Origin and functional heterogeneity of fibroblasts.

The inherent plasticity and resiliency of fibroblasts make this cell type a conventional tool for basic research. But where do they come from, are all fibroblasts the same, and how do they function in disease? The first fibroblast lineages in mammalian development emerge from the ooze of primary mesenchyme during gastrulation. They are cells that efficiently create and negotiate the extracellular matrix of the mesoderm in order to migrate and meet their developmental fate. Mature fibroblasts in epithelial tissues live in the interstitial spaces between basement membranes that spatially delimit complex organ structures. While the function of resident fibroblasts in healthy tissues is largely conjecture, the accumulation of fibroblasts in pathologic lesions offers insight into biologic mechanisms that control their function; fibroblasts are poised to coordinate fibrogenesis in tissue injury, neoplasia, and aging. Here, we examine the developmental origin and plasticity of fibroblasts, their molecular and functional definitions, the epigenetic control underlying their identity and activation, and the evolution of their immune regulatory functions. These topics are reviewed through the lens of fate mapping using genetically engineered mouse models and from the perspective of single-cell RNA sequencing. Recent observations suggest dynamic and heterogeneous functions for fibroblasts that underscore their complex molecular signatures and utility in injured tissues.

S100A4 Is a Biomarker and Regulator of Glioma Stem Cells That Is Critical for Mesenchymal Transition in Glioblastoma.

Glioma stem cells (GSC) and epithelial-mesenchymal transition (EMT) are strongly associated with therapy resistance and tumor recurrence, but the underlying mechanisms are incompletely understood. Here, we show that S100A4 is a novel biomarker of GSCs. S100A4+ cells in gliomas are enriched with cancer cells that have tumor-initiating and sphere-forming abilities, with the majority located in perivascular niches where GSCs are found. Selective ablation of S100A4-expressing cells was sufficient to block tumor growth in vitro and in vivo We also identified S100A4 as a critical regulator of GSC self-renewal in mouse and patient-derived glioma tumorspheres. In contrast with previous reports of S100A4 as a reporter of EMT, we discovered that S100A4 is an upstream regulator of the master EMT regulators SNAIL2 and ZEB along with other mesenchymal transition regulators in glioblastoma. Overall, our results establish S100A4 as a central node in a molecular network that controls stemness and EMT in glioblastoma, suggesting S100A4 as a candidate therapeutic target. Cancer Res; 77(19); 5360-73. ©2017 AACR.

Phosphatidylinositol 3-kinase signaling determines kidney size.

Kidney size adaptively increases as mammals grow and in response to the loss of 1 kidney. It is not clear how kidneys size themselves or if the processes that adapt kidney mass to lean body mass also mediate renal hypertrophy following unilateral nephrectomy (UNX). Here, we demonstrated that mice harboring a proximal tubule-specific deletion of Pten (Pten(ptKO)) have greatly enlarged kidneys as the result of persistent activation of the class I PI3K/mTORC2/AKT pathway and an increase of the antiproliferative signals p21(Cip1/WAF) and p27(Kip1). Administration of rapamycin to Pten(ptKO) mice diminished hypertrophy. Proximal tubule-specific deletion of Egfr in Pten(ptKO) mice also attenuated class I PI3K/mTORC2/AKT signaling and reduced the size of enlarged kidneys. In Pten(ptKO) mice, UNX further increased mTORC1 activation and hypertrophy in the remaining kidney; however, mTORC2-dependent AKT phosphorylation did not increase further in the remaining kidney of Pten(ptKO) mice, nor was it induced in the remaining kidney of WT mice. After UNX, renal blood flow and amino acid delivery to the remaining kidney rose abruptly, followed by increased amino acid content and activation of a class III PI3K/mTORC1/S6K1 pathway. Thus, our findings demonstrate context-dependent roles for EGFR-modulated class I PI3K/mTORC2/AKT signaling in the normal adaptation of kidney size and PTEN-independent, nutrient-dependent class III PI3K/mTORC1/S6K1 signaling in the compensatory enlargement of the remaining kidney following UNX.

EGFR signaling promotes TGFß-dependent renal fibrosis.

The mechanisms by which angiotensin II (Ang II) promotes renal fibrosis remain incompletely understood. Ang II both stimulates TGFß signaling and activates the EGF receptor (EGFR), but the relative contribution of these pathways to renal fibrogenesis is unknown. Using a murine model with EGFR-deficient proximal tubules, we demonstrate that upstream activation of EGFR-dependent ERK signaling is critical for mediating sustained TGFß expression in renal fibrosis. Persistent activation of the Ang II receptor stimulated ROS-dependent phosphorylation of Src, leading to sustained EGFR-dependent signaling for TGFß expression. Either genetic or pharmacologic inhibition of EGFR significantly decreased TGFß-mediated fibrogenesis. We conclude that TGFß-mediated tissue fibrosis relies on a persistent feed-forward mechanism of EGFR/ERK activation through an unexpected signaling pathway, highlighting EGFR as a potential therapeutic target for modulating tissue fibrogenesis.

Transcriptional networks in epithelial-mesenchymal transition.

BACKGROUND: Epithelial-mesenchymal transition (EMT) changes polarized epithelial cells into migratory phenotypes associated with loss of cell-cell adhesion molecules and cytoskeletal rearrangements. This form of plasticity is seen in mesodermal development, fibroblast formation, and cancer metastasis. METHODS AND FINDINGS: Here we identify prominent transcriptional networks active during three time points of this transitional process, as epithelial cells become fibroblasts. DNA microarray in cultured epithelia undergoing EMT, validated in vivo, were used to detect various patterns of gene expression. In particular, the promoter sequences of differentially expressed genes and their transcription factors were analyzed to identify potential binding sites and partners. The four most frequent cis-regulatory elements (CREs) in up-regulated genes were SRY, FTS-1, Evi-1, and GC-Box, and RNA inhibition of the four transcription factors, Atf2, Klf10, Sox11, and SP1, most frequently binding these CREs, establish their importance in the initiation and propagation of EMT. Oligonucleotides that block the most frequent CREs restrain EMT at early and intermediate stages through apoptosis of the cells. CONCLUSIONS: Our results identify new transcriptional interactions with high frequency CREs that modulate the stability of cellular plasticity, and may serve as targets for modulating these transitional states in fibroblasts.

VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization.

Increased numbers of S100A4(+) cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4(+) cancer cells have been examined, the functional role of S100A4(+) stromal cells in metastasis is largely unknown. To study the contribution of S100A4(+) stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4(+) stromal cells. Depletion of S100A4(+) stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4(+) stromal cells are attributable to local non-bone marrow-derived S100A4(+) cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4(+) fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4(+) fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4(+) fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4(+) fibroblasts in providing the permissive "soil" for metastatic colonization, a challenging step in the metastatic cascade.

The Jeremiah Metzger lecture. The origin of fibroblasts and the terminality of epithelial differentiation.

For 142 years the fibroblast has lived a nomadian existence among the interstitial spaces of the metazoan body plan. The cell surface of fibroblasts lacks specific identifying markers and its parental lineage has been shrouded in mystery. Over the last 15 years much has changed. We know now that fibroblasts derive from non-motile epithelial or endothelial cells through a process called epithelial-mesenchymal transition (EMT). In this lecture I discuss the mechanisms of EMT producing fibroblasts, and the inevitable conclusion that epithelia and endothelia, rather than being terminally differentiated, are in a state of nuclear diapause and ready to change phenotype in response to the demands of tissue repair.

Dystroglycan in the diagnosis of FSGS.

BACKGROUND AND OBJECTIVES: alpha- and beta-dystroglycan (DG), which link the actin cytoskeleton of the podocyte to the glomerular basement membrane, are maintained in FSGS but decreased in minimal change disease (MCD). Fibrosis has been linked to increased fibroblast-specific protein-1 (FSP1) and epithelial-mesenchymal transition. We studied DG, FSP1, and podocyte differentiation in FSGS variants and cases of suspected FSGS. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We studied renal biopsies with FSGS, not otherwise specified (NOS), tip lesion, or collapsing variants (COLL), versus secondary FSGS or cases without segmental sclerotic lesions where a diagnosis of MCD versus FSGS could not be established (undefined [UNDEF]) and compared the expression of DG, FSP1, and podocyte Wilms' tumor antigen (WT1). RESULTS: WT1 is markedly decreased in NOS versus normal and correlates with the extent of sclerosis. alpha- and beta-DG are maintained in most primary and secondary FSGS cases. In contrast, alpha-DG is significantly decreased in UNDEF, supporting a diagnosis of MCD. Furthermore, follow-up shows remission or decreased proteinuria in four of six of these UNDEF cases in response to therapy. Interstitial FSP1 is numerically highest in COLL but is only rarely found in tubules or podocytes in any other forms of FSGS. CONCLUSIONS: We conclude that increased FSP1 may be a marker of the aggressive course of collapsing FSGS. Furthermore, DG staining is a useful adjunct to assist in distinction of FSGS versus MCD in biopsies without defining lesions.

Epithelial-mesenchymal transition as a potential explanation for podocyte depletion in diabetic nephropathy.

BACKGROUND: Depletion of glomerular podocytes is an important feature of progressive diabetic nephropathy. Although the most plausible explanation for this podocyte depletion is detachment from the glomerular basement membrane after cellular apoptosis, the mechanism is unclear. Fibroblast-specific protein 1 (FSP1; encoded by the S100A4 gene) is a member of the S100 family of calcium-binding proteins and is constitutively expressed in the cytoplasm of tissue fibroblasts or epithelial cells converted into fibroblasts by means of epithelial-mesenchymal transition. STUDY DESIGN: Retrospective cross-sectional analysis. SETTINGS & PARTICIPANTS: 109 patients with type 2 diabetes mellitus, of whom 43 (39%) underwent kidney biopsy. PREDICTOR: Clinical stage (4 categories) and histological grade (5 categories) of diabetic nephropathy. OUTCOME: FSP1 expression in podocytes in urine and glomeruli in kidney biopsy specimens. MEASUREMENTS: Immunohistochemistry, real-time polymerase chain reaction, and in situ hybridization. RESULTS: 38 of 109 patients (35%) were normoalbuminuric, 16 (15%) had microalbuminuria, 8 (7%) had macroalbuminuria, and 47 (43%) had decreased kidney function. Approximately 95% of podocytes in urine sediment were not apoptotic, and 86% expressed FSP1. The number of FSP1-positive podocytes in urine sediment was significantly larger in patients with macroalbuminuria than in those with normoalbuminuria (P = 0.03). Intraglomerular expression of FSP1 occurred almost exclusively in podocytes from patients with diabetes, and the number of FSP1-positive podocytes was larger in glomeruli showing diffuse mesangiopathy than in those showing focal mesangiopathy (P = 0.01). The number also was larger in glomeruli with nodular lesions than in those without nodular lesions (P < 0.001). FSP1-positive podocytes selectively expressed Snail1 and integrin-linked kinase, a known trigger for epithelial-mesenchymal transition. LIMITATIONS: Nonrepresentative study population. CONCLUSIONS: These results suggest that the appearance of FSP1 in podocytes of patients with diabetes is associated with more severe clinical and pathological findings of diabetic nephropathy, perhaps because of induction of podocyte detachment through epithelial-mesenchymal transition-like phenomena.

Biomarkers for epithelial-mesenchymal transitions.

Somatic cells that change from one mature phenotype to another exhibit the property of plasticity. It is increasingly clear that epithelial and endothelial cells enjoy some of this plasticity, which is easily demonstrated by studying the process of epithelial-mesenchymal transition (EMT). Published reports from the literature typically rely on ad hoc criteria for determining EMT events; consequently, there is some uncertainty as to whether the same process occurs under different experimental conditions. As we discuss in this Personal Perspective, we believe that context and various changes in plasticity biomarkers can help identify at least three types of EMT and that using a collection of criteria for EMT increases the likelihood that everyone is studying the same phenomenon - namely, the transition of epithelial and endothelial cells to a motile phenotype.

Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis.

RATIONALE: Lung fibroblasts are key mediators of fibrosis resulting in accumulation of excessive interstitial collagen and extracellular matrix, but their origins are not well defined. OBJECTIVES: We aimed to elucidate the contribution of lung epithelium-derived fibroblasts via epithelial-mesenchymal transition (EMT) in the intratracheal bleomycin model. METHODS: Primary type II alveolar epithelial cells were cultured from Immortomice and exposed to transforming growth factor-beta(1) and epidermal growth factor. Cell fate reporter mice that permanently mark cells of lung epithelial lineage with beta-galactosidase were developed to study EMT, and bone marrow chimeras expressing green fluorescent protein under the control of the fibroblast-associated S100A4 promoter were generated to examine bone marrow-derived fibroblasts. Mice were given intratracheal bleomycin (0.08 unit). Immunostaining was performed for S100A4, beta-galactosidase, green fluorescent protein, and alpha-smooth muscle actin. MEASUREMENTS AND MAIN RESULTS: In vitro, primary type II alveolar epithelial cells undergo phenotypic changes of EMT when exposed to transforming growth factor-beta(1) and epidermal growth factor with loss of prosurfactant protein C and E-cadherin and gain of S100A4 and type I procollagen. In vivo, using cell fate reporter mice, approximately one-third of S100A4-positive fibroblasts were derived from lung epithelium 2 weeks after bleomycin administration. From bone marrow chimera studies, one-fifth of S100A4-positive fibroblasts were derived from bone marrow at this same time point. Myofibroblasts rarely derived from EMT or bone marrow progenitors. CONCLUSIONS: Both EMT and bone marrow progenitors contribute to S100A4-positive fibroblasts in bleomycin-induced lung fibrosis. However, neither origin is a principal contributor to lung myofibroblasts.

Stable expression of HIF-1alpha in tubular epithelial cells promotes interstitial fibrosis.

Chronic hypoxia accelerates renal fibrosis. The chief mediator of the hypoxic response is hypoxia-inducible factor 1 (HIF-1) and its oxygen-sensitive component HIF-1alpha. HIF-1 regulates a wide variety of genes, some of which are closely associated with tissue fibrosis. To determine the specific role of HIF-1 in renal fibrosis, we generated a knockout mouse in which tubular epithelial expression of von Hippel-Lindau tumor suppressor (VHL), which acts as a ubiquitin ligase to promote proteolysis of HIF-1alpha, was targeted. We investigated the effect of VHL deletion (i.e., stable expression of HIF-1alpha) histologically and used the anti-HIF-1alpha agent [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] (YC-1) to test whether inhibition of HIF-1alpha could represent a novel approach to treating renal fibrosis. The area of renal fibrosis was significantly increased in a 5/6 renal ablation model of VHL-/- mice and in all VHL-/- mice at least 60 wk of age. Injection of YC-1 inhibited the progression of renal fibrosis in unilateral ureteral obstruction model mice. In conclusion, HIF-1alpha appears to be a critical contributor to the progression of renal fibrosis and could be a useful target for its treatment.

Stromal transforming growth factor-beta signaling mediates prostatic response to androgen ablation by paracrine Wnt activity.

Mechanisms of androgen dependence of the prostate are critical to understanding prostate cancer progression to androgen independence associated with disease mortality. Transient elevation of transforming growth factor-beta (TGF-beta) occurs after androgen ablation. To determine the role of TGF-beta on prostate response to androgen ablation, conditional TGF-beta type II receptor knockout mouse models of the epithelia (Tgfbr2(NKX3.1KO)) and stromal fibroblasts (Tgfbr2(fspKO)) were used. After castration, the prostates of Tgfbr2(NKX3.1KO) mice had apoptosis levels similar to those expected for control Tgfbr2(floxE2/floxE2) mice. Prostates of Tgfbr2(fspKO) mice, however, had reduced regression and high levels of proliferation associated with canonical Wnt activity throughout the glandular epithelia regardless of androgen status. In contrast, Tgfbr2(floxE2/floxE2) prostates had epithelial canonical Wnt activity only in the surviving proximal ducts after castration. In vitro studies showed that androgen antagonist, bicalutamide, transiently elevated both Tgfbr2(floxE2/floxE2) and Tgfbr2(fspKO) stromal expression of Wnt-2, Wnt-3a, and Wnt-5a. The neutralization of Wnt signaling by the expression of secreted frizzled related protein-2 (SFRP-2) resulted in decreased LNCaP prostate epithelial cell proliferation in stromal conditioned media transfer experiments. In vivo tissue recombination studies using Tgfbr2(fspKO) prostatic stromal cells in combination with wild-type or SV40 large T antigen expressing epithelia resulted in prostates that were refractile to androgen ablation. The expression of SFRP-2 restored the Tgfbr2(fspKO)-associated prostate responsiveness to androgen ablation. These studies reveal a novel TGF-beta, androgen, and Wnt paracrine signaling axis that enables prostatic regression of the distal ducts after androgen ablation while supporting proximal duct survival.

Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.

Cardiac fibrosis, associated with a decreased extent of microvasculature and with disruption of normal myocardial structures, results from excessive deposition of extracellular matrix, which is mediated by the recruitment of fibroblasts. The source of these fibroblasts is unclear and specific anti-fibrotic therapies are not currently available. Here we show that cardiac fibrosis is associated with the emergence of fibroblasts originating from endothelial cells, suggesting an endothelial-mesenchymal transition (EndMT) similar to events that occur during formation of the atrioventricular cushion in the embryonic heart. Transforming growth factor-beta1 (TGF-beta1) induced endothelial cells to undergo EndMT, whereas bone morphogenic protein 7 (BMP-7) preserved the endothelial phenotype. The systemic administration of recombinant human BMP-7 (rhBMP-7) significantly inhibited EndMT and the progression of cardiac fibrosis in mouse models of pressure overload and chronic allograft rejection. Our findings show that EndMT contributes to the progression of cardiac fibrosis and that rhBMP-7 can be used to inhibit EndMT and to intervene in the progression of chronic heart disease associated with fibrosis.

A proximal activator of transcription in epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site-1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box-binding factor-A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, beta-catenin, ZO 1, vimentin, alpha1(I) collagen, and alpha-smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.

Role of EGF receptor activation in angiotensin II-induced renal epithelial cell hypertrophy.

For determination of the molecular mechanisms underlying the induction of epithelial cell hypertrophy by angiotensin II (Ang II), a well-characterized porcine renal proximal tubular cell line LLCPKcl4, which does not express endogenous Ang II receptor subtypes, was transfected with cDNA encoding Ang II subtype 1 receptor (AT1R/Cl4). Ang II transactivated the EGF receptor (EGFR) in these AT1R/Cl4 cells, which was blocked by the selective AT1R antagonist losartan but not by the selective AT2R antagonist PD123319. Ang II did not transactivate EGFR in empty vector-transfected LLCPKcl4 cells (Vector/Cl4). Ang II elicited release of soluble heparin-binding EGF-like growth factor (HB-EGF) from AT1R/Cl4 cells, and Ang II-induced EGFR activation was prevented by pretreatment with the specific HB-EGF inhibitor CRM197 or the metalloproteinase inhibitors batimastat or phenanthroline, none of which had any effect on EGFR activation by exogenously administered EGF. Ang II stimulated protein synthesis and cell hypertrophy in AT1R/Cl4 cells without increasing cell number, and signaling studies revealed that Ang II stimulated phosphorylation of the 40S ribosomal protein S6 and the eukaryotic translation initiation factor 4E-binding protein 1, the two downstream target proteins of the mammalian target of rapamycin, which is a central regulator of protein synthesis and cell size. Ang II-induced mammalian target of rapamycin activation, [3H]leucine incorporation, and cellular hypertrophy were inhibited by pretreatment with either batimastat or CRM197 or by pretreatment with rapamycin or the EGFR tyrosine kinase inhibitor AG1478. Ang II also stimulated Smad 2/3 phosphorylation, which was blocked by a selective TGF-beta receptor I kinase inhibitor but not by CRM197. With blockade of TGF-beta receptor, Ang II-mediated hypertrophy was converted into cell proliferation, which was blocked by CRM197. In summary, this is the first demonstration that HB-EGF shedding-dependent EGFR transactivation, along with activation of TGF-beta signaling pathways, mediates Ang II-induced renal tubular epithelial cell hypertrophy.

Fibroblast-specific protein 1 is a specific prognostic marker for renal survival in patients with IgAN.

BACKGROUND: There is little direct evidence that fibroblasts are involved in the progression of the renal interstitial fibrosis in human glomerulonephritis. With the availability of a new specific marker for fibroblasts, we determined the presence of fibroblasts in kidneys with IgA nephropathy (IgAN) and correlated their numbers with various clinical parameters. In particular, we also prospectively asked if the number of fibroblasts in the renal interstitium correlates with prognosis. METHODS: Cells positive for fibroblast-specific protein 1 (FSP1) were localized in renal biopsy specimens using immunohistochemistry with anti-FSP1 antibody. Clinical features were analyzed by one-way analysis of variance (ANOVA) with the Bonferroni correction. To assess the prognostic impact of the number of FSP1+ fibroblasts on renal survival in 142 patients with normal serum creatinine, the relationship between covariates to renal survival were evaluated univariately using the log-rank test and multivariately using Cox proportional hazards. RESULTS: Fibroblasts identified by their expression of FSP1 accumulate in areas showing severe interstitial fibrosis. Some tubular epithelial cells undergoing epithelial-mesenchymal transition (EMT) in fibrotic areas also express FSP1. Numbers of FSP1+ fibroblasts directly correlate with serum creatinine (r = 0.74, P < 0.0001) and inversely correlate with estimated creatinine clearance (r = -0.54, P < 0.0001), and by multivariate analysis, the clinical factors influencing renal survival are urinary protein excretion [> or = 1.0 g/day, relative risk (RR) = 4.20, P= 0.032], hypertension (RR 5.85, P = 0.0027), and > or = 20 FSP1+ fibroblasts per high power field (HPF) (RR 7.39, P = 0.0015). Staining for FSP1+ fibroblasts is largely nonoverlapping with alpha-smooth muscle actin+ (alpha-SMA) cells in the interstitium. CONCLUSION: The target protein FSP1 identifies human fibroblasts and tubular epithelium undergoing EMT, and distinguishes them from the diaspora of alpha-SMA+ vascular smooth muscle cells. FSP1+ fibroblasts are critically related to the progression of IgAN; consequently, staining FSP1 in renal biopsy specimens provides a valuable histologic index of progression.

Antibodies against macrophages that overlap in specificity with fibroblasts.

BACKGROUND: Fibroblasts can be misidentified as macrophages because both cell types share antigens that are associated with popular antibodies targeting the monocyte/macrophage lineage. With the recent description of fibroblast-specific protein 1 (FSP1), we revisited the specificity of antibodies directed against macrophages to determine systematically which antibodies best distinguish both cell types in fibrotic tissues. METHODS: Tissue fibrosis was produced in mice carrying the GFP transgene encoding green fluorescent protein under the control of the FSP1 promoter. Single cell suspensions from these marked tissues were submitted for flow cytometry using antibodies against Mac-1, Mac-2, Mac-3, F4/80, CD68, major histocompatibility complex (MHC) class II, and CD45, and cDNA amplification of mRNA encoding the above target antigens was performed using specific primer sets in sorted pools of cells. Fibrotic tissues were also stained by immunohistochemistry with the same antibodies and examined under confocal microscopy. RESULTS: Comparison overlap between FSP1(+) fibroblasts with each of the macrophage markers demonstrated that all antimacrophage antibodies (Mac-1, Mac-2, Mac-3, CD68, MHC class II, and CD45) except one (F4/80) recognize both cell types. CONCLUSION: Antibodies directed against F4/80 clearly distinguish macrophages from FSP1(+) fibroblasts in fibrotic tissues and is the preferred antibody in mice.