Hydroxyproline Deposition Research Articles

Hapten-immune pulmonary interstitial fibrosis (HIPIF) in mice requires both CD4+ and CD8+ T lymphocytes

We present a description and analysis of a mouse model for pulmonary interstitial fibrosis that is induced by a specific immune response to a small reactive chemical group called trinitrophenyl. We describe the model, and then we examine the cellular mechanism for the induction of the fibrosis. The specific increase in hydroxyproline reached a peak by day 7 and persisted through day 28 in all animals that were sensitized to and challenged with the hapten. Distinct patterns of fibrosis that were seen histologically correlated with antigenic pretreatment and were dependent on T lymphocytes. We also report that the inflammatory and fibrotic responses could be adoptively transferred with immune lymphocytes but not with immune serum. In vivo administration of anti-CD4 and anti-CD8 monoclonal antibodies to sensitized mice prevented the development of immune-mediated lung inflammation and was effective in reducing hydroxyproline deposition. We conclude that (activated) T lymphocytes contribute to the pathogenesis of pulmonary fibrotic diseases. The possibility arises that haptens in the environment may promote sensitization of individuals via their skin or lungs and cell-mediated immune responses to haptenated antigens within the lung may promote pulmonary fibrosis.

The Effect of In Vivo T Helper and T Suppressor Lymphocyte Depletion on Wound Healing

The role of T lymphocytes in wound healing is still not well-defined. Because it had been previously shown that in vivo depletion of T cells leads to impaired wound healing, the effect of depleting T cell subsets on subsequent fibroplasia was studied. T helper/effector cells were depleted by the use of the monoclonal antibody GK1.5, reactive against the L3T4 antigen (CD4). T suppressor/cytotoxic lymphocytes were depleted by using the 2.43 monoclonal antibody reactive against the Lyt 2 antigen (CD8). In the first experiment, Balb/c mice were treated with the antibodies starting at 24 hours before wounding was performed, and weekly thereafter. Depletion of the T helper/effector cells had no effect on wound-breaking strength or hydroxyproline deposition in sponge granulomas, whereas depletion of T suppressor/cytotoxic cells significantly enhanced both of these healing parameters. In a second experiment, T cell subset depletion was started on Days 0, 3, 7, 10, and 14 postwounding, and treatments were continued weekly thereafter. Once again, depletion of T helper/effector cells had no effect on wound healing, whereas depletion of T suppressor/cytotoxic cells markedly increased both wound-breaking strength and collagen synthesis. In conclusion, the data show that T suppressor/cytotoxic cells have a counter-regulatory role in wound healing, whereas the T cell subset responsible for up-regulating wound healing remains to be identified.

A subcutaneous implant for wound healing studies in humans

A method for studying wound healing in humans is described. The technique is based on the production of a standard subcutaneous injury during implantation of a retrievable sponge. The injury is produced by introducing a small device (PVA implant) which consists of a 5.7-cm piece of perforated silicone tubing containing two pieces of polyvinyl alcohol sponge. The sponge provides a site for attracting inflammatory cells with subsequent fibroblast infiltration. The sterile PVA implant is inserted subcutaneously in the upper arm by means of a 12-gauge needle and remains there for 14 days. Upon removal, one sponge has hydroxyproline deposition quantitated using a high-performance liquid chromatography technique. The other sponge may be processed for light and electron microscopy or specialty staining. Other collagen determinations such as percentage neutral salt-soluble collagen are also possible. Using the PVA implant has made it possible to follow the kinetics of collagen deposition in the rat. There was a marked increase in collagen accumulation from Day 2 (0.89 nmole/mg sponge) to Day 14 (18 nmole/mg sponge) in the rat. Collagen deposition was also measured in human control subjects (5.07 nmole/mg sponge, n = 12) and compared to trauma patients (2.04 nmole/mg sponge, n = 5). Histologic staining showing fibroblast infiltration and collagen deposition correlated well with the biochemical findings. This implant, coupled with recent HPLC technology, provides a safe, acceptable technique to study human would healing parameters and overcomes many of the limitations of previous methods.

Attenuation of pulmonary fibrosis in mice by aminophylline

Cyclic nucleotides have been shown in vitro to regulate fibroblast proliferation and/or collagen production. We have reported previously that propranolol, which decreases the cAMP/cGMP ratio. potentiates the amount of fibrosis produced in a damaged lung. The purpose of this study was to determine if elevations in the cAMP/cGMP ratio may attenuate collagen production by fibroblasts following lung damage. Lung injury was induced in mice by either butylated hydroxytoluene (BHT) (350 or 400mg/kg intraperitoneally) or bleomycin (4 units/kg intratracheally). The mice were treated with a phosphodiesterase inhibitor, aminophylline (20 /kg twice daily), prior to induction of lung injury and for the duration of the study. Cyclic nucleotide changes in the lung were also determined during lung injury, with and without aminophylline. The administration of aminophylline, which increased the cAMP/cGMP ratio, resulted in attenuation of the increase in total lung collagen normally seen after injury, while having no effect on collagen levels in the undamaged lung. The results are compatible with the hypothesis that elevation of whole lung cAMP/cGMP ratio early in the damage and repair process correlates with decreased hydroxyproline deposition.

Cell Surfaces in Plant-Microorganism Interactions

Ethylene production and cell wall hydroxyproline-rich glycoprotein (HRGP) biosynthesis are greatly enhanced in melon (Cucumin melo cv. Cantaloup charentais) seedlings infected with Colletotrichum lagenarium. Short-term experiments performed in the presence of specific inhibitors of the ethylene pathway from methionine, namely l-canaline and amino-ethoxyvinylglycine, indicate that under non-toxic conditions, both ethylene and [(14)C]hydroxyproline deposition in the cell wall of infected tissues are significantly lowered. On the contrary, treatment of healthy tissues with 1-aminocyclopropane 1-carboxylic acid, a natural precursor of ethylene, stimulates both the production of the hormone and the incorporation of [(14)C]hydroxyproline into cell wall proteins.The data provide the first evidence of the in vivo effect of ethylene on the cell wall hydroxyproline-rich glycoprotein biosynthesis in plants.

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix.

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti-60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.