Loss of spatial organization and destruction of the pericellular matrix in early osteoarthritis in vivo and in a novel in vitro methodology.

OBJECTIVES: Current repair procedures for articular cartilage (AC) cannot restore the tissue's original form and function because neither changes in its architectural blueprint throughout life nor the respective biological understanding is fully available. We asked whether two unique elements of human cartilage architecture, the chondrocyte-surrounding pericellular matrix (PCM) and the superficial chondrocyte spatial organization (SCSO) beneath the articular surface (AS) are congenital, stable or dynamic throughout life. We hypothesized that inducing chondrocyte proliferation in vitro impairs organization and PCM and induces an advanced osteoarthritis (OA)-like structural phenotype of human cartilage. METHODS: We recorded propidium-iodine-stained fetal and adult cartilage explants, arranged stages of organization into a sequence, and created a lifetime-summarizing SCSO model. To replicate the OA-associated dynamics revealed by our model, and to test our hypothesis, we transduced specifically early OA-explants with hFGF-2 for inducing proliferation. The PCM was examined using immuno- and auto-fluorescence, multiphoton second-harmonic-generation (SHG), and scanning electron microscopy (SEM). RESULTS: Spatial organization evolved from fetal homogeneity, peaked with adult string-like arrangements, but was completely lost in OA. Loss of organization included PCM perforation (local micro-fibrillar collagen intensity decrease) and destruction [regional collagen type VI (CollVI) signal weakness or absence]. Importantly, both loss of organization and PCM destruction were successfully recapitulated in FGF-2-transduced explants. CONCLUSION: Induced proliferation of spatially characterized early OA-chondrocytes within standardized explants recapitulated the full range of loss of SCSO and PCM destruction, introducing a novel in vitro methodology. This methodology induces a structural phenotype of human cartilage that is similar to advanced OA and potentially of significance and utility.

Immunoregulatory functions for murine intraepithelial lymphocytes: gamma/delta T cell receptor-positive (TCR+) T cells abrogate oral tolerance, while alpha/beta TCR+ T cells provide B cell help.

Past work has shown that a subset of effector T cells with unique characteristics could abrogate hapten- or antigen-induced tolerance, and the reconstitution of this immune response has been termed contrasuppression. We have studied contrasuppression in a model of oral tolerance (OT) in which adoptively transferred antigen-specific T contrasuppressor (Tcs) cells reverse OT and result in antibody responses to the eliciting antigen. In the present study, we show that murine intraepithelial lymphocytes (IELs) from mice orally immunized with sheep red blood cells (SRBC) contain T cells that exhibit Tcs cell activity. This effect was mediated by CD3+ gamma/delta T cell receptor-positive (TCR+), but not alpha/beta TCR+ T cells, and gamma/delta TCR+ Tcs cells were associated with both the CD4-,CD8+ and CD4-,CD8- (double-negative) IEL fractions. The CD4-,CD8+ gamma/delta TCR+ IELs were further separated into Vicia villosa-adherent and -nonadherent fractions. Adoptive transfer of V. villosa-adherent gamma/delta TCR+ T cells to mice with OT to SRBC resulted in splenic IgA, IgM, and IgG subclass anti-SRBC responses, while V. villosa-nonadherent gamma/delta TCR+ T cells were without activity. The gamma/delta TCR+ IELs did not support in vitro antibody responses in B cell cultures, while alpha/beta TCR+ IELs were effective T helper cells. Further, cytokine production by the gamma/delta TCR+ IELs was examined, and the gamma/delta TCR+ V. villosa-adherent fraction, which possessed contrasuppressor function, contained low levels of IL-5 mRNA and small numbers of IL-5-producing cells when compared with alpha/beta TCR+ IELs and V. villosa-nonadherent gamma/delta TCR+ IELs. Our results now show that mouse IELs contain two distinct types of T cells that function in the immune response, e.g., alpha/beta TCR+ T cells that produce IL-5 and function as helper cells, and gamma/delta TCR+ T cells that restore antibody responses in mice that had been orally tolerized with antigen.

Transforming growth factor beta1 and laminin-111 cooperate in the induction of interleukin-16 expression in synovial fibroblasts from patients with rheumatoid arthritis.

OBJECTIVES: In synovial tissues of patients with rheumatoid arthritis (RA), strong expression of laminins and integrins co-localises with increased expression of inflammatory cytokines. Synovial fibroblasts (SF) contribute to the pathogenesis of RA through increased expression of cytokines and chemoattractant factors, one of which is interleukin-16 (IL16). A study was undertaken to investigate the regulatory pathways of IL16 in SF from patients with RA (RA-SF) and osteoarthritis (OA-SF). METHODS: SF were seeded in laminin-coated flasks and activated by the addition of cytokines. The expression of IL16 was investigated by quantitative RT-PCR, immunoblotting and ELISA; its biological activity was determined by a cell migration assay. Cell-matrix interactions were investigated by cell binding and attachment assays. Relevant intracellular signalling pathways were studied by immunoblotting and with pharmacological blocking reagents. RESULTS: Stimulation of SF with transforming growth factor beta(1)(TGF-beta(1)) and growth on laminin-111 (LM-111) significantly increased the expression of IL16. Binding to LM-111 induced significantly more IL16 mRNA in RA-SF than in OA-SF (p<0.05). The IL16 cytokine was detected in supernatants of TGF-beta(1)-activated and in LM-111+TGF-beta(1)-activated RA-SF (38 to 62 pg/ml), but not in supernatants of OA-SF. This IL16 regulation involved p38MAPK, ERK1/2 and SMAD2 signalling, but not NFkappaB. CONCLUSIONS: Binding of RA-SF to LM-111 in the presence of TGF-beta(1) triggers a significant IL16 response and thus may contribute to the infiltration of CD4+ lymphocytes into synovial tissues. This mode of IL16 induction represents a novel pathway leading to IL16 production in RA-SF but not in OA-SF, which operates independently of NFkappaB signalling.

Hypoxia reduces the inhibitory effect of IL-1beta on chondrogenic differentiation of FCS-free expanded MSC.

OBJECTIVE: Mesenchymal stromal cells (MSC) are a promising tool for tissue engineering of the intervertebral disc (ID). The IDs are characterized by hypoxia and, after degeneration, by an inflammatory environment as well. We therefore investigated the effects of inflammation induced with interleukin (IL)-1beta and of hypoxia (2% O(2)) on the chondrogenic differentiation of MSC. METHODS: Bone-marrow-derived MSC (bmMSC) were cultured in a fetal-calf-serum-free medium and characterized according to the minimal criteria for multipotent MSC. Chondrogenic differentiation of MSC was induced following standard protocols, under hypoxic conditions, with or without IL-1beta supplementation. After 28 days of differentiation, micromasses were analyzed by histochemical staining and immunohistochemistry and by determining the mRNA level of chondrogenic marker genes utilizing quantitative RT-PCR. RESULTS: Micromasses differentiated under IL-1beta supplementation are smaller and express less extracellular matrix (ECM) protein. Micromasses differentiated under hypoxia appear larger in size, display a denser ECM and express marker genes comparable to controls. The combination of hypoxia and IL-1beta supplementation improved chondrogenesis compared to IL-1beta supplementation alone. Micromasses differentiated under standard conditions served as controls. CONCLUSION: Inflammatory processes inhibit the chondrogenic differentiation of MSC. This may lessen the regenerative potential of MSC in situ. Thus, for the cell therapy of IDs using MSC to be effective it will be necessary to manage the inflammatory conditions in situ. In contrast, hypoxic conditions exert beneficial effects on chondrogenesis and phenotype stability of transplanted MSC, and may improve the quality of the generated ECM.

Co-activation of synovial fibroblasts by laminin-111 and transforming growth factor-beta induces expression of matrix metalloproteinases 3 and 10 independently of nuclear factor-kappaB.

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-beta induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription-polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-beta induced significant increases in MMP-3 mRNA (12.35-fold, p < 0.001) and protein (mean 62 ng/ml, sixfold, p < 0.008) and in expression of MMP-10 mRNA (11.68-fold, p < 0.05) and protein (54 ng/ml, 20-fold, p > or = 0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-beta response. No phosphorylation of nuclear factor-kappaB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-beta suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-kappaB phosphorylation.

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Regulatory functions for murine intraepithelial lymphocytes in mucosal responses.

Our studies have given direct evidence that CD3+, CD4-, CD8+ T cells in the IELs can be separated into at least two subsets that produce IFN-gamma and/or IL-5 and may exhibit Th1- and Th2-type functions in the epithelium and in the underlying lamina propria region. Further, it was also shown that TCR1+ T cells in IELs are capable of producing IFN-gamma and/or IL-5. In addition to production of these cytokines, IELs derived from mice orally primed with TD antigen contain a subset of T cells which possess antigen-specific immunoregulatory function. CD3+, CD4-, CD8+ T cells isolated from IEL of TD antigen-primed mice abrogated systemic unresponsiveness to secondary-type responses including those of the IgA isotype upon adoptive transfer to mice with OT. Our studies further suggested that these CD3+, CD4-, CD8+ IELs use the gamma-delta form of TCR (TCR1) for their immunoregulatory function.

Efficient generation of transgenic BALB/c mice using BALB/c embryonic stem cells.

BALB/c is one of the most widely used and best characterized mouse strains in immunology. For various applications, it is necessary to generate BALB/c transgenic mice. However, using the conventional microinjection technique it is extremely inefficient to produce transgenic BALB/c mice since the one-cell stage BALB/c embryos are highly vulnerable to pronuclear DNA microinjection. To overcome this problem, we have investigated the generation of Egr-1 (early growth response gene) transgenic mice via the transfection of BALB/c embryonic stem cells. Transfectants carrying Egr-1 constructs comprising either the immunoglobulin heavy chain or the MHC class II promoter/enhancer system were injected into C57BL/6 host blastocysts resulting in chimeric mice. For both type of expression vectors, transgenic offspring of the germline chimeras expressed recombinant Egr-1 in lymphoid tissues containing B cells. This demonstrates the successful generation of Egr-1 transgenic BALB/c mice using transfected ES cell.

Immunoregulatory confluence: T cells, Fc receptors and cytokines for IgA immune responses.

IgA isotype responses are regulated by at least two compartments including those of CD4+ Th2 type cells and cytokines produced by these cells. Interaction of CD4+ Th cells and APC via TCR and Ag-MHC II leads to activation of Th2 type cells. This would allow for secretion of cytokines, especially IL-5 and IL-6 which are key cytokines for the terminal differentiation of B cells into Ig secreting cells. Further, expression of Fc alpha RII on CD4+ Th2 cells could be important for the recruitment of sIgA+ B cells which would allow selective interactions of Th2 cells and sIgA + B cells via Fc alpha RII. This could lead to selectively transfer of IL-5 and IL-6 to sIgA + B cells from CD4+ Th2 cells.

[Cell-based therapy to treat stress urinary incontinence: which cell type at what cost?]. / Zellbasierte Therapie der Belastungsinkontinenz: Welche Zelle zu welchen Kosten?

In Germany, 6-8 million woman and men suffer urinary incontinence, which represents 12.5 % of the population. It is estimated that by the middle of this century, it will increase to almost 30 %. The primary reason will be primarily related to the aging population but also to patient awareness and seeking a solution. In addition to the cost which is covered by the health insurance, the patient will spend more than half a billion euro/year out-of-pocket, not to mention the social stigma associated with urinary incontinence. The current common treatment options are symptomatic but do not restore functionality. One option might be tissue engineering or stem cell therapy. This article describes the likelihood that this therapy will change the approach in treating stress urinary incontinence. Boundaries and legal aspects are highlighted as well as approximated cost. These treatment costs might be currently higher than the standard treatment options, but the investment to reduce these costs are paid indirectly by society.

[Laminin-dependent inflammatory response in synovial fibroblasts of rheumatoid arthritis patients]. / Laminin-modulierte Entzündungsreaktionen in synovialen Fibroblasten von Rheumapatienten.

Elevated expression of matrix-metalloproteinases (MMP) contributes to cartilage destruction in rheumatoid arthritis. We report on a novel pathway of inflammatory activation of synovial fibroblasts that is induced by TGF-beta and laminin (extracellular matrix) and leads to increased expression of the proteases MMP-3 and MMP-10. Neither costimulation by the central inflammatory cytokines TNF-alpha and IL-1beta nor NFkB signalling is needed for this pathway.

[Expression analysis of different collagens and cytokines in cartilage cells derived from arthrotic hip and knee joints]. / Expressionsanalyse verschiedener Kollagene und Zytokine in Knorpelzellen aus arthrotisch veränderten Hüft- und Kniegelenken.

AIM: Osteoarthritis (OA) is characterized by an irreversible destruction of articular cartilage. This is associated with a multiplicity of factors, causing an increased catabolic metabolism in cartilage. However, the prevalence of the OA is very variable in different joints. Therefore , we conducted a comparative analysis of chondrocytes derived from knee and hip joints with respect to their expression of inflammatory factors, such as IL-1beta, IL-1beta-receptorantagonist, iNOS, components of cartilage matrix (collagen I, II, and VI) as well as vimentin. METHODS: Different cytokines and proteins were detected by immune-histochemical staining of cartilage samples ex vivo. Further, chondrocytes were isolated from OA knee and hip joints, expanded in vitro and gene expression patterns were investigated by quantitative RT-PCR. RESULTS: Chondrocytes from knee and hip joints of OA patients express collagenes I, II and VI, IL-1beta and IL-1beta-RA, iNOS as well as Vimentin. A significant difference in gene expression patterns was not found in chondrocytes from the hip joints versus the knee joint ex vivo or in primary culture cells in vitro. However, in vitro the expression of type I collagen exceeded the expression of type II collagen. The IL-1beta-expression was high ex vivo, remained low during primary culture but was significantly elevated after primary culture in hip chondrocytes. CONCLUSION: Osteoarthritic gene expression patterns in cells derived from hip or knee joints ex vivo and in primary culture were not significantly different. We conclude that the rather frequent occurrence of OA in these joints in comparison to the ankle joint may be associated with a close physiological relation of cells in these joints. However, future studies which will include ankle cartilage must be investigated in further detail.

[Characterisation of human meniscus cells]. / Charakterisierung von humanen Meniskuszellen.

AIM: The meniscus of the human knee joint has an important function for the shock absorption, stability and power transmission from the upper to the lower leg. After meniscus injury often a partial or complete resection is necessary. Only injuries in the outer third may heal spontaneously or upon primary suture due to the vascularisation in these segments. After partial or total meniscectomy osteoarthritis of the knee joint is common in a large number of patients. The goal of our investigations was to establish meniscus cell cultures and to characterise the fibrochondrocytes (meniscus cells) in vitro. METHODS: We examined the expression of different growth factors, cytokines and proteins in human menisci from surgical preparations using immunohistochemistry and RT-PCR analysis. RESULTS: Human meniscus cells express the collagens I , II, III, and VI, the matrix metalloproteinases-1, -2, -3, -8, and -13, BMP-2, and -4, TGFbeta1, VEGF, IGF-I, and -II, FGF-2, endostatin, iNOS, vimentin, TIMP-1, and -2, aggrecan, IL-1beta, IL-6, and IL-18. Staining with the monoclonal antibody AS.02 in all examined cells confirmed their mesenchymal origin. CONCLUSION: New strategies for the treatment of meniscus damage can be derived from these results and further advances for the tissue engineering of meniscus tissue can be obtained.

Biocompatibility correlation of polymeric materials using human osteosarcoma cells.

Metal implants are the preferred materials to generate articular prostheses, plates, or bone pegs in orthopedic surgery. Although titanium and titanium alloys show a relatively good biocompatibility, clinical experience revealed that coating of the metallic implant surface may increase the biocompatibility. In a search for optimum bone implant surfaces, we determined polarity and contact angle parameters of a variety of polymers and substances and correlated the findings in a biocompatibility assay using an in vitro bone cell model. We report that an optimum adherence of SAOS-2 cells to such surfaces and a good vitality for polymers are characterized by water-based contact angles of 80 degrees and 20 degrees for advancing and receding probes, respectively.

[Frequency of terminally differentiated fibroblasts in the synovial membrane of rheumatoid arthritis patients]. / Häufigkeit terminal differenzierter Fibroblasten in der Synovialmembran von Rheumapatienten.

The metabolic activation of synovial fibroblasts (SF) and their expression of matrix degrading enzymes and inflammatory cytokines contributes to the pathology of rheumatoid arthritis (RA). It is remarkable that SF of RA patients do not proliferate at higher rates when compared to SF of other patients, but they are resistant to apotposis inducing signals. The chronic inflammation in RA causes fibrosis of the synovial tissue and fibrosis has been associated with terminal differentiation. Therefore we investigated if there are increased numbers of terminally differentiated fibroblasts in the RA synovium and if there is a correlation between terminal differentiation of SF and increased levels of expression of interleukins and matrix metalloproteinases. We analyzed specimen of four RA patients, two patients with osteoarthritis (OA) and two healthy donors suffering from joint injuries. By use of RT-PCR techniques we examined mRNA expression of two genes in SF which are associated with terminal differentiation, p16INK4a and p21-cip. In addition, we labelled differentiated fibroblasts using the SA-beta-galaktosidase assay and investigated differences in protein expression patterns of factor PIVa and the tropomyosin 1 and 2 molecules. We report that the number of terminally differentiated fibrolasts are not increased in the synovial membrane of RA patients. On the contrary we show that the synovia of the much younger patients has higher levels of terminally differentiated fibroblasts. Consequently, the fibrosis of synovial tissues in RA patients at later stages of disorder is not associated with proliferation and differentiation of the fibroblasts but rather a consequence of chronic inflammation.

Transforming growth factor-beta and IL-1 beta act in synergy to enhance IL-6 secretion by the intestinal epithelial cell line, IEC-6.

Intestinal epithelial cells are a potentially important source for a number of cytokines that may modulate the immune response at the intestinal mucosa. We have recently begun to study the mechanisms that regulate IL-6 production by intestinal epithelial cells using the nontransformed crypt-like rat intestinal epithelial cell line IEC-6 as a model. Culture of the IEC-6 cells with human rIL-1 beta resulted in an enhanced secretion of IL-6 by the cells. RT-PCR analysis of IL-1 beta-treated cells showed an enhanced level of IL-6 mRNA at 4 h, suggesting that IL-1 beta enhanced IL-6 gene expression. In a previous study, transforming growth factor-beta (TGF-beta 1) was also found to enhance IL-6 secretion by the IEC-6 cells and because both IL-1 beta and TGF-beta may be present in inflamed mucosal tissue, the effect of adding both cytokines together was next investigated. Culture of the IEC-6 cells with both TGF-beta 1 and IL-1 beta resulted in a synergistic enhancement of IL-6 secretion that was seen even at high levels of both TGF-beta and IL-1 beta. IL-6 mRNA levels from cells treated with both TGF-beta 1 and IL-1 beta were also determined to be enhanced when compared to that of cells treated with IL-1 beta or TGF-beta 1 only, as determined by RT-PCR analysis. Pretreatment of the IEC-6 cells with TGF-beta 1 for 2 or 3 days before addition of the IL-1 beta induced the IEC-6 cells to differentiate and become more sensitive to stimulation by IL-1 beta. Subsequent experiments determined that TGF-beta enhanced the capacity of the IEC-6 cells to bind labeled IL-1 beta indicating that TGF-beta may have enhanced the expression of IL-1 receptors on the cells. These results suggest that the intestinal epithelial cell may represent an important source of IL-6 in inflammatory responses at the intestinal mucosa and that TGF-beta could potentiate this function.

Selective induction of Th2 cells in murine Peyer's patches by oral immunization.

We have used three different methods to determine the T helper (Th) cell response, including Th1 and Th2 types, in murine Peyer's patches (PP) following oral immunization with sheep red blood cells (SRBC). These include: (i) use of cytokine-specific (IFN-gamma and IL-5), single cell assays to estimate the frequencies of Th1 and Th2 cells respectively, (ii) cytokine-specific mRNA--cDNA dot blot and Northern gel hybridizations to detect levels of specific mRNA, and (iii) T cell cloning techniques to determine the frequency of Th1 and Th2 clones. Mice were immunized with SRBC by either the oral or i.p. route. The PP and splenic (SP) CD3+ and CD3+ CD4+ T cell subsets were isolated and cultured with antigen, feeder cells, and IL-2, and were assessed at various intervals (days 0, 1, 3, and 6) for numbers of T cells producing either IFN-gamma or IL-5 by use of an enzyme-linked immunospot (ELISPOT) procedure. Cultures of T cells from PP or SP of mice given SRBC by the oral route had a high frequency of IL-5 spot forming cells (SFC), with lower numbers of IFN-gamma SFC. However, cultures of CD3+ T cells and CD3+ CD4+ Th cells from spleens of i.p. immunized mice exhibited predominantly IFN-gamma SFC, with smaller but significant numbers of IL-5 SFC. This distinct pattern of cytokine production was supported by mRNA analysis where high IL-5 specific mRNA levels were noted in PP T cell cultures of orally primed mice, while IFN-gamma mRNA was predominant in the SP CD3+ T cell and CD3+ CD4+ Th cell cultures from i.p. immunized mice. When the frequencies of IFN-gamma or IL-5 SFC were assessed among cloned Th cells from orally- or systemically-immunized mice, 74% of Th cell clones from PP of mice orally immunized with SRBC were IL-5 producers (Th2 type), while 67% of Th cell clones from SP of mice immunized by the i.p. route were IFN-gamma producers (Th1 type). Our studies show that higher frequencies of IFN-gamma producing Th1-type cells occur in SP of mice given antigen by the systemic route, while oral immunization results in predominantly IL-5 producing, Th2-type cells in PP.