Clive R. Roberts

Anti-IL-4 treatment prevents dermal collagen deposition in the tight-skin mouse model of scleroderma

The tight‐skin ( Tsk  /+) mutant mouse, a putative murine model of scleroderma, is characterized primarily by the excessive deposition of collagen and other extracellular matrix molecules in the dermis, and also by a developmentally acquired defect in pulmonary architecture. Passive transfer experiments have suggested an etiologic role for the immune system in Tsk  /+ dermal pathology. In addition, CD4+ T lymphocytes have been shown to be required for the excessive accumulation of dermal collagen in these mice. As IL‐4, a product of differentiated CD4+ T cells, is capable of regulating the synthesis of various matrix molecules (including type I collagen) by fibroblasts in vitro, we investigated the potential role of IL‐4 in mediating Tsk  /+ dermal fibrosis. Confirming that Tsk  /+ cells are capable of responding to IL‐4, we found receptors for this cytokine on Tsk  /+ embryonic fibroblasts and a dermal fibroblast cell line derived from these mice. Furthermore, IL‐4 receptors on Tsk  /+ fibroblasts were functional since IL‐4 stimulation in vitro increased type I collagen secretion from these cells. These results demonstrated the potential for IL‐4 to be directly involved in the excessive deposition of dermal collagen in Tsk  /+ mice. Critical insight into the role played by IL‐4 in mediating the dermal phenotype, however, was obtained following the administration of neutralizing anti‐IL‐4 antibodies to Tsk  /+ mice. This treatment prevented the development of dermal fibrosis, leading to normalization of dermal collagen content. Given the requirement for CD4+ T cells in Tsk  /+ dermal fibrosis, our results suggest that Th2 cells and/or factors elaborated by this T cell subset may play a key role in regulating dermal collagen content in this strain.

Domain Interactions in the Gelatinase A·TIMP-2·MT1-MMP Activation Complex THE ECTODOMAIN OF THE 44-kDa FORM OF MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE DOES NOT MODULATE GELATINASE A ACTIVATION

On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly285, is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown. We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry. A strong interaction between the TIMP-2 C domain (Glu153-Pro221) and the gelatinase A hemopexin C domain (Gly446-Cys660) was demonstrated by the yeast two-hybrid system. Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex. Using recombinant MT1-MMP hemopexin C domain (Gly285-Cys508), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found. Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells. The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2. Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.

Ultrastructure and tensile properties of human tracheal cartilage.

The cartilage of the walls of the trachea and bronchi acts to keep these airways open despite intrathoracic pressure differences during breathing that would otherwise collapse them and limit air flow. Changes in biomechanical properties and composition of airway cartilage may contribute to altered lung function in obstructive lung diseases. To investigate the relationship between collagen organization and equilibrium tensile modulus within the structure of airway cartilage, we used scanning electron microscopy (SEM), histochemistry and equilibrium tensile testing to analyze tracheal cartilage from 10 humans aged 17-81 yr. We show that the surfaces of tracheal cartilage matrix are collagen-rich and surround a proteoglycan-rich core. Collagen fibrils in the superficial zones are oriented in the plane of the cartilage surface. In deeper layers of the cartilage, collagen fibrils are oriented less regularly. Equilibrium tensile modulus of 100 microm thick strips of cartilage was measured and was found to decrease with depth; from 13.6 +/- 1.5 MPa for the ablumenal superficial zone to 4.6 +/- 1.7 MPa in the middle zone (means +/- S.D., n = 10, p < 0.001). Stress-strain curves were linear for strains up to 10% with minimal residual strain. This is consistent with a model in which collagen fibres in the outer layers of the cartilage resist tensile forces, and hydrated proteoglycans in the central zone resist compression forces as the cartilage crescent bends.

Matrix Metalloproteinase Processing of CXCL11/I-TAC Results in Loss of Chemoattractant Activity and Altered Glycosaminoglycan Binding

The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5–73), -(5–63), and -(5–58) forms. NH2-terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5–73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5–73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.

Matrix metalloproteinase 8 deficiency in mice exacerbates inflammatory arthritis through delayed neutrophil apoptosis and reduced caspase 11 expression

OBJECTIVE Neutrophil accumulation is balanced by both cell infiltration and cell clearance, the controls of which are pivotal in the pathogenesis of rheumatoid arthritis (RA) and other chronic inflammatory diseases. Of the neutrophil-specific proteases, matrix metalloproteinase 8 (MMP-8; also known as neutrophil collagenase or collagenase 2) is traditionally viewed as being crucial for collagen degradation and hence cell migration and infiltration. This study was undertaken to examine the role of MMP-8 in a murine model of spontaneous RA. METHODS MMP-8(-/-) mice were backcrossed onto the Fas-defective MRL/lpr background, a mouse strain characterized by systemic autoimmunity including spontaneous autoimmune arthritis. Arthritis was induced with Freunds complete adjuvant and clinical disease and histologic parameters were assessed. RESULTS MMP-8(-/-) mice had earlier and more severe joint inflammation than their MMP-8(+/+) counterparts, coupled with a massive accumulation of neutrophils in synovial tissue, an unexpected result considering the commonly held view that MMP-8 has important extracellular matrix-degradative functions. Protease and protease inhibitor analysis of MMP-8(-/-) mouse neutrophils by CLIP-CHIP microarray revealed very little additional change in protease levels except for low expression of the apoptosis initiator caspase 11. This was confirmed at the protein level in unstimulated, lipopolysaccharide-treated, and interferon-γ-treated MMP-8(-/-) mouse neutrophils. Downstream of caspase 11, the activity of the apoptosis executioner caspase 3 was consequently reduced in MMP-8(-/-) mouse neutrophils, translating to reduced neutrophil apoptosis and cell accumulation compared with wild-type mouse cells. CONCLUSION Our findings indicate that MMP-8 is not essential for neutrophil migration in arthritis and likely other autoimmune diseases. Rather, MMP-8 is important for normal rates of neutrophil apoptosis and hence regulates cell clearance. Because MMP-8 deficiency leads to an exaggerated accumulation of neutrophil infiltrates due to delayed apoptosis and concurrent pathologic changes associated with dramatically increased neutrophil infiltration, MMP-8 is antiinflammatory and therefore a drug antitarget in the treatment of arthritis.

Aggrecan Components Differentially Modulate Nerve Growth Factor-Responsive and Neurotrophin-3-Responsive Dorsal Root Ganglion Neurite Growth

Aggrecan is one of the major chondroitin sulfate proteoglycans (CSPGs) expressed in the central nervous system. The signaling pathways activated downstream of cell interaction with aggrecan and with CSPGs in general and the importance of chondroitin sulfate–glycosaminoglycan side chains in their inhibition are unclear. Therefore, to analyze the effect of different components of aggrecan in inhibiting neurite growth, neurite outgrowth was quantified in an in vitro model in which chick dorsal root ganglion (DRG) explants were grown on substrates containing aggrecan bound to hyaluronan and link protein as a macromolecular aggregate, aggrecan monomers, hyaluronan, or ChABC‐treated aggrecan. Aggrecan aggregate, aggrecan monomer, and hyaluronan inhibited neurite outgrowth from nerve growth factor (NGF)– and neurotrophin‐3 (NT3)–responsive DRG neurons. Aggrecan inhibition was dependent on its chondroitin sulfate–glycosaminoglycans, as ChABC digestion alleviated neurite inhibition because of aggrecan. Growth cones displayed full or partial collapse on aggrecan aggregate, hyaluronan, and ChABC‐treated aggrecan. Inhibition of Rho kinase (ROCK) with Y27632 increased neurite growth on some but not all of the aggrecan components tested. With NGF in the culture medium, Y27632 increased neurite outgrowth on aggrecan aggregate, monomers, and ChABC‐treated aggrecan, but not on hyaluronan. The ROCK inhibitor also increased NT3‐responsive outgrowth on aggrecan aggregate and hyaluronan, but not on ChABC‐treated aggrecan. This study showed that the matrix proteoglycan aggrecan and its components have multiple effects on neurite outgrowth and that some of these effects involve the Rho/ROCK pathway.

Matrix Metalloproteinase–9 in Myeloid Cells: Implications for Allergic Inflammation

Matrix metalloproteinase–9 (MMP–9; 92 kDa gelatinase) is utilized by myeloid and lymphoid cells for migration across basement membranes. Although eosinophils are commonly seen infiltrating asthmatic airways, the role of basophils in allergic inflammation is debated. This study was undertaken to evaluate the content of MMP–9 in purified basophils compared with eosinophils and neutrophils. Peripheral blood basophils were isolated to greater than 95% purity using negative selection with antibody–coated magnetic beads using a 12–antibody cocktail. Eosinophils of greater than 98% purity were obtained by negative selection and neutrophils by positive selection using anti–CD16 magnetic beads. MMP–9 activity was assessed by gelatin zymography of cell lysates. Under parallel conditions, neutrophils contained 1,000–fold more MMP–9 than eosinophils. No activity was detected from 2×105 basophils. Immunocytochemistry with an anti–MMP–9 antibody showed bright staining of all neutrophils, lesser staining of eosinophils and no detectable staining of basophils. The failure to find MMP–9 in basophils may explain their paucity in asthmatic airway inflammation or suggest they secrete other enzymes capable of degrading type IV collagen.

Influence of repetitive mechanical loading on MMP2 activity in tendon fibroblasts

Matrix metalloproteinase2 has been implicated in tendon pathology caused by repetitive movements. However, its activity in the early stages of the tendons response to overuse, and its presence in the circulation as a possible indicator of tendon degradation, remain unknown. Human tendon cells were repetitively stretched for 5 days, and the rabbit Achilles tendon complex underwent repetitive motion 3× per week for 2 weeks. Quantitative polymer chain reaction analysis was performed to detect matrix metalloproteinase2/14 and tissue inhibitor of matrix metalloproteinase2 messenger ribonucleic acid of cells and rabbit tissue, and matrix metalloproteinase2 protein levels were determined with an enzyme linked immunoassay. Matrix metalloproteinase2 activity was examined using zymography of the conditioned media, tendon and serum. Immunohistochemistry was used to localize matrix metalloproteinase2 in tendon tissue, and the density of fibrillar collagen in tendons was examined using second harmonic generation microscopy. Tendon cells stretched with high strain or high frequency demonstrated increased matrix metalloproteinase2 messenger ribonucleic acid and protein levels. Matrix metalloproteinase2 activity was increased in the rabbit Achilles tendon tissue at weeks 1 and 2; however, serum activity was only increased at week 1. After 2 weeks of exercise, the collagen density was lower in specific regions of the exercised rabbit Achilles tendon complex. Matrix metalloproteinase2 expression in exercised rabbit Achilles tendons was detected surrounding tendon fibroblasts. Repetitive mechanical stimulation of tendon cells results in a small increase in matrix metalloproteinase2 levels, but it appears unlikely that serum matrix metalloproteinase2 will be a useful indicator of tendon overuse injury.

IDENTIFICATION OF BIOMARKERS FOR EARLY TENDON DEGENERATION USING AN IN-VIVO RABBIT MODEL

Introduction Tendinopathy is a common and costly diagnosis in workers with Activity-related Soft Tissue Disorder (ASTD), and in recreational and professional athletes. The associated pain results in reduced physical activity, reduced productivity, missed work days, and substantial costs to the government. Workers whose tendons are exposed to prolonged repetitive activity (eg, manual workers like fish processors) and/or high mechanical loads (eg, construction workers) are at high risk for the development of ASTD-associated tendinopathy. Pain is usually one of the first symptoms of tendinopathy; however imaging studies have demonstrated that significant degenerative tendon alterations can develop in response to mechanical loading, months before there are any overt symptoms. Goal: To identify circulation markers of early tendon degeneration. Research questions: (1) Will fatigue damage in tendons, caused by different loading regimes, correlate with changes in expression of genes involved in tendon degeneration? (2) Can we track the development of early phase tendon degeneration in vivo using ultrasound and measure indicators of tendon degradative activity in the circulation? It is hypothesised that different loading regimes will lead to characteristic, degradative differences in gene expression. Ultrasound will be able to visualise and follow early onset, degradative changes in the strained tendons. Methods Rabbit Achilles tendons will be subjected to varying loading regimens using the in-vivo Backman model. After set periods of exercise, microdialysis and blood samples will be taken and analysed for genes involved in degradation of tendons. At the same intervals, ultrasound images will be taken to track changes in the cross sectional area of the tendon. After the end of the full exercise period, the tendon tissue will be examined for characteristic degradative changes using microscopy. Results It is expected that the exercise regimen will lead to significant changes in gene expression in the tendon.1 Blood and microdialysis samples are expected to show significant changes in for example, collagen type I and III, matrix metalloproteinases (MMPs), tissue inhibitors for MMPs (TIMPs), and other genes involved.2 It is expected that the tissue will exhibit characteristic morphological changes seen in degradative tendon tissue, for example, rounded nuclei of tenocytes and a looser, kinked pattern of the collagen fibrils.3 Discussion Treatment for tendinopathies in workers can be a long and costly process which is not always successful, due to the degenerative nature of chronic tendinopathy. Detecting the early stages of tendinopathy could allow for the possibility of interventions to decrease or prevent tendon pain and/or rupture, and reduce healthcare and insurance costs.

26 – Airway Remodelling

Publisher Summary Structural changes in the airway walls involving extracellular-matrix remodeling are prominent features of asthma. These changes are driven by the mediators released as a consequence of chronic allergic inflammation. Changes in a matrix have the capacity to influence airway function in asthma. However, it is not clear how each of the many changes that occur in the airway wall contribute to alter airway function in asthma. Conversely, geometric considerations would result in exaggerated airway narrowing for a given degree of smooth muscle shortening as the airway wall is thickened by the deposition of these molecules internal to the smooth muscle. Elastin and cartilage degradation in the airway walls would be expected to result in decreased airway wall stiffness and increased airway narrowing for a given amount of force generated by the smooth muscle. Degradation of matrix associated with the smooth muscle may both decrease the stiffness of the parallel elastic component and uncouple smooth muscle from the load provided by lung recoil, thereby allowing exaggerated smooth-muscle shortening. An increase in muscle mass may be associated with an increase, a decrease, or no change in smooth-muscle contractility. If an increase in muscle mass is not associated with any other phenotypic changes, it would be expected to contribute to exaggerated airway narrowing.