Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function

Wounds heal through a highly regulated, self-limited inflammatory response, however, precise inflammatory mediators have not been fully delineated. In this study, we report that in a mouse model of excisional skin wound healing the chemokine CX3CL1 and its receptor CX3CR1 were both highly induced at wound sites; CX3CL1 colocalized with macrophages and endothelial cells, whereas CX3CR1 colocalized mainly with macrophages and fibroblasts. Loss of CX3CR1 function delayed wound closure in both CX3CR1 knockout (KO) mice and in wild-type mice infused with anti-CX3CR1-neutralizing Ab. Conversely, transfer of bone marrow from donor wild-type mice, but not from donor CX3CR1 KO mice, restored wound healing to normal in CX3CR1 KO-recipient mice. Direct effects of CX3CR1 disruption at the wound site included marked reduction of macrophages and macrophage products, such as TGF-beta1 and vascular endothelial growth factor. Consistent with this, we observed reduced alpha-smooth muscle actin (a marker for myofibroblasts) and collagen deposition in skin from wounded CX3CR1 KO mice, as well as reduced neovascularization. Together, the data support a molecular model of skin wound repair in which CX3CR1 mediates direct recruitment of bone marrow-derived monocytes/macrophages which release profibrotic and angiogenic mediators.     

Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma

The mechanisms contributing to airway wall remodeling in asthma are under investigation to identify appropriate therapeutic targets. Bronchial myofibroblasts would represent an important target because they play a crucial role in the genesis of subepithelial fibrosis, a characteristic feature of the remodeling process, but their origin is poorly understood. We hypothesized that they originate from fibrocytes, circulating cells with the unique characteristic of expressing the hemopoietic stem cell Ag CD34 and collagen I. In this study we show that allergen exposure induces the accumulation of fibrocyte-like cells in the bronchial mucosa of patients with allergic asthma. These cells are CD34-positive; express collagen I and alpha-smooth muscle actin, a marker of myofibroblasts; and localize to areas of collagen deposition below the epithelium. By tracking labeled circulating fibrocytes in a mouse model of allergic asthma, we provide evidence that fibrocytes are indeed recruited into the bronchial tissue following allergen exposure and differentiate into myofibroblasts. We also show that human circulating fibrocytes acquire the myofibroblast phenotype under in vitro stimulation with fibrogenic cytokines that are produced in exaggerated quantities in asthmatic airways. These results indicate that circulating fibrocytes may function as myofibroblast precursors and may contribute to the genesis of subepithelial fibrosis in asthma.     

The stroma reaction myofibroblast: a key player in the control of tumor cell behavior

The cooperation between epithelial and mesenchymal cells is essential for embryonic development and probably plays an important role in pathological phenomena such as wound healing and tumor progression. It is well known that many epithelial tumors are characterized by the local accumulation of connective tissue cells and extracellular material; this phenomenon has been called the stroma reaction. One of the cellular components of the stroma reaction is the myofibroblast, a modulated fibroblast which has acquired the capacity to neoexpress alpha-smooth muscle actin, the actin isoform typical of vascular smooth muscle cells, and to synthesize important amounts of collagen and other extracellular matrix components. It is now well accepted that the myofibroblast is a key cell for the connective tissue remodeling which takes place during wound healing and fibrosis development. Myofibroblasts are capable of remodeling connective tissue but also interact with epithelial cells and other connective tissue cells and may thus control such phenomena as tumor invasion and angiogenesis. In this review we discuss the mechanisms of myofibroblast evolution during fibrotic and malignant conditions and the interaction of myofibroblasts with other cells in order to control tumor progression. On this basis we suggest that the myofibroblast may represent a new important target of antitumor therapy.     

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.     

Peripheral blood fibrocytes: differentiation pathway and migration to wound sites

Fibrocytes are a distinct population of blood-borne cells that display a unique cell surface phenotype (collagen I+/CD11b+/CD13+/CD34+/CD45RO+/MHC class II+/CD86+) and exhibit potent immunostimulatory activities. Circulating fibrocytes rapidly enter sites of tissue injury, suggesting an important role for these cells in wound repair. However, the regulatory processes that govern the differentiation of blood-borne fibrocytes and the mechanisms that underlie the migration of these cells to wound sites are currently not known. We report herein that ex vivo cultured fibrocytes can differentiate from a CD14+-enriched mononuclear cell population and that this process requires contact with T cells. Furthermore, we demonstrate that TGF-beta1 (1-10 ng/ml), an important fibrogenic and growth-regulating cytokine involved in wound healing, increases the differentiation and functional activity of cultured fibrocytes. Because fibrocytes home to sites of tissue injury, we examined the role of chemokine/chemokine receptor interactions in fibrocyte trafficking. We show that secondary lymphoid chemokine, a ligand of the CCR7 chemokine receptor, acts as a potent stimulus for fibrocyte chemotaxis in vitro and for the homing of injected fibrocytes to sites of cutaneous tissue injury in vivo. Finally, we demonstrate that differentiated, cultured fibrocytes express alpha smooth muscle actin and contract collagen gels in vitro, two characteristic features of wound-healing myofibroblasts. These data provide important insight into the control of fibrocyte differentiation and trafficking during tissue repair and significantly expand their potential role during wound healing.     

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.     

Circulating peripheral blood fibrocytes in human fibrotic interstitial lung disease

Fibrotic interstitial lung diseases are illnesses of unknown cause characterized by progressive decline in lung function. Fibrocytes are bone marrow-derived, circulating progenitor cells capable of differentiating into diverse mesenchymal cell types. Prior work has shown fibrocytes to traffic to the lung via the CXCL12-CXCR4 chemokine axis in an animal model of pulmonary fibrosis. We therefore assessed the relevance of fibrocytes in patients with fibrotic interstitial lung disease. We found enhanced expression of CXCL12 in both the lungs and plasma of patients with lung fibrosis. CXCL12 levels were associated with an order of magnitude higher number of circulating fibrocytes in the peripheral blood of these patients. Most of the circulating fibrocytes in patients with interstitial lung diseases were negative for the myofibroblast marker alpha-smooth muscle actin, suggesting a relatively undifferentiated phenotype. Taken together, these data suggest that fibrocytes are involved in the pathogenesis of human lung fibrosis.     

Calpain activity is essential in skin wound healing and contributes to scar formation

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.     

Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells

Prior studies have demonstrated the expression of a contractile actin isoform, alpha-smooth muscle actin, in bone marrow stromal cells. One objective of the current study was to correlate contractility with alpha-smooth muscle actin expression in human bone marrow stroma-derived mesenchymal stem cells. A second objective was to determine the effects of transforming growth factor-beta1, platelet derived growth factor-BB, and a microfilament-modifying agent on alpha-smooth muscle actin expression and alpha-smooth muscle actin-enabled contraction. Adult human bone marrow stromal cells were passaged in monolayer and their inducibility to chondrocytic, osteoblastic, and adipogenic phenotypes was demonstrated. Western blot analysis was employed along with densitometry to quantify the alpha-smooth muscle actin content of the cells and their contractility was evaluated by their contraction of a type I collagen-glycosaminoglycan sponge-like matrix into which they were seeded. Transforming growth factor-beta1 (1 ng/ml) significantly increased and platelet-derived growth factor-BB (10 ng/ml) decreased alpha-smooth muscle actin expression and the contractility of the cells. Cytochalasin D also blocked cell contraction. There was a notably high correlation of cell-mediated contraction normalized to the DNA content of the matrices with alpha-smooth muscle actin content of the cells by linear regression analysis (R(2) = 0.88). These findings lay the groundwork for considering the role of alpha-smooth muscle actin-enabled contraction in mesenchymal stem cells and in their connective tissue cell progeny.     

Transplantation with bone marrow stromal cells promotes wound healing under chemotherapy through altering phenotypes

Stem cell transplantation is a promising strategy for delayed wound healing caused by chemotherapy. However, the fate of stem cells under chemotherapy has not been fully elucidated. Herein we characterized human fetal bone marrow stromal cells (hBMSCs) during wound healing in mice treated with cyclophosphamide (CTX). The isolated hBMSCs expressed the phenotype of CD11b(low)/CD14(low)/CD34(low)/CD45(low)/CD29(high)/CD44(high)/CD90(high)/CD105(high)/CD146(high)/STRO-1(low). Following in vitro exposure to CTX, hBMSCs showed decreased cell growth in a dose- and time-dependent manner, accompanied by increased expressions of collagen-I/III, and CD31. After transplantation, wounds closed as early as 8 days and were positive for α-smooth muscle actin (α-SMA), implicating the enhanced re-epithelialization and wound contraction. Moreover, proliferating cell nuclear antigen (PCNA) and CD31 showed co-localization with α-SMA, suggesting the differentiation of hBMSCs into epithelial cells and myofibroblasts/fibroblasts. Taken together, our results indicate hBMSCs can accelerate wound healing under chemotherapy through altering their phenotypes.     

Nicotinic acetylcholine receptor alpha7 subunit is time-dependently expressed in distinct cell types during skin wound healing in mice

Recent studies have shown that nicotinic acetylcholine receptor alpha7 subunit (nAChRα7) plays an important role in regulation of inflammation, angiogenesis and keratinocyte biology, but little is known about its expression after the skin is wounded. A preliminary study on time-dependent expression and distribution of nAChRα7 was performed by immunohistochemistry, Western blotting and RT-PCR during skin wound healing in mice. After a 1-cm-long incision was made in the skin of the central dorsum, mice were killed at intervals ranging from 6 h to 14 days post-injury. In uninjured skin controls, nAChRα7 positive staining was observed in epidermis, hair follicles, sebaceous glands, vessel endothelium and resident dermal fibroblastic cells. In wounded specimens, a small number of polymorphonuclear cells, a large number of mononuclear cells (MNCs) and fibroblastic cells (FBCs) showed positive reaction for nAChRα7 in the wound zones. Simultaneously, nAChRα7 immunoreactivity was evident in endothelial-like cells of regenerated vessels and neoepidermis. By morphometric analysis, an up-regulation of nAChRα7 expression was verified at the inflammatory phase after skin injury and reached a peak at the proliferative phase of wound healing. The expression tendency was further confirmed by Western blotting and RT-PCR assay. By immunofluorescent staining for co-localization, the nAChRα7-positive MNCs and FBCs in skin wounds were identified as macrophages, fibrocytes and myofibroblasts. A number of nAChRα7-positive myofibroblasts were also CD45 positive, indicating that they originated from differentiation of fibrocytes. The results demonstrate that nAChRα7 is time-dependently expressed in distinct cell types, which may be closely involved in inflammatory response and repair process during skin wound healing.     

The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo

Myofibroblasts are specialized fibroblasts responsible for granulation tissue contraction and the soft tissue retractions occurring during fibrocontractive diseases. The marker of fibroblast-myofibroblast modulation is the neo expression of alpha-smooth muscle actin (alpha-SMA), the actin isoform typical of vascular smooth muscle cells that has been suggested to play an important role in myofibroblast force generation. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. When the NH2-terminal sequence of alpha-SMA Ac-EEED is delivered to cultured myofibroblast in the form of a fusion peptide (FP) with a cell penetrating sequence, it inhibits their contractile activity; moreover, upon topical administration in vivo it inhibits the contraction of rat wound granulation tissue. The NH2-terminal peptide of alpha-skeletal actin has no effect on myofibroblasts, whereas the NH2-terminal peptide of beta-cytoplasmic actin abolishes the immunofluorescence staining for this isoform without influencing alpha-SMA distribution and cell contraction. The FPs represent a new tool to better understand the specific functions of actin isoforms. Our findings support the crucial role of alpha-SMA in wound contraction. The alpha-SMA-FP will be useful for the understanding of the mechanisms of connective tissue remodeling; moreover, it furnishes the basis for a cytoskeleton-dependent preventive and/or therapeutic strategy for fibrocontractive pathological situations.     

IL-13 induces skin fibrosis in atopic dermatitis by thymic stromal lymphopoietin

Skin fibrotic remodeling is a major feature in human atopic dermatitis (AD). Inflammation and tissue fibrosis are common consequences of Th2 responses. Elevated IL-13 and thymic stromal lymphopoietin (TSLP) have been found in the AD skin lesions. Fibrocytes can be recruited to inflamed tissues to promote wound healing and fibrosis. Dermal transgenic expression of IL-13 causes an AD-like phenotype with fibrosis and increased TSLP. However, the role of TSLP in fibrotic remodeling is unknown. In this study, we investigated the role of TSLP and fibrocytes in the generation of IL-13-induced skin fibrosis. In AD lesion, cessation of IL-13 transgene expression resulted in reduced skin inflammation but with no effect on further progression of fibrosis. This was accompanied by markedly increased CD34(+)/procollagen 1(+) fibrocytes. Furthermore, fibrocytes express TSLP receptor (TSLPR), and TSLP directly promotes PBMC-derived fibrocytes to produce collagen. Neutralization of TSLP or genetic deletion of TSLPR in IL-13 transgenic mice resulted in a significant reduction in fibrocytes and in skin fibrosis. Furthermore, reduction of fibrosis by depletion of TSLP was independent of IL-13. Interestingly, the number of fibrocytes was highly increased in the skin samples of AD patients. These data indicate that the progression of skin fibrosis in IL-13-induced AD occurs via TSLP/TSLPR-dependent but IL-13-independent novel mechanisms by promoting fibrocyte functions.     

Effects of compound 48/80 and exogenous histamine on wound healing in mice

Compound 48/80 has previously been shown to improve wound healing in rats, presumably through stimulation of histidine decarboxylase activity and mobilization of histamine from mast cells. In the present study, C57Bl/6 mice were wounded by dorsal skin incision followed by treatment with compound 48/80, exogenous histamine, or the combination of 48/80 plus histamine. Skin-breaking strength was significantly increased over saline-injected controls by the combined treatment with 48/80 and histamine. Neither 48/80 or histamine alone had any influence on wound healing. Histamine content of skin at the wound site was significantly reduced by 48/80 treatment, but was unaffected by 48/80 plus histamine or histamine given alone. In contrast, stomach and leg muscle histamine levels were significantly increased beyond those of unwounded, wounded saline- or 48/80-injected mice. These results were also confirmed in CD mice, and are in contrast to findings in rats in which treatment with 48/80 alone significantly improved wound healing of similarly injured animals.     

IGF-I and procollagen alpha1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease

This study tested the hypothesis that insulin-like growth factor I (IGF-I) expression is increased at sites of fibrosis in diseased intestine of patients with Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection assay in uninvolved and involved intestine of 13 CD patients (10 ileum, 3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ hybridization histochemistry compared the localization of IGF-I and procollagen alpha1(I) mRNAs. Masson's trichrome staining and immunohistochemistry for IGF-I precursor, alpha-smooth muscle actin (A), vimentin (V), desmin (D), and c-kit were used to examine the mesenchymal cell subtypes that express IGF-I and collagen in uninvolved and involved ileum and colon of CD patients and "normal" ileum and colon from noninflammatory controls. IGF-I mRNA was elevated in involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen alpha1(I) mRNA showed overlapping distribution within fibrotic submucosa and muscularis propria of involved CD ileum and colon. In involved CD intestine, increased IGF-I precursor expression localized to mesenchymal cells in regions of tissue disorganization and fibrosis in muscularis mucosa, submucosa, and muscularis propria. In these regions, there were increased numbers of V(+) cells relative to normal or uninvolved intestine. Increased IGF-I expression was localized to cells with a phenotype typical of fibroblasts (V(+)/A(-)/D(-)), myofibroblasts (V(+)/A(+)/D(+)), and, to a lesser extent, cells with normal enteric smooth muscle phenotype (V(-)/A(+)/D(+)). We conclude that increased IGF-I expression in multiple mesenchymal cell subtypes and increased numbers of cells with fibroblast/myofibroblast phenotype are involved in fibrosis associated with CD.     

Blood-derived small Dot cells reduce scar in wound healing

Wounds in fetal skin heal without scar, however the mechanism is unknown. We identified a novel group of E-cadherin positive cells in the blood of fetal and adult mice and named them "Dot cells". The percentage of Dot cells in E16.5 fetal mice blood is more than twenty times higher compared to adult blood. Dot cells also express integrin beta1, CD184, CD34, CD13low and Sca1low, but not CD45, CD44, and CD117. Dot cells have a tiny dot shape between 1 and 7 microm diameters with fast proliferation in vitro. Most of the Dot cells remain positive for E-cadherin and integrin beta1 after one month in culture. Transplantation of Dot cells to adult mice heals skin wounds with less scar due to reduced smooth muscle actin and collagen expression in the repair tissue. Tracking GFP-positive Dot cells demonstrates that Dot cells migrate to wounds and differentiate into dermal cells, which also express strongly to FGF-2, and later lose their GFP expression. Our results indicate that Dot cells are a group of previously unidentified cells that have strong wound healing effect. The mechanism of scarless wound healing in fetal skin is due to the presence of a large number of Dot cells.     

The Mobilization and Recruitment of C-Kit+ Cells Contribute to Wound Healing after Surgery

Delayed wound healing is a serious clinical problem in patients after surgery. A recent study has demonstrated that bone marrow-derived c-kit-positive (c-kit⁺) cells play important roles in repairing and regenerating various tissues and organs. To examine the hypothesis that surgical injury induces the mobilization and recruitment of c-kit+ cells to accelerate wound healing. Mice were subjected to a left pneumonectomy. The mobilization of c-kit+ cells was monitored after surgery. Using green fluorescent protein (GFP⁺) bone marrow-transplanted chimera mice, we investigated further whether the mobilized c-kit+ cells were recruited to effect wound healing in a skin puncture model. The group with left pneumonectomies increased the c-kit⁺ and CD34⁺ stem cells in peripheral blood 24 h after surgery. At 3 days after surgery, the skin wound size was observed to be significantly smaller, and the number of bone marrow-derived GFP⁺ cells and GFP⁺/c-kit+ cells in the wound tissue was significantly greater in mice that had received pneumonectomies, as compared with those that had received a sham operation. Furthermore, some of these GFP⁺ cells were positively expressed specific markers of macrophages (F4/80), endothelial cells (CD31), and myofibroblasts (αSMA). The administration of AMD3100, an antagonist of a stromal-cell derived factor (SDF)-1/CXCR4 signaling pathway, reduced the number of GFP⁺ cells in wound tissue and completely negated the accelerated wound healing. Surgical injury induces the mobilization and recruitment of c-kit+ cells to contribute to wound healing. Regulating c-kit+ cells may provide a new approach that accelerates wound healing after surgery.     

IL-7 enhances the responsiveness of human T cells that develop in the bone marrow of athymic mice

The beige/nude/xid/human (bnx/hu) model of human hematopoiesis provides a unique opportunity to study extrathymic human T lymphocyte development in an in vivo system. Purified human hematopoietic stem cells develop into mature T lymphocytes and immature progenitors in the bone marrow of athymic bnx mice. The human T cells are all TCR alpha beta(+) and display a restricted TCRV beta repertoire. In the current studies, we examined the effects of systemic human IL-7 (huIL-7) administration on the phenotype and the activation status of the bnx/hu T cells. In the majority of the mice that did not have huIL-7 administration, a higher frequency of human CD3(+)/CD8(+) than CD3(+)/CD4(+) T cells developed in the bone marrow. This phenomenon is also frequently observed in human bone marrow transplant recipients. Extremely low levels of IL-2 were expressed by human CD3(+) cells isolated from these mice, in response to PMA plus ionomycin and to CD3 and CD28 cross-linking. IL-4 was not expressed by cells exposed to either stimulus, demonstrating a profound inability of the bnx/hu T cells to produce this cytokine. Systemic production of huIL-7 from engineered stromal cells transplanted into the mice increased the human CD4 to CD8 ratios, and increased the ratio of memory to naive CD4(+) and CD8(+) T cells. The human CD3(+) cells recovered from mice that had systemic huIL-7 and equivalent numbers of CD3(+)/CD4(+) and CD3(+)/CD8(+) cells in the marrow were still unable to produce IL-4 in response to any condition tested, but were capable of normal levels of IL-2 production following stimulation.     

Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/alpha-smooth muscle actin or H-2b/alpha-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3+/-4.4% and 12.7+/-9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7+/-9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8+/-17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIalpha-positive ratio was 2.2+/-1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3+/-0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.