Modulation of the immune response by extracellular matrix proteins

Inflammation entrains a focused and coordinated response from many different elements. Soluble factors such as chemokines and cytokines direct the recruitment, differentiation, and fate of leukocytes. Cells and pathogens are killed and consumed, yet where the response is effective, inflammation will melt away, leaving a healthy functioning tissue. All this commonly takes place in an environment known as the extracellular matrix (ECM). The ECM is not a passive partner in the process and recent work demonstrates the important role that proteins found in this environment play in connecting different parts of the immune response together. In this review we will focus on these connections and the proteins that make them. One emerging trend that we will highlight is the ability of endogenous molecules to interact with receptors that are better known as sensors of the molecular fingerprints of infection. We propose that this may be particularly relevant in the context of autoimmunity, since the provision of such signals may be crucial in breaking tolerance.     

Modulation of growth factor action by the extracellular matrix

Growth factors were historically defined as molecules produced by the body to regulate cell growth and proliferation. After the identification of their receptors and the intracellular signaling machinery they activate, it is now clear that they are involved in the regulation of multiple processes essential for development and normal tissue function. The present review gives a brief overview of growth factor action. Receptor activation and signaling are discussed, highlighting the role of extracellular matrix interactions.     

Evaluation effects of chitosan for the extracellular matrix production by fibroblasts and the growth factors production by macrophages.

Chitosan is reported as an accelerator of wound healing. Histological findings of previous reports indicate that chitosan accelerates the reformation of connective tissue, however the details of the mechanism are not clear. In this study, firstly L929 mouse fibroblasts were cultured with chitosan and the production of extracellular matrix (ECM) was evaluated in vitro. Type I and III collagens and fibronectin were secreted by L929 with or without chitosan; however there was no significant difference in the amount of ECM between the control and the chitosan groups. Secondly, macrophages were stimulated with chitosan, and then transforming growth factor-beta 1 (TGF-beta1) and platelet-derived growth factor (PDGF) messenger ribonucleic acid (mRNA) expressions and production of their proteins were assayed in vitro. As a result, chitosan promoted the production of TGF-beta1 and PDGF. These results indicate that chitosan does not directly accelerate ECM production by fibroblast and the ECM production may increase by the growth factors.     

Proliferation of rat pleural mesothelial cells in response to hepatocyte and keratinocyte growth factors

The proliferative response of cultured pulmonary mesothelial cells (MCs) to epithelial cell mitogens such as keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) is investigated. A cell line of rat pleural MCs and freshly prepared rat visceral and parietal MCs were studied. Both KGF and HGF stimulated thymidine uptake in the cell line when cultured for 2 d in serum-free conditions; the growth increase was magnified when tumor necrosis factor (TNF)-alpha was also added to the cultures. Adding asbestos fibers alone to MCs in culture did not enhance DNA synthesis by these cells. The MCs were also shown to synthesize significant amounts of HGF but much less KGF when cultured for 2 d. When freshly prepared MCs were examined, normal cell growth was more rapid in the parietal cells, which also had a more epithelial-type morphology. The addition of HGF and KGF resulted in increased DNA synthesis in each cell type, but no effect of added TNF-alpha was found. The results indicate that pulmonary MCs have the potential to proliferate in response to cytokines such as HGF and KGF that are usually associated with epithelial cell regeneration after injury.     

Effects of growth factors on extracellular matrix production by vocal fold fibroblasts in 3-dimensional culture

Culturing cells in 3-dimensional (3D) systems is important in tissue engineering and in fundamental studies of cellular mechanisms that are sensitive or specific to the 3D environment. To guide the engineering of artificial vocal fold lamina propria tissue, we developed 3D cultures containing human vocal fold fibroblasts (hVFFs) dispersed in a synthetic peptide hydrogel matrix. Growth factors were added to the culture to examine their influence on extracellular matrix (ECM) synthesis, cell proliferation, and matrix contraction. The hVFF-hydrogel constructs were treated with transforming growth factor-beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF), or hepatocyte growth factor (HGF), and the culture was maintained for 21 days. TGF-beta1 induced matrix contraction and enhanced collagen and sulfated glycosaminoglycan production, bFGF effectively increased cell proliferation, and HGF stimulated synthesis of hyaluronic acid and elastin with less collagen accumulation than other conditions. Of the growth factors tested, HGF appears to be most useful for stimulating essential tissue components for restoring vocal fold pliability. The results also suggest that multiple growth factors might be employed sequentially or in combination to program the makeup of cell-hydrogel constructs for vocal fold tissue repair.     

Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.     

Modulation of squamous carcinoma cell growth, morphology, adhesiveness and extracellular matrix production by interferon-gamma and tumor necrosis factor-alpha

Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were examined for effects on human squamous carcinoma cells. IFN-gamma inhibited proliferation at concentrations between 100 and 1,500 units/ml and the inhibitory effects were potentiated by TNF-alpha. Inhibition of cell growth was not accompanied by cytotoxicity. Combined treatment with the two cytokines also inhibited cell-substrate adhesion and altered the morphology of the cells. The treated cells were large and flattened. These morphological features are similar to those that have been previously described for normal keratinocytes induced to differentiate by a variety of means. The changes in biological properties were accompanied by alterations in production of extracellular matrix components - e.g., fibronectin and thrombospondin. Synthesis of both components was decreased following treatment. The cytokine-induced alterations in squamous carcinoma cell properties were fully reversible. These findings indicate that malignant squamous epithelial cells may be similar to their normal counterparts in their responses to IFN-gamma and TNF-alpha. In the malignant cells, however, these cytokines do not appear to induce permanent phenotypic changes.     

Modulation of Borrelia burgdorferi stringent response and gene expression during extracellular growth with tick cells

Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.     

Modulation of the growth and guidance of rat brain stem neurons using patterned extracellular matrix proteins

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information regarding the physiological characteristics of neuronal cells behaviour in vitro. However, neuronal patterning using long term embryonic brain slice cultures has not been comprehensively demonstrated to-date. Structuring was performed by micro contact printing of laminin. The slice cultures were evaluated by means of phase contrast microscopy at 3-22 days in culture. We were able to consistently achieve outgrowth of neurons, neurites and filopodia from brain stem slices cultured on ECM proteins structures of grid- and line-shapes. We believe that brain slice cultures on patterned substrates is a favourable approach to study functional synapses in vitro under defined conditions. The use of appropriate structures and the subsequent cell patterning may help to gain further understanding of axonal, dendritic and synaptic signal transductions and processes.     

Comparative study on motility of the cultured fetal and neonatal dermal fibroblasts in extracellular matrix.

One of the differences between fetal and adult skin healing is the ability of fetal wounds heal without contraction and scar formation. Extracellular matrix (ECM) provides a substratum for cells adhesion, migration, and proliferation and can directly influence the form and function of cells. As motility is essential for many important biological events, including wound healing, inflammatory response, embryonic development, and tumor metastasis, this study was designed to compare the motilities cultured dermal fetal and neonatal fibroblasts in the extracellular matrix. The motility of cultured fetal and neonatal fibroblasts was compared using a video-microscopy system that was developed in combination with a self-designed CO2 mini-incubator. To determine migration speed, cells were viewed with a 4X phase-contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. We found that cultured fetal fibroblasts move faster than neonatal fibroblast on type I collagen (fetal fibroblast, 15.1 micrometer/hr; neonatal fibroblast, 13.7 micrometer/hr), and in fibronectin (fetal fibroblast, 13.2 micrometer/hr; neonatal fibroblast, 13.0 micrometer/hr) and hyaluronic acid (fetal fibroblast, 11 micrometer/hr; neonatal fibroblast, 9.8 micrometer/hr).     

Modulation of extracellular matrix gene expression by heparin and endothelial cell growth factor in human smooth muscle cells

We have previously shown that human vascular smooth muscle cells grown for several passages in the presence of heparin and endothelial cell growth factor (ECGF) exhibit profound alterations in the synthesis of extracellular matrix proteins. In the present study, we demonstrate that the mRNA steady-state levels for various matrix macromolecules were altered in the presence of heparin and ECGF, but not ECGF alone. Specifically, the expression of types I and IV collagens, fibronectin, and decorin proteoglycan genes were markedly inhibited, whereas that of versican proteoglycan and beta-actin genes were unaffected. The effects were fully reversible. The suppression of the collagen gene expression was related to decreased collagen production by cells incubated with heparin and ECGF. Thus, heparin and ECGF synergistically elicit a coordinate and selective inhibition of matrix gene expression in human smooth muscle cells.     

An in vitro model of urothelial regeneration: Effects of growth factors and extracellular matrix proteins

Although the cellular turnover of resting urothelium is very low, its regenerative capacity is known to be outstanding. In organotypic mouse urothelial cultures closely mimicking the differentiation and multilayering of normal urothelium, we examined the cell biological mechanisms underlying urothelial regeneration and the specific role of growth factors and several extracellular matrix (ECM) components. Exposure to epidermal growth factor (EGF) and acidic fibroblast growth factor (aFGF) and culture on laminin resulted in enhanced expansion of the urothelium. Microscopy and assessment of proliferative activity revealed that enhanced urothelial expansion due to EGF could be attributed to increased proliferative activity and an increase in cell numbers, whereas aFGF-stimulated expansion must be considered the consequence of increased cellularity and migration. Laminin-enhanced urothelial expansion was shown to be the result of spreading of the entire urothelial organotypic culture. This was associated with a considerable decrease in the number of cell layers. A synergistic effect of growth factors and laminin was not found. This organotypic urothelial cell culture model seems to be very useful in studying strategies to improve urothelial regeneration.     

Organization of extracellular matrix in epiphyseal growth plate

Three cations of varying size and charge density, egg-white lysozyme, protamine and ruthenium red, were used to stain the extracellular matrix of epiphyseal cartilage growth plate. With these stains, it was possible to distinguish three types of proteoglycans or materials associated with them, which may well have as their major differences the type of cross linking to the tissue. One type was stained by ruthenium red and protamine but not by lysozyme, was extractable with 3 M guanidinium chloride and was relatively uniformly dispersed throughout the matrix of the growth plate. The other two types were stained by all three cations, were not extractable with 3 M guanidinium chloride and were intimately associated with fibrils. One of these was found on the collagen fibrils, was relatively scanty in the resting zone near the articular surface, relatively restricted to the extralacunar area in the columnar zones and appeared to diminish in amount in the hypertrophic zone. This material often had a 640-Å periodic array on the surface of collagen fibrils. The third type also was stained by all three cations and was not extractable with 3 M guanidinium chloride. It was distinguished from the other class of lysozyme-reactive matrix components by the larger volume of distribution occupied by the stained material. It also had a different distribution in that it was widely dispersed in the resting zone, was restricted to the lacuna in the columnar zone and was absent in the hypertrophic zone. Thus, cartilage matrix as well as the chondrocytes undergo differentiation in the epiphyseal growth plate.     

Expression of adhesion molecules and extracellular matrix proteins in glioblastomas: relation to angiogenesis and spread

We studied the immunohistochemical expression of inducible adhesion molecules, integrins and extracellular matrix proteins in 10 cases of glioblastoma multiforme in order to investigate their angiogenesis, local invasiveness, poor metastasizing properties and their lack of tumour infiltrating leukocytes. In glioblastomas endothelial proliferations represent the majority of vascular structures; they were positive for endothelial markers (vWF, CD31, VE-cadherin) and negative for macrophage markers (CD68, PAM-1). Immunohistologically, they were subtyped into: 1 solid-glomeruloid ICAM-1, α₂β₁, α₃β₁, α₅β₁ negative; 2 channelled-branching ICAM-1 negative and α₂β₁, α₃β₁, α₅β₁ positive; 3 channelled-telangiectatic ICAM-1, α₂β₁, α₃β₁, α₅β₁ positive. In channelled proliferations, the expression and distribution of tenascin and merosin in the basal membrane was similar to that of normal brain vessels. The expression of all these molecules might indicate different steps of maturation of endothelial proliferations. The majority of endothelial proliferations may be immunohistologically considered as incomplete vascular structures; this might account for the low metastasizing tendency and low recruitment of leukocytes by these tumours. Neoplastic astrocytes were GFAP-1, ICAM-1, VCAM-1, α₂β₁, α₃β₁ and α₅β₁ immunoreactive and α₆β₄ negative; this allows them to interact with extracellular matrix proteins and might, in part, explain the tendency of glioblastomas to infiltrate locally.     

Progressive lung cell reactions and extracellular matrix production after a brief exposure to asbestos.

Inhaled chrysotile asbestos fibers have been shown to deposit initially on the first alveolar duct bifurcations. In brief accidental exposure to asbestos, this would be the most likely site of a significant cellular or fibrotic reaction. The characteristics and progression of tissue reactions occurring at first alveolar duct bifurcations after a single brief asbestos exposure was defined using morphometric techniques. Seven-week-old rats were exposed, nose only, for 1 hour to chrysotile asbestos fibers. After the exposure, the animals were kept in air for 2 days or 1 month, and then their lungs were fixed by vascular perfusion or by intratracheal instillation of 2% glutaraldehyde. The first bifurcations of seven alveolar ducts in each animal were isolated from plastic-embedded tissue and thin-sectioned for electron-microscopic analysis. Two days after exposure, the volume of epithelium and interstitium in the duct bifurcations had increased by 78% and 28%, respectively (P less than 0.05). The total number and volume of alveolar macrophages on the bifurcations increased about 10 times (P less than 0.05), whereas the number and volume of interstitial macrophages increased threefold (P less than 0.05). Statistically significant increases in the numbers of Type I (82%) and Type II (29%) epithelial cells also occurred. One month after the 1-hour exposure, the volume of epithelium and the number of Type I and Type II cells were still greater than control values, but these differences no longer achieved statistical significance. The volume of the interstitium, on the other hand, increased 67% (P less than 0.05), and this was accompanied by a persistently high number of interstitial macrophages, accumulation of myofibroblasts/smooth muscle cells, and an increased volume of interstitial matrix. These results demonstrate that a brief exposure to chrysotile asbestos causes a rapid response that involves an influx of macrophages to the first alveolar duct bifurcations and alterations in the alveolar epithelium. These acute structural changes are followed by a progressive response manifested by increased numbers of interstitial cells and localized interstitial fibrosis.     

Arterial enlargement in response to high flow requires early expression of matrix metalloproteinases to degrade extracellular matrix

This study investigated the effects of high flow and shear stress on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) during flow-induced arterial enlargement using a model of arteriovenous fistula (AVF) creation on the carotid artery with the corresponding jugular vein in Japanese white male rabbits. Flow increased 8-fold 7 days after AVF. Endothelial cells (EC) and smooth muscle cells (SMC) proliferated with internal elastic lamina (IEL) degradation in response to high flow and shear stress. Expression of MMP-2 mRNA peaked at 2 days (1700-fold) and maintained high level expression. MMP-9 mRNA gave a 10.8-fold increase within 2 days and decreased later. Their proteins were detected in EC and SMC. Membrane type-1-MMP (MT1-MMP) mRNA increased 121-fold at 3 days and maintained high expression. TGF-beta1 was increased after AVF. Two-peak up-regulation of Egr-1 mRNA was recognized at 1 and 5 days of AVF. These results suggest that high flow and shear stress can mediate EC and SMC to express MMP-2 and MMP-9, which degrade cell basement membranes and IEL to induce arterial enlargement. The disproportional increase in MT1-MMP and TIMP-2 might contribute to MMP-2 activation. Egr-1 and TGF-beta1 might play important roles in this process.     

Modulation of mesangial cell migration by extracellular matrix components. Inhibition by heparinlike glycosaminoglycans.

Extension of mesangial cells (MC) into the pericapillary space is a pathologic response seen in several forms of glomerulonephritis. This process may involve both cytoplasmic extension by MC and actual cellular migration. For investigation of whether extracellular matrix factors could modulate this process, the migratory responses of rat MC were quantitatively examined using a cell culture model. Denuding ("wounding") a portion of a confluent culture of MC was followed by migration of mesangial cells into the denuded area. The expected proliferative response to this treatment was blocked by irradiation. The migratory response began within 8 hours of wounding and continued for at least 80 hours. The MC migratory response was specifically inhibited in a dose-dependent and reversible manner by heparin and heparinlike glycosaminoglycans (GAGs). Chondroitin sulfates and hyaluronic acid did not significantly inhibit MC migration. Glomerular basement membrane heparinlike GAGs may normally prevent MC extension into the pericapillary space. Changes in the density or composition of these substances during glomerular inflammatory processes could permit the development of MC pericapillary extensions and thereby lead to further alterations in basement membrane integrity.     

Effect of growth factors and extracellular matrix materials on the proliferation and differentiation of microencapsulated myoblasts

An alternative approach to gene therapy via non-autologous somatic gene therapy is to implant genetically-engineered cells protected from immune rejection with microcapsules to deliver a therapeutic gene product. This delivery system may be optimized by using myoblast cell lines which can undergo terminal differentiation into myotubes, thus removing the potential problems of tumorigenesis and space restriction. However, once encapsulated, myoblasts do not proliferate or differentiate well. We now report the use of extracellular matrix components and growth factors to improve these properties. Addition of matrix material collagen, merosin or laminin all stimulated myoblast proliferation, particularly when merosin and laminin were combined; however, none, except collagen, stimulated differentiation. Inclusion of basic fibroblast growth factor (bFGF) within the microcapsules in the presence of collagen stimulated proliferation of C₂C₁₂ myoblasts, as well as differentiation into myotubes. Inclusion of insulin growth factor (IGF-II) in the microcapsules had no effect on proliferation but accelerated myoblasts differentiation. When the above matrix material and growth factors were provided in combination, the use of merosin and laminin together with bFGF and IGF-II stimulated myoblast proliferation but had no effect on differentiation. In contrast, the cocktail containing bFGF, IGF-II and collagen induced increased myoblasts proliferation and subsequent differentiation. Hence, the combination of bFGF, IGF-II and collagen appears optimal in improving proliferation and differentiation in encapsulated myoblasts.