Biomarkers for skin involvement and fibrotic activity in scleroderma.

Systemic sclerosis (scleroderma, SSc) is characterized as a severe and very heterogeneous disease with a bright variation of skin and organ manifestations in individual patients. The pathogenesis is still not fully elucidated; however, it is known that this disease starts with an initial vascular damage, which then leads to an inflammatory process and finally promotes the development of an accumulation of collagen and other extracellular matrix (ECM) components. As a result of the heterogeneous characteristics of this multisystem, autoimmune disease, it is always a challenge to identify high-risk patients and to monitor the fibrotic activity also in response to therapies. This can be achieved by several physical methods including the mRSS, the durometer and ultrasound determination of skin thickness. However, this also requires the use of laboratory biomarkers, which are easily detectable and that reflect the inflammatory and/or fibrotic activity. As skin correlates well with the extent of fibrosis also in other organs, we focused in this review on biomarkers which reflect skin involvement of scleroderma patients. These include growth factors, cytokines and proteases as well as their inhibitors. Moreover, several ECM proteins, especially the collagens have been determined in skin biopsies and in blood/serum samples. Determination of proteins has been supported by mRNA levels using PCR techniques and expression analysis of gene expression patterns. This review summarizes all non-invasive physical and laboratory examinations, which permit a better understanding of the fibrotic activity of the disease, can be effectively used to assess potential therapeutic response and help to find better treatment options.

Apoptosis in v-myc-transfected MSU-1.1 fibroblasts is induced by cell-matrix contact and differs from that of normal dermal fibroblasts.

In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3-4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF. MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors alpha1beta1 and alpha2beta1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.l fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF.

Expression of monocyte chemoattractant protein-1 in the lesional skin of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease with unknown etiology characterized by excessive deposition of collagen in the skin as well as various internal organs. One of the characteristic histological features is the presence of infiltrating mononuclear cells in the dermis in its early stage. As well as T cells, macrophages are implicated to play an important role in the initial pathologic changes associated with SSc by releasing fibrogenic cytokines, including transforming growth factor-beta or platelet-derived growth factor. However, the precise mechanism for increased monocyte/macrophage recruitment in the lesional skin of SSc is still not completely elucidated. Monocyte chemoattractant protein-1 (MCP-1) is a predominant monocyte chemoattractant secreted by various cells types including mononuclear cells, fibroblasts, smooth muscle cells, endothelial cells, or keratinocytes. In this study, we examined the expression of MCP-1 protein and mRNA in the lesional skin of seven patients with SSc by immunohistochemistry and in situ hybridization. Results of immunohistochemistry showed that MCP-1 was detected on infiltrating mononuclear cells and fibroblastic cells in scleroderma skin, whereas normal skin showed only minimal MCP-1 expression. We demonstrated the expression of MCP-1 mRNA in infiltrating mononuclear cells and keratinocytes in scleroderma and contact dermatitis skin. In addition, signals were also detected in fibroblasts in the lesional skin of scleroderma, whereas fibroblasts in normal skin and contact dermatitis skin did not express MCP-1 mRNA. These findings suggest that MCP-1 plays a role in recruiting monocyte/macrophages in the lesional skin of scleroderma and that activated fibroblasts in scleroderma are involved in this process.

Pseudoscleroderma associated with lung cancer: correlation of collagen type I and connective tissue growth factor gene expression.

Pseudoscleroderma as a paraneoplastic syndrome is a rare disease. We report here a patient with lung cancer (undifferentiated squamous cell carcinoma), who developed acrosclerosis. Using in situ hybridization, marked expression of alpha1(I)-collagen and connective tissue growth factor (CTGF) mRNA was found in fibroblasts scattered throughout the dermis. However, transforming growth factor (TGF)-beta1 expression was not detected. The pattern of CTGF gene expression and collagen synthesis was similar to that in systemic scleroderma. The absence of TGF-beta1 mRNA could indicate that tumour-derived factors induce the expression of CTGF.

Mast cells enhance contraction of three-dimensional collagen lattices by fibroblasts by cell-cell interaction: role of stem cell factor/c-kit.

Reorganization of the extracellular matrix is important in many biological and pathophysiological processes, including tissue remodelling, wound healing, or cancer metastasis. The ability of cultured fibroblasts to reorganize and contract three-dimensional type I collagen gels is regarded as an in vitro model for this process. In tissue fibrosis, complex interactions among fibroblasts, inflammatory cells and the extracellular matrix are taking place. Mast cells have often been discussed to play a role in several fibrotic conditions including scleroderma, scar formation, or wound healing. In this study, we examined the effects of mast cells on contraction of collagen lattices. The results demonstrate that co-culture of dermal fibroblasts with a human mast cell line (HMC-1) significantly enhanced contraction of the three-dimensional collagen lattices, whereas mast cells alone failed to contract the gel. Addition of culture supernatants of mast cells did not enhance the speed of gel contraction, indicating the importance of cell-cell contact. Morphological analysis showed that mast cells were incorporated into the lattices. Histological examination also demonstrated that within the lattices, mast cells were localized in close contact to, or attached to, fibroblasts. As fibroblasts and mast cells are known to attach via stem cell factor (SCF)/c-kit interaction when co-cultured in monolayers, we also examined the effect of antibodies against SCF and c-kit in this system. Addition of both antibodies inhibited gel contraction up to 70%. In contrast, antibodies against interleukin-4 (IL-4) and IL-4 receptor did not affect gel contraction. These results indicate that mast cells enhance fibroblast-mediated contraction of collagen lattices via direct cell-cell contact, mediated in part by SCF/c-kit interactions.

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

Interactions of fibroblasts with the extracellular matrix: implications for the understanding of fibrosis.

The cellular organization and the compartmentalization in multicellular organisms is mediated by the extracellular matrix (ECM). This structure is composed by a wide variety of different macromolecules which carry distinct domains with defined structural and/or biological activities. Cells are known to interact with these molecules via specific receptors. Following activation, these receptors transduce signals either directly to the intracellular cytoskeleton or via different signalling cascades. Cell-matrix interactions, therefore, not only control the shape and orientation of cells but can also directly regulate cellular functions, including migration, differentiation, proliferation, and the expression of different genes. These cell-matrix interactions have been elucidated in detail for several biological processes, especially morphogenesis and differentiation, but also play an important role during pathological situations, e.g. wound healing and tumor progression. Although much less investigated, similar mechanisms are thought to regulate the biological behavior of fibroblastic cells, the final target cells in fibrosis. The activity of these cells depends in various ways on the presence of ECM molecules. First, some of the molecules are known to bind to and modulate the activity of those growth factors and cytokines, which lead to the activation of fibroblasts during the early phases of fibrosis. Second, deposition of large amounts of ECM molecules alters the environment and the mechanical load on the cells which are embedded in this matrix. Third, ECM molecules directly modulate fibroblast metabolism via certain integrin receptors. This review summarizes recent developments in all three domains. It mainly focuses on the direct role of ECM molecules in the biosynthetic activity of fibroblasts.

A compound heterozygote patient with Ehlers-Danlos syndrome type VI has a deletion in one allele and a splicing defect in the other allele of the lysyl hydroxylase gene.

We report the first deletion mutation and the first splicing defect in the lysyl hydroxylase gene in a compound heterozygote patient with Ehlers-Danlos syndrome type VI with markedly reduced lysyl hydroxylase activity. Northern analysis of the RNA isolated from skin fibroblasts of the patient demonstrated the presence of a truncated lysyl hydroxylase mRNA. PCR and sequence analysis confirmed the truncation and indicated that the cells contain two types of shortened mRNAs, one lacking the sequences corresponding to exon 16 and the other lacking that corresponding to exon 17 of the lysyl hydroxylase gene. Analysis of genomic DNA revealed deletion of the penultimate adenosine from the 3' end of intron 15 from one allele. This defect was probably responsible for the skipping of exon 16 sequences from the transcript. The other allele, inherited from the mother, contains an Alu-Alu recombination with a deletion of about 3,000 nucleotides from the gene; this abnormality explains the lack of exon 17 sequences. The identified mutations in exon 16 and exon 17 do not alter the reading frame of the transcripts.

Expression of type VI collagen mRNA during wound healing.

During the highly regulated process of wound healing the expression of the interstitial collagens I and III is increased in a time-dependent fashion. Although ultrastructural and in vitro studies suggest a physiologic role of collagen VI in the organization of extracellular matrix deposition, nothing is known about its role in wound healing. Therefore, we studied collagen VI gene expression during wound healing in humans compared to that of collagens I and III. The presence of specific alpha 1(VI) and alpha 3(VI) mRNA species in scar tissue was demonstrated by Northern blot analysis. Quantification of mRNA expression by dot blot analysis and in situ hybridization indicated that like for the interstitial collagens I and III collagen VI gene expression was increased during wound healing, reaching its maximum 2 weeks after initial insult. In the late phase of wound healing like alpha 1(I) the alpha 1(VI) gene expression was not down regulated significantly. In contrast, a reduction of alpha 3(VI) collagen gene expression was observed, as was for the alpha 1(III) collagen gene, indicating a non-coordinate regulation of these chains. Collagen VI gene expression could be localized to fibroblast-like cells and to endothelial cells of newly formed vessels. Collagen VI gene expression was undetectable in smooth muscle cells and myoepithelial cells of eccrine glands. These results indicate that collagen VI gene expression is regulated in a time-dependent fashion and that fibroblasts and endothelial cells appear to play an important role in collagen VI synthesis during wound healing.

Interleukin-6 expression by fibroblasts grown in three-dimensional gel cultures.

We investigated the expression and biological activity of interleukin-6 (IL-6) by human fibroblasts cultured as monolayers and within three-dimensional type I collagen lattices. In the course of contracting the gel to a dense tissue-like structure, the cells upregulated their levels of IL-6 mRNA as well as IL-6 biological activity. While there was little mRNA and protein activity (6,500 U/ml) in monolayer cultures, fibroblasts in the 3D system showed a 13-fold increase in IL-6 mRNA on day 3. IL-6 protein was increased 6-fold (38,000 U/ml) on day 4. Stimulation of fibroblast cultures with IL-1 alpha resulted in enhanced IL-6 production in both systems, but the fibroblasts embedded into the 3D network continued to exhibit higher levels.

Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.

The ability of cultured human fibroblasts to reorganize and contract three dimensional collagen I gels is regarded as an in vitro model for the reorganization of connective tissue during wound healing. We investigated whether adhesion receptors of the integrin family are involved. It was found that synthesis and transcription of the alpha 2 beta 1 integrin (but not of alpha 1 beta 1 or alpha 3 beta 1) is selectively upregulated when fibroblasts are seeded into type I collagen gels. Time course experiments revealed that high synthetic levels of alpha 2 beta 1 parallel the gel contraction process and return to "baseline" levels after the contraction has subsided. Furthermore, function-blocking mAbs directed to the alpha 2 and beta 1 chain of integrins inhibited gel contraction. Remodelling of connective tissue can be important for tumor cells during invasion and formation of metastases. Therefore, we tested human melanoma cell lines for this function. Five out of nine melanoma lines contracted collagen gels in vitro. Among these, two highly aggressive melanoma cell lines (MV3 and BLM) most efficiently contracted gels almost reaching the rate of normal adult fibroblasts. In these cells, synthesis of alpha 2 beta 1 was also significantly upregulated when seeded into collagen I gels. Moreover, function blocking anti-alpha 2 in conjunction with anti-beta 1 chain mAbs completely inhibited gel contraction for several days. Other melanoma cells (530) with lower metastatic potential which were not able to contract gels, showed no induction of alpha 2 beta 1 synthesis in gel culture. Our results suggest an important role of integrin alpha 2 beta 1 in the contraction of collagen I by normal diploid fibroblasts during wound healing and in the reorganization of collagen matrices by highly aggressive human melanoma cells.

TGF beta-1 and TNF alpha expression in the epidermis of patients with epidermodysplasia verruciformis.

In epidermodysplasia verruciformis (EV), the infection with specific human papillomaviruses (HPV) might be under control of the local immunosurveillance mechanisms related to cytokines produced by epidermal cells. We have investigated by in situ hybridization the expression of mRNA coding for TGF beta-1 and TNF alpha in the skin of patients with EV (n = 4) as compared to the skin lesions of patients with other premalignant (actinic keratosis; n = 5) or malignant (squamous cell carcinoma; n = 4) skin lesions, and to the skin of healthy individuals (n = 5). The expression of TGF beta-1 and TNF alpha mRNA was higher in the epidermis of EV patients as compared to the control skin from healthy individuals. The increased expression of mRNA for both cytokines was confirmed by northern blot analysis of RNA isolated from the skin lesions of the patient with EV. No specific signals for TGF beta-1 and TNF alpha were detected in actinic keratosis, and in cases of squamous cell carcinomas only single neoplastic cells were positive for TGF beta-1. It is conceivable that in EV TGF beta-1 and TNF alpha can be involved in the regulation of the growth and differentiation of HPV-infected keratinocytes and in the persistence of HPV-induced skin lesions.

[Polymerase chain reaction--principle and application in medicine]. / Die Polymerasekettenreaktion--Prinzip und Anwendung in der Medizin.

The polymerase chain reaction, a new molecular technique, is of increasing importance in many areas of medical research and diagnosis. Within hours DNA sequences can be amplified millionfold with very high specificity, making detection and further analysis possible. Using multiple rounds of exponential amplification, even one copy of a gene of interest can be detected by agarose gel electrophoresis and other methods. Furthermore, minute amounts of DNA even from tissue damaged by embedding in paraffin can be analysed. Polymerase chain reaction methodology has already gained significance in many areas of medical research, e.g. diagnosis of inherited diseases, detection of viral DNA in clinical samples, cancer research and diagnosis, and characterization of gene expression.