg of cell lysates as above.
FIGURE 3
FIGURE 3
Fibrocytes produce LTs. Fibrocytes and fibroblasts were purified from WT (C57BL/6) mice and cultured at 105/ml overnight in SFM. The next morning, A23187, a calcium ionophore was added for 1 h at 5 μM. Supernatants were collected and analyzed for the production of CysLTs by specific EIA, n = 4. Ionophore-stimulated fibrocytes produced significantly more CysLTs than did fibroblasts, p = 0.002 by Student’s t test.
FIGURE 4
FIGURE 4
LTs regulate fibrocyte proliferation. A, Fibrocytes were purified from lung mince cultures at day 14 from either WT (C57BL/6) or 5-LO−/− mice. Fibrocytes were cultured at 2 × 105/ml in complete medium for 48 h and proliferation was measured via [3H]thymidine incorporation over the final 16 h, n = 6, p = 0.0025 by Student’s t test. B, Fibrocytes were purified from WT (129SvEv) mice or strain-matched 5-LO−/− mice (C) and cultured at 2 × 105/ml in SFM with or without 1–100 nM LTD4 or LTC4 for 24 h. Proliferation was measured over the final 16 h as previously described, n = 6; *, p < 0.05 by ANOVA.
FIGURE 5
FIGURE 5
Exogenous LTD4 can augment fibrocyte proliferation in a CysLT1-dependent manner. Fibrocytes were purified from lung mince cultures of WT (C57BL/6) mice and cultured at 2 × 105/ml in SFM in the presence or absence of 10 nM LTD4 and either MK571 at 10 nM (A) or Ly171883 at 1 μM (B) for 48 h, n = 6. LTD4 was able to significantly stimulate fibrocyte proliferation in both experiments. MK571 and Ly171883 both significantly reduced LTD4-stimulated proliferation (p < 0.05 for both by ANOVA).
FIGURE 6
FIGURE 6
LTD4 stimulates fibrocyte chemotaxis in vitro. Fibrocytes were purified from lung mince cultures of WT (C57BL/6) mice. In brief, 1 × 106 serum-starved fibrocytes were plated in the top well of Boyden chambers and chemotaxis through 8-μm gelatin-coated filters was measured in response to fibronectin (100 mg/ml) or 10 nM LTD4. Ten high-powered fields were counted for each condition. LTD4 stimulation of chemotaxis of fibrocytes is equivalent to that of fibronectin and statistically increased over control (SFM alone), p < 0.05.
FIGURE 7
FIGURE 7
Fewer fibrocytes were cultured from the lungs of FITC-treated 5-LO−/− mice. WT (129SvEv) or 5-LO−/− mice were challenged with FITC on day 0. On day 5 after FITC treatment, BAL was performed and cell pellets were cultured for 14 days to enumerate fibrocytes. Fewer fibrocytes were cultured from the BAL of 5-LO−/− mice compared with WT mice, n = 6–7/group, p = 0.002 by Student’s t test.
FIGURE 8
FIGURE 8
LTs are not necessary for fibrocyte recruitment in response to FITC in vivo. WT (C57BL/6) or 5-LO−/− mice were treated with FITC on day 0. On day 5 after FITC treatment, lungs were subjected to collagenase/DNase digest and cells were stained for expression of CD45 and collagen 1. Similar percentages of CD45+ col 1+ fibrocytes were noted in both groups. Similarly, absolute numbers were not different either. Similar results were obtained when we analyzed the recruitment of fibrocytes to WT mice treated with FITC in the presence or absence of MK571, analyzed BAL cells rather than lung digests, or analyzed WT and 5-LO−/− mice on the 129SvEv background. Thus, LTs do not appear to be essential for fibrocyte recruitment in vivo. Graphs are representative of n = 3 mice/group.
FIGURE 9
FIGURE 9
LTD4 stimulates human fibrocyte proliferation. Fibrocytes were isolated from buffy coats of n = 5 normal volunteers grown in the presence of complete medium with 20% FCS for 14 days before being trypsinized and plated at 2 × 105cells/ml in 96-well flat-bottom plates in the presence of SFM or SFM + 10 nM LTD4 for 48 h. Proliferation was measured by incorporation of [3H]thymidine over the final 16 h. Addition of LTD4 significantly stimulated the proliferation of fibrocytes from each subject (p = 0.01 by paired t test). The mean of each group is shown as the black horizontal line.
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