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Basic and translational research
Extended report
Caveolin-1 regulates leucocyte behaviour in fibrotic lung disease
  1. Elena Tourkina1,
  2. Mathieu Richard1,
  3. James Oates1,
  4. Ann Hofbauer1,
  5. Michael Bonner1,
  6. Pal Gööz1,
  7. Richard Visconti2,
  8. Jing Zhang2,
  9. Sergei Znoyko1,
  10. Corey M Hatfield1,
  11. Richard M Silver1,
  12. Stanley Hoffman1,2
  1. 1Division of Rheumatology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA
  2. 2Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina, USA
  1. Correspondence to Elena Tourkina, Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 912 MSC 637, Charleston, SC 29425, USA; tourkine{at}musc.edu

Abstract

Objectives Reduced caveolin-1 levels in lung fibroblasts from patients with scleroderma and the lungs of bleomycin-treated mice promote collagen overexpression and lung fibrosis. This study was undertaken to determine whether caveolin-1 is deficient in leucocytes from bleomycin-treated mice and patients with scleroderma and to examine the consequences of this deficiency and its reversal.

Methods Mice or cells received the caveolin-1 scaffolding domain (CSD) peptide to reverse the pathological effects of reduced caveolin-1 expression. In bleomycin-treated mice, the levels of caveolin-1 in leucocytes and the effect of CSD peptide on leucocyte accumulation in lung tissue were examined. To validate the results in human disease and to identify caveolin-1-regulated molecular mechanisms, monocytes and neutrophils were isolated from patients with scleroderma and control subjects and caveolin-1, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, CXC chemokine receptor 4 (CXCR4) and matrix metalloproteinase 9 (MMP-9) expression/activation were evaluated. These parameters were also studied in monocytes treated with cytokines or CSD peptide.

Results Leucocyte caveolin-1 is important in lung fibrosis. In bleomycin-treated mice, caveolin-1 expression was diminished in monocytes and CSD peptide inhibited leucocyte recruitment into the lungs. These observations are relevant to human disease. Monocytes and neutrophils from patients with scleroderma contained less caveolin-1 and more activated ERK, JNK and p38 than those from control subjects. Treatment with CSD peptide reversed ERK, JNK and p38 hyperactivation. Scleroderma monocytes also overexpressed CXCR4 and MMP-9, which was inhibited by the CSD peptide. Cytokine treatment of normal monocytes caused adoption of the scleroderma phenotype (low caveolin-1, high CXCR4 and MMP-9 and signalling molecule hyperactivation).

Conclusions Caveolin-1 downregulation in leucocytes contributes to fibrotic lung disease, highlighting caveolin-1 as a promising therapeutic target in scleroderma.

https://doi.org/10.1136/ard.2009.117580

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    Introduction

    Scleroderma is a complex autoimmune disease involving fibrosis of the skin, lungs and other organs. Indeed, lung fibrosis is the primary cause of morbidity and mortality in scleroderma. Despite the fact that it is an autoimmune disease, little is known about the potential dysregulation of signalling molecules in cells of the immune system in scleroderma. Activated alveolar macrophages and polymorphonuclear cells (PMNs) are frequently observed in bronchoalveolar lavage fluid (BALF) from patients with scleroderma lung disease,1 2 and the presence of these cells in BALF has been used to identify patients at high risk for progression of pulmonary fibrosis. Activated alveolar macrophages and PMNs are also present in high levels in the BALF and lung tissue in mice treated with bleomycin, an animal model of scleroderma. Thus, monocytes/macrophages and PMNs are particularly reasonable candidates to be immune cells in which signalling is altered during fibrotic lung disease.

    Caveolin-1 serves as a central molecule in signalling cascades because it is a scaffolding protein that binds to a variety of kinases and thereby regulates their activity. The caveolin-1 scaffolding domain (CSD) peptide, when attached to the Antennapedia internalisation sequence, is able to enter cells and mimic the activity of full-length caveolin-1.3 4 While many papers have reported the importance of caveolin-1 in inflammation, most of these papers have focused on caveolin-1 in endothelial cells where it is important in signalling cascades involving mediators such as intercellular adhesion molecule, lipopolysaccharide and nitric oxide.3,–,9 Less is known about the functions of caveolin-1 in cells of the immune system. Caveolin-1 is present in various classes of leucocytes including monocytes/macrophages, PMNs, mast cells and lymphocytes.10,–,14 It inhibits the expression of proinflammatory cytokines in macrophages by regulating the activation of mitogen-activated protein kinase (MAPK) family members.15 In vivo experiments demonstrate roles for caveolin-1 in neutrophil activation, transendothelial migration and resultant lung inflammation,16 and in inflammatory cell accumulation in the lungs of bleomycin-treated mice.17

    Caveolin-1 levels are significantly reduced in the fibrotic lung tissue of bleomycin-treated mice and of patients with scleroderma and idiopathic pulmonary fibrosis, and in fibroblasts derived from patient lung tissue.17,–,19 This study demonstrates that the deficit in caveolin-1 in mice and humans with fibrotic lung disease is not limited to lung fibroblasts; peripheral blood cells are also deficient in caveolin-1.

    Materials and methods

    Detailed methods are presented in the online supplement for:

    • bleomycin-induced lung injury and CSD peptide treatment

    • mouse leucocyte staining

    • human monocyte and PMN isolation

    • human leucocyte staining

    • gelatin zymography

    • statistical analyses.

    Results

    CSD peptide inhibits monocyte and neutrophil accumulation in the lungs of bleomycin-treated mice

    We previously showed that the CSD peptide has a general inhibitory effect on the accumulation of inflammatory cells in the lungs of bleomycin-treated mice.17 To identify specific inflammatory cell types affected by CSD peptide, we have now quantified neutrophils and monocytes/macrophages. Bleomycin causes a massive increase in the number of these cells in lung tissue which is almost completely inhibited by CSD peptide (figure 1), both 3 and 7 days after bleomycin treatment. In accordance with the literature, more neutrophils are present 3 days after bleomycin treatment than 7 days after, whereas more monocytes/macrophages are present 7 days after bleomycin treatment.

    Figure 1
    Figure 1

    Caveolin-1 scaffolding domain (CSD) peptide inhibits the recruitment of monocytes/macrophages and neutrophils into the lungs of bleomycin-treated mice. Sections from saline-treated mice receiving the CSD peptide or control scrambled (Scr) peptide were harvested 7 days after treatment. Sections from bleomycin-treated mice receiving the CSD peptide or Scr peptide were harvested 3 or 7 days after treatment. Representative images are shown of sections stained with monoclonal antibodies Gr-1 (A) to detect neutrophils and Mac-3 (C) to detect monocytes/macrophages and counterstained with the nuclear stain 4',6-diamidino-2-phenylindole. Bar = 10 µm. The number of cells per mm2 stained with Gr-1 (B) or with Mac-3 (D) was determined in images from six mice in each category, five fields per mouse. ***p<0.001.

    Because CSD peptide reverses the effect of bleomycin on the accumulation of monocyte/macrophages and neutrophils in lung tissue, we tested the possibility that bleomycin inhibits the expression of caveolin-1 in monocytes and neutrophils, both in the peripheral circulation and in lung tissue. Seven days after bleomycin treatment we observed a major decrease in caveolin-1 in circulating monocytes but not in neutrophils (figure 2). We also observed modest, but statistically significant, decreases in caveolin-1 expression in monocyte/macrophages and neutrophils in the lung tissue of bleomycin-treated mice (see figure S1 in the online supplement).

    Figure 2
    Figure 2

    Bleomycin inhibits caveolin-1 (Cav-1) expression in circulating monocytes. Peripheral blood was collected from mice 7 days after treatment with bleomcyin or saline vehicle. Cells were collected by cytospin and stained with 4′,6-diamidino-2-phenylindole (DAPI), with anti-caveolin-1, and with either Mac-3 or Gr-1. Representative images are shown of staining in Mac-3-positive (A) and Gr-1-positive (B) cells. Bar = 10 µm. (C) Caveolin-1 staining intensity in randomly chosen Mac-3-positive cells (Cav/Mono) and Gr-1-positive cells (Cav/Neut) from saline- and bleomycin-treated mice was quantified (average ± SEM). In each case the data were obtained from 20–60 cells from 3–7 mice. **p<0.01.

    Signalling molecule profiles in monocytes and PMNs from patients with scleroderma and controls

    To validate the relevance to human disease of our results in the bleomycin model, we evaluated caveolin-1 levels in monocytes and PMNs isolated from the blood of patients with scleroderma and control subjects. The clinical features of these patients are summarised in table S1 in the online supplement. Among the most noteworthy observations were: duration 7.6±6.8 years; limited cutaneous disease in 6 patients, diffuse cutaneous disease in 11 patients, undifferentiated connective tissue disease in 1 patient; organ involvement (lungs, 18/18; gastrointestinal, 18/18; heart, 11/18; kidneys, 1/18).

    Caveolin-1 levels were 41% as high in monocytes from patients with scleroderma as in those from control subjects (figure 3A,D; p<0.001). We also confirmed this deficit in caveolin-1 expression in monocytes from patients with scleroderma by immunofluorescent microscopy (figure 3F). When the activation of MAPK family members regulated by caveolin-1 was examined, we found that activated extracellular signal-regulated protein kinase (ERK), p38 and c-Jun N-terminal kinases (JNK) were present at much higher levels in monocytes in patients with scleroderma than in monocytes from control subjects (p<0.001), even though total ERK, p38 and JNK were present at similar levels (figure 3A,D). Two additional molecules important in inflammation, COX-2 and the chemokine receptor CXC chemokine receptor 4 (CXCR4), were also present at much higher levels in monocytes from patients with scleroderma than in those from controls (p<0.001).

    Figure 3
    Figure 3

    Signalling molecules differ between normal and scleroderma leucocytes. The expression/activation of the indicated proteins in freshly isolated leucocytes from healthy donors (norm, normal) and patients with scleroderma (SSc) was determined by western blotting (50 µg total protein per lane for monocytes, 100 µg for polymorphonuclear cells (PMNs)) and quantified by densitometry. (A) Representative western blot performed using monocytes enriched by adherence. (B) Representative western blot from one of three independent experiments performed using monocytes enriched by immunodepletion rather than adherence. (C) Representative western blot performed using PMNs. (D) Densitometric quantification (average ± SEM) of the data from seven independent experiments similar to (A) using seven different subjects in each category. The level of each protein in normal monocytes was set to 100 arbitrary units. (E) Densitometric quantification (average ± SEM) of the data from seven independent experiments similar to (C) using seven different subjects in each category. The level of each protein in normal PMNs was set to 100 arbitrary units. The expression of caveolin-1 in isolated monocytes (F) and PMNs (G) was evaluated by immunofluorescent microscopy. Bars = 5 µm. Note that, in both normal and scleroderma monocytes, caveolin-1 is present primarily in a punctate pattern forming a ring around the perimeter of the cells and to a lesser extent over the rest of the cell. However, the ring is much brighter and more continuous in normal monocytes than in scleroderma monocytes. Note that, in normal PMNs, caveolin-1 is present in a punctate pattern at high levels over the nucleus and at a moderate level at the cell surface. In contrast, scleroderma PMNs show no staining at the cell surface and only occasional punctate staining over the nucleus. ***p<0.001. CSD, caveolin-1 scaffolding domain; CXCR4, CXC chemokine receptor 4; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; Scr, scrambled.

    To rule out the possibility that the differences between normal and scleroderma monocytes in caveolin-1, pERK, pJNK, pp38 and CXCR4 expression were induced by the adherence step used in their isolation, monocytes were also enriched by negative selection (to remove T cells, B cells, natural killer cells, erythrocytes and granulocytes). This approach yielded a cell population that was found by both flow cytometry and immunocytochemistry of cells collected by cytospin to contain about 95% Mac-1-positive monocytes, an improvement over enrichment by adherence which we found to yield about 75% Mac-1-positive monocytes. The results obtained with negative selection (figure 3B) were indistinguishable from those obtained with adherence. These observations indicate that the properties we have attributed to SSc monocytes are indeed the properties of these cells and not of contaminating cells in the preparation.

    To determine whether the decrease in caveolin-1 expression observed in scleroderma monocytes is also observed in other types of immune cells, we examined caveolin-1 expression in PMNs, T cells and B cells. As for monocytes from patients with scleroderma, caveolin-1, ERK, JNK, p38 and COX-2 expression or activation were also altered in PMNs from patients with scleroderma. Caveolin-1 levels were 12% as high in PMNs from patients with scleroderma as in normal PMNs (figure 3C,E; p<0.001). This deficit in caveolin-1 expression was confirmed by immunofluorescent microscopy (figure 3G). Activated ERK, p38 and JNK were present at much higher levels in PMNs from patients with scleroderma than in control cells (p<0.001), even though total ERK, p38 and JNK were present at similar levels (figure 3C,E). COX-2 levels were also upregulated (p<0.001). There was also a small but statistically significant decrease in the caveolin-1 level in T cells from patients with scleroderma (see figure S2 in the online supplement). In contrast, there was no difference in caveolin-1 expression between normal and scleroderma B cells (see figure S2 in the online supplement).

    Activated monocytes from control subjects are similar to those from patients with scleroderma

    To determine whether cytokines present in the blood and tissues of patients with scleroderma activate monocytes by decreasing caveolin-1 expression, monocytes from control subjects were treated with the classic proinflammatory cytokine tumour necrosis factor α (TNFα) or the profibrotic cytokine transforming growth factor β (TGFβ). Each treatment caused an almost complete loss of caveolin-1 (about 90%) (figure 4). Each treatment also increased the activation of ERK and JNK and CXCR4 expression. Only TNFα increased p38 activation; neither TGFβ nor TNFα increased COX-2 expression (figure 4). Thus, much of the phenotype of scleroderma monocytes (low caveolin-1, high pERK, pJNK, pp38 and CXCR4) is mimicked by treating normal monocytes with TGFβ and/or TNFα.

    Figure 4
    Figure 4

    Cytokine treatment of monocytes alters caveolin-1 (Cav-1) and CXC chemokine receptor 4 (CXCR4) expression and extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 activation. Normal peripheral blood mononuclear cells were plated in six-well plates (2 × 107 cells/well), incubated for 1 h and washed with Hank's balanced salt solution. Attached monocytes were further incubated for 3 h with RPMI 1640 containing 10 ng/ml of tumour necrosis factor α (TNFα) or transforming growth factor β (TGFβ) or no addition. The expression/activation of the indicated proteins was determined by western blotting (50 µg total protein per lane) of cell extracts and quantified by densitometry. Actin served as a loading control. (A) Representative blot. (B) Densitometric quantification (average±SEM) of the data from three independent experiments similar to (A) using three different normal subjects. The level of each protein in untreated monocytes was set to 100 arbitrary units. ***p<0.001; **p<0.01; *p<0.05.

    Mechanism of signalling molecule activation in monocytes from patients with scleroderma

    To confirm that the hyperactivation of MAPK family members in monocytes from patients with scleroderma is due to decreased caveolin-1 expression/function, monocytes from control subjects and patients with scleroderma were treated with the CSD peptide to upregulate caveolin-1 function. CSD peptide inhibited ERK, JNK and p38 activation (p<0.05, p<0.001 and p<0.001, respectively), while total ERK, JNK and p38 expression was unaffected (figure 5). Treatment with CSD peptide decreased CXCR4 expression in monocytes from patients with scleroderma but not in those from control subjects where this molecule is expressed only at low levels. CSD peptide treatment did not affect COX-2 expression in monocytes from patients with scleroderma or control subjects, indicating that the upregulation of COX-2 in scleroderma monocytes occurs via a signalling mechanism that is independent of caveolin-1 and MAPK.

    Figure 5
    Figure 5

    Caveolin-1 scaffolding domain (CSD) peptide inhibits extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinases (JNK) and p38 activation in normal and scleroderma monocytes. Peripheral blood mononuclear cells from normal subjects and patients with scleroderma were plated in six-well plates (2 × 107 cells/well), incubated for 1 h and washed with Hank's balanced salt solution. Attached monocytes were incubated for 3 h in RPMI 1640 containing 5 µM of the CSD peptide (+) or scrambled control peptide (−). The expression/activation of the indicated proteins was determined by western blotting (50 µg total protein per lane). Actin served as the loading control. Similar results were obtained in three independent experiments performed using cells isolated from different subjects. CXCR4, CXC chemokine receptor 4.

    CSD peptide regulates the inflammatory function of monocytes

    To evaluate whether caveolin-1 can regulate inflammatory cell function, the effect of CSD peptide on the release of matrix metalloproteinase (MMP-9) by normal monocytes, normal monocytes treated with TGFβ and monocytes from patients with scleroderma was determined. MMP-9 release was enhanced by about 50% in both scleroderma monocytes and TGFβ-treated normal monocytes (figure 6). Treatment with CSD peptide decreased MMP-9 release to the same low level in normal monocytes, scleroderma monocytes and TGFβ-treated normal monocytes (figure 6). These results demonstrate that caveolin-1 regulates the immune function of monocytes and that the deficient expression of caveolin-1 in scleroderma monocytes is directly responsible for their altered function.

    Figure 6
    Figure 6

    Caveolin-1 scaffolding domain (CSD) peptide inhibits matrix metalloproteinase 9 (MMP-9) release from transforming growth factor β (TGFβ)-activated normal monocytes and scleroderma monocytes. Peripheral blood mononuclear cells from normal subjects and patients with scleroderma were plated in six-well plates (2 × 107 cells/well), incubated for 1 h and washed with Hank's balanced salt solution. Attached monocytes were incubated in six-well plates for 1 h at 37°C in RPMI 1640 supplemented with 10 ng/ml TGFβ or no addition. The cells were then incubated an additional 3 h in fresh RPMI 1640 containing 5 µM of either CSD peptide or control scrambled (Scr) peptide. Monocytes from patients with scleroderma without TGFβ priming were also treated with CSD or control peptide for 3 h. The level of MMP-9 in equal aliquots of culture medium was determined by gelatin zymography and quantified by densitometry. (A) Representative zymogram. (B) Densitometric quantification (average±SEM) of the data from four independent experiments similar to (A) using four different subjects in each category. The level of MMP-9 secreted by normal monocytes that were not treated with TGFβ and were treated with control peptide was set to 100 arbitrary units. ***p<0.001; *p<0.05.

    Discussion

    The reduced level of caveolin-1 present in fibroblasts from fibrotic lung tissue has been shown to be directly responsible for the hyperactivation of several signalling molecules and the overexpression of collagen in vitro and to contribute to the progression of lung fibrosis in vivo.17 18 The present study is the first demonstration that caveolin-1 levels are also reduced in leucocytes from humans and animals with fibrotic lung disease, and that reduced leucocyte caveolin-1 expression regulates signalling and cell behaviour in a manner that promotes the progression of lung fibrosis.

    CSD peptide has been observed to reverse the effects of caveolin-1 depletion in many systems by substituting for the full-length molecule.17 19 20 We demonstrate here that treatment with CSD peptide inhibits the accumulation of monocytes/macrophages and neutrophils in the lung tissue of bleomycin-treated mice. Consistent with the idea that bleomycin treatment causes the loss of caveolin-1 from leucocytes and that CSD peptide treatment compensates for the loss of caveolin-1, we found lower caveolin-1 levels in lung monocytes/macrophages, lung neutrophils and circulating monocytes from bleomycin-treated mice than from control mice. On the other hand, we did not detect reduced caveolin-1 levels in circulating neutrophils from bleomycin-treated mice. There are several reasonable explanations for the fact that CSD peptide reversed neutrophil accumulation in lung tissue in bleomycin-treated mice yet we did not detect low caveolin-1 levels in circulating neutrophils. (1) The highest level of monocyte/macrophage accumulation in lung tissue was 7 days after bleomycin treatment whereas the highest level of neutrophil accumulation was at 3 days. We only measured caveolin-1 at 7 days. It is likely that at 3 days we would have detected diminished caveolin-1 in circulating neutrophils. (2) The population of neutrophils with reduced caveolin-1 may migrate extremely efficiently into the lungs whereas the population of neutrophils that remains in the circulation may not have reduced caveolin-1. (3) CSD peptide may inhibit neutrophil migration into injured lung tissue via an indirect mechanism in which it does not simply substitute for reduced caveolin-1 in circulating neutrophils. Given our results with leucocytes from patients with scleroderma in whom we did see reduced caveolin-1 levels in both circulating monocytes and PMNs, we favour the first explanation.

    In addition to a lower level of caveolin-1 expression in PMNs from patients with scleroderma, we also observed an altered caveolin-1 distribution. In PMNs from normal subjects, caveolin-1 is present in a punctate pattern at high levels over the nucleus and at a moderate level at the cell surface. In contrast, PMNs from patients with scleroderma show no staining at the cell surface and only occasional punctate staining over the nucleus. Given that caveolin-1 cycles back and forth from cell-surface caveolae to the Golgi complex via recycling endosomes,21 we speculate that caveolin-1 trafficking is altered in PMNs from patients with scleroderma.

    Experiments using monocytes and PMNs isolated from patients with scleroderma and control subjects have validated the importance of caveolin-1 in the control of leucocyte behaviour in human disease and allowed us to study molecular mechanisms leading to the loss of caveolin-1 and resulting from the loss of caveolin-1. We observed in both monocytes and PMNs from patients with scleroderma that caveolin-1 expression was diminished and that, as observed in other cell types in which caveolin-1 expression is diminished, several members of the MAPK family of signalling molecules (ERK, JNK, p38) were hyperactivated. In addition, COX-2 expression was increased in both cell types and expression of the pro-migratory chemokine receptor CXCR4 and of the inflammatory mediator MMP-9 was increased in monocytes from patients with scleroderma. While it may seem counterintuitive for increased MMP-9 activity to be associated with a fibrotic disease, this is a standard observation.22 One could imagine several reasons why upregulating MMP expression does not reverse fibrosis. It may be that fragments of collagen generated by MMPs signal cells to produce even more collagen. Another possibility is that MMPs secreted by monocytes do not have access to or are not effective in degrading the mature highly-crosslinked collagen that makes up most of the collagen in fibrotic lesions.

    The proinflammatory cytokine TNFα and the profibrotic cytokine TGFβ have major roles in inflammation and fibrosis in scleroderma1 and have been shown to inhibit caveolin-1 expression in other cell types.18 19 23 We therefore examined the possibility that these treatments might inhibit caveolin-1 expression in monocytes. Indeed, when normal monocytes were treated with TNFα or TGFβ, they adopted several aspects of the phenotype of monocytes from patients with scleroderma (low caveolin-1, hyperactivation of ERK and JNK, high CXCR4). However, these treatments did not upregulate COX-2, only TNFα activated p38, and only TGFβ upregulated MMP-9 secretion.

    To determine whether the low level of caveolin-1 in monocytes from patients with scleroderma is upstream from the other observed alterations in phenotype, these cells were treated with the CSD peptide. This treatment reversed the hyperactivation of ERK, JNK and p38 and the upregulation of CXCR4 and MMP-9 expression, but did not significantly affect COX-2 expression. In summary, our results suggest that the TGFβ-rich and TNFα-rich milieu in the blood and tissues of patients with scleroderma leads to the reduction of caveolin-1 expression in monocytes. In turn, the low level of caveolin-1 in monocytes from patients with scleroderma promotes the migration of these cells into target tissues by increasing CXCR4 expression and promotes tissue damage by upregulating MMP-9 expression. In contrast, the upregulation of COX-2 in monocytes from patients with scleroderma occurs via a caveolin-1-independent mechanism. While our observation that monocytes and PMNs from patients with scleroderma overexpress COX-2 suggests that COX-2 inhibitors might be a useful treatment for lung inflammation/fibrosis, this approach may fail because COX-2 inhibitors are likely to promote the proliferation and production of collagen by lung fibroblasts.24 25

    In addition to helping create a milieu in injured lung tissue that promotes tissue damage and the activation of fibroblasts to secrete collagen and other extracellular matrix proteins, monocytes also have a direct role in fibrosis. While historically it was believed that the overexpression of collagen in fibrotic diseases occurred when resident fibroblasts became activated, it is now recognised that a subpopulation of activated collagen-producing fibroblasts arise via the differentiation of monocytes into circulating CD45+ CXCR4+ collagen+ cells known as fibrocytes that traffic into injured lung tissue where they further differentiate into fibroblasts.26,–,28 Given that: (1) scleroderma monocytes and fibroblasts are deficient in caveolin-1; (2) scleroderma monocytes overexpress CXCR4; (3) the overexpression of CXCR4 by scleroderma monocytes is reversed by the CSD peptide; and (4) TGFβ and TNFα inhibit caveolin-1 expression and promote CXCR4 expression in normal monocytes, it is an attractive idea that the low level of caveolin-1 in monocytes from patients with scleroderma promotes their expression of CXCR4, their differentiation into fibrocytes, the migration of fibrocytes into damaged lung tissue and their further differentiation into fibroblasts.

    Our previous studies showed that manipulating caveolin-1 expression and function has profound effects on the behaviour of fibroblasts and epithelial cells in vitro and in vivo.17 29 The current study shows that the CSD peptide can also reverse the effects of the deficient expression of caveolin-1 in monocytes and PMNs from bleomycin-treated mice and patients with scleroderma. Thus, the beneficial effects of CSD peptide treatment in bleomycin-treated mice may result from a combination of its effects on both inflammation and fibrosis and may involve several cell types including fibroblasts, epithelial cells, monocytes and PMNs. Because inflammation and fibrosis are intertwined in the bleomycin model, it would be impossible to deduce the relative contribution of each cell type to the effects of the CSD peptide without performing deletions of particular cell types (eg, monocytes, PMNs, fibrocytes). In summary, the combined observations suggest that treatment with CSD peptide may be beneficial for scleroderma and other human conditions characterised by interstitial lung disease because of its effects on several cell types.

    Acknowledgments

    The authors thank Dr William Sessa (Yale University, New Haven, Connecticut, USA) and Dr Pascal Bernatchez (University of British Columbia, Vancouver, Canada) for advice and discussion on the use of the CSD peptide.

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    Supplementary materials

    Footnotes

    • Funding This work was supported by the following grants: Scleroderma Foundation (to ET), NIH NIAMS grant K01 AR054143 (to ET), NIH NIAMS grant R03 AR 056767 (to ET), NIH NHLBI grant R01 HL73718 (to SH), NIH NIAMS grant P60 AR049459 (Multidisciplinary Clinical Research Center to RMS) and NIH NCRR Construction Grant C06 RR015455.

    • Competing interests None.

    • Ethics approval This study was conducted with the approval of the Medical University of South Carolina Institutional Review Board.

    • Provenance and peer review Not commissioned; externally peer reviewed.