Human blood monocytes interact with type I collagen through alpha x beta 2 integrin (CD11c-CD18, gp150-95).

Human blood monocytes are attracted into connective tissues during early steps of inflammation and wound healing, and locally interact with resident cells and extracellular matrix proteins. We studied the effects of type I collagen on monocyte adhesion and superoxide anion production, using human monocytes elutriated from peripheral blood and type I collagen obtained from rat tail tendon. Both acid-soluble and pepsin-digested type I collagens promoted the adhesion of monocytes, whereas only acid-soluble collagen with intact telopeptides induced the production of superoxide. Adhesion and activation of monocytes on acid-soluble type I collagen depended on the presence of divalent cations. mAbs directed against integrin subunits CD11c and CD18 specifically inhibited adhesion and activation of monocytes on type I collagen. Protein membrane extracts obtained from monocytes were submitted to affinity chromatography on collagen I-Sepharose 4B, and analyzed by Western blotting using specific anti-integrin subunit Abs. In the case of both acid-soluble and pepsin-digested collagens, two bands were revealed with mAbs against CD11c and CD18 integrin subunits. Our results demonstrate that monocytes interact with type I collagen through CD11c-CD18 (alpha x beta 2) integrins, which promote their adhesion and activation. For monocyte activation, specific domains of the type I collagen telopeptides are necessary. Interactions between monocytes and collagen are most likely involved in the cascade of events that characterize the initial phases of inflammation.

Regulation of matrix metalloproteinase-2 (gelatinase A, MMP-2), membrane-type matrix metalloproteinase-1 (MT1-MMP) and tissue inhibitor of metalloproteinases-2 (TIMP-2) expression by elastin-derived peptides in human HT-1080 fibrosarcoma cell line.

Soluble kappa-elastin peptides were shown to stimulate the expression of MMP-2 (but not MMP-9) by human fibrosarcoma HT-1080 cells, both at the protein and mRNA levels; maximal effect being observed at a concentration of 25 microg/ml of kappa-elastin. The stimulatory effect could be reproduced using Val-Gly-Val-Ala-Pro-Gly (VGVAPG) peptide, an elastin-derived hydrophobic hexapeptide which represented the elastin receptor binding sequence of tropoelastin. Furthermore, treatment of cells with lactose (30 mM), which dissociated 67-kDa elastin binding protein (EBP) from cell surfaces, completely abolished this effect, suggesting that the elastin receptor could mediate such a response. Using a specific monoclonal antibody, 67-kDa EBP was detected in HT-1080 membrane preparations by Western immunoblotting. Following treatment with 25 microg/ml kappa-elastin or 200 microg/ml VGVAPG, increased levels of the active 62-kDa form of MMP-2 were found in HT-1080 cell extracts. Stimulation of MT1-MMP mRNA expression by treatment with elastin-derived peptides (EDPs) was shown by competitive polymerase chain reaction (PCR). A reverse zymography analysis revealed that EDPs also stimulated TIMP-2 (but not TIMP-1) production by HT-1080 cells. Competitive PCR confirmed increased TIMP-2 mRNA expression by such treatment. These results suggest that occupancy of the 67-kDa elastin receptor by elastin-derived peptides enhanced both expression and activation of proMMP-2 and consequently, could promote the invasive/metastatic ability of tumor cells expressing this receptor.

Changes in serum lipoprotein(a) levels in children with corticosensitive nephrotic syndrome.

Variations in lipoprotein(a) [Lp(a)] levels were evaluated during the course of the nephrotic syndrome in 20 children (17 males, 3 females, aged 2-16 years), to evaluate the use of this parameter in the prognosis and monitoring of the disease. One patient was in relapse, 12 in remission, and 7 alternated between remission and relapse. Results were compared with those obtained in a control population of 100 age-matched children. Lp(a) was measured by a previously described immunonephelometric technique. Serum Lp(a) levels were increased in children with relapsing nephrotic syndrome compared with controls (median value of 419 mg/l vs. 86 mg/l). The median Lp(a) level in patients with nephrotic syndrome in remission was different from controls (270 mg/l under steroid therapy and 163 mg/l without steroid therapy), but remained within the reference range. Of the patients in relapse, 66% had Lp(a) levels above the generally accepted limit for cardiovascular risk of 300 mg/l, compared with 16% of controls, 44% of patients with nephrotic syndrome in remission under steroid therapy, and 18% of patients with nephrotic syndrome in remission without steroid therapy. In 2 patients, Lp(a) was measured repeatedly and was significantly higher during the acute phase of the disease (up to sixfold basal level). Changes in Lp(a) levels correlated with cholesterol levels, but the kinetics and the extent of variations were different. These data suggest that measurement of Lp(a) is useful for monitoring the nephrotic syndrome in children, particularly for detecting complications.

Modulation of protein synthesis by extracellular matrix: potential involvement of two nucleolar proteins, nucleolin and fibrillarin.

Fibroblasts cultivated in a collagen matrix exhibit a large decrease in the synthesis of most proteins, depending on transcriptional and posttranscriptional controls. We have previously shown that ribosomal RNA content and half-life were decreased in collagen lattice cultures. Here, we cultivated human dermal fibroblasts in monolayers and in lattices and studied by competitive RT-PCR analysis the expression of the nucleolar proteins nucleolin and fibrillarin, two key factors in ribosome processing and association. Nucleolin expression was found increased, and fibrillarin expression decreased, in collagen-lattice vs monolayer-cultured fibroblasts, with some variability according to the strains (+25 to +250% and -40 to -60%, respectively). These data suggest that a possible trouble of the association between neosynthesized rRNA and nucleolar proteins is, at least partly, responsible for the inhibition of protein synthesis induced by the extracellular matrix.

The binding of type I collagen to lymphocyte function-associated antigen (LFA) 1 integrin triggers the respiratory burst of human polymorphonuclear neutrophils. Role of calcium signaling and tyrosine phosphorylation of LFA 1.

Monoclonal antibodies to the alpha L beta 2 integrin inhibit the binding of type I collagen to PMN (polymorphonuclear neutrophil leukocytes) as well as the subsequent stimulation of superoxide production and enzyme secretion-elicited by this collagen. Pepsinized collagen still binds PMN but no longer stimulates them. The I domain of the alpha chain of the integrin is involved in the binding. Two sequences of the alpha 1(I) polypeptide chain of collagen participate in the process. Experiments of competitive inhibition by synthetic peptides showed that the sequence RGD (915-917) is used for binding to the cells and DGGRYY (1034-1039) serves to stimulate PMN. Experiments of radioactive labeling of the cells and affinity chromatography on Sepharose-collagen confirmed the presence in PMN extracts of two proteins, 95 and 185 kDa, respectively, corresponding to the molecular weights of the beta 2 and alpha L chains of the integrin and recognized by their specific monoclonal antibodies. The transduction pathways depending on the alpha L beta 2 integrin do not involve a G protein (ruled out by the use of cholera and pertussis toxins), whereas the cytoskeleton was found to participate in the process, as evidenced by inhibition by cytochalasin B. After collagen stimulation, cytoplasmic inositol trisphosphate and calcium ion increased sharply for less than 2 min. The use of the inhibitors staurosporine and calphostin C demonstrated that protein kinase C was involved. Evaluation of the activity of this enzyme showed that, upon stimulation of PMN with collagen I, it was translocated to plasma membrane. Acrylamide gel electrophoresis of the protein bands corresponding to the integrin alpha L beta 2, followed by immunoblotting using monoclonal antibodies to phosphotyrosine, permitted us to demonstrate that, prior to stimulation by type I collagen, there was no phosphorylation, whereas after stimulation, both alpha L and beta 2 chains were stained by anti-phosphotyrosine antibodies. The adhesion of PMN to pepsinized type I collagen triggered tyrosine phosphorylation of the beta 2 chain of the integrin, without stimulating O2-. production by these cells, whereas their stimulation by complete type I collagen induced the tyrosine phosphorylation of both alpha L and beta 2 subunits. The tyrosine phosphorylation of both integrin subunits during transduction of stimuli is a heretofore undescribed phenomenon that may correspond to a new system of transmembrane communication.

Stimulation of sulphated glycosaminoglycan and decorin production in adult dermal fibroblasts by recombinant human interleukin-4.

Interleukin-4 (IL-4) is a pleiotropic cytokine expressed by inflammatory cells. Previous work from our laboratory has shown that it stimulates collagen synthesis in fibroblasts. Here we report the effects of recombinant human IL-4 on glycosaminoglycan (GAG) and proteoglycan synthesis in normal dermal fibroblasts from adult donors. IL-4 (10 and 100 units/ml) induced a dose-dependent increase of [3H]glucosamine and [35S]sulphate incorporation into total GAGs. The analysis of the different GAG fractions indicated the enhanced synthesis of dermatan/chondroitin sulphates. IL-4 had no effect on hyaluronan synthesis. The increase of sulphated GAG synthesis was correlated with an increase of proteoglycans in the culture medium. Decorin was identified as the major chondroitin/dermatan sulphate-containing proteoglycan in the culture medium of fibroblasts. Its synthesis was strongly stimulated by IL-4. Both the core-protein synthesis and mRNA expression were enhanced, indicating that the cytokine acted, at least in part, at the pre-translational level. These results indicate that IL-4 is able to modulate not only collagen, but also proteoglycan, production by human fibroblasts. Their implications in physiopathological processes such as wound healing or fibrosis is suggested.

Protein synthesis in collagen lattice-cultured fibroblasts is controlled at the ribosomal level.

Fibroblasts cultivated in three-dimensional lattices exhibit a large decrease of protein synthesis, mainly through transcriptional control. However, no previous work was devoted to a potential ribosomal regulation. We evaluated ribosomal ribonucleic acid (RNA) in monolayer- and collagen lattice-cultured fibroblasts. After one week of culture, total RNA was 60% lower in lattice-cultured fibroblasts than in monolayer-cultured cells. The decrease was identical for 18 S and 28 S rRNA subfractions. The half-life of RNA was much shorter in collagen lattice-cultured fibroblasts than in monolayers. These results suggest that protein synthesis in lattice-cultured fibroblasts is partly regulated at the ribosomal level.

Adhesion of human neutrophils to and activation by type-I collagen involving a beta 2 integrin.

We previously demonstrated that the alpha 1(I) polypeptide chain of collagen can bind and activate polymorphonuclear neutrophils (PMN). In the present experiments, performed in culture grade 96-well plastic plates coated with collagen, fibronectin, or other proteins, adhesion was assessed by staining the adhering cells after 30 min with crystal violet and measuring absorbance at 560 nm, and activation of PMNs was assessed by measuring the amount of O2-formed. Adhesion occurred at 17 and 37 degrees C but activation at 37 degrees C only. Monoclonal antibody anti-CD 18 inhibited adhesion, showing that the receptor of collagen I on PMNs is a beta 2 integrin. On the other hand, adhesion of PMNs to fibronectin was inhibited by monoclonal antibodies to CD18 and to CD11b.

Activation of human neutrophils by type I collagen. Requirement of two different sequences.

Contact between type I collagen purified from several species and human polymorphonuclear neutrophils (PMNs) triggers the production of O2.- by these cells. The activity of collagen is located in the alpha 1(I)-CB6 cyanogen bromide-cleaved (CB)-peptide, which is the C-terminal CB-peptide of the alpha 1(I) chain. Experiments based on the competitive inhibition of O2.- production by simultaneous incubation of PMNs with type I collagen and synthetic peptides identical to the conserved sequences of this collagen demonstrated that the binding of collagen to PMNs and the subsequent activation of these cells depend on the simultaneous presence of two sequences: Arg-Gly-Asp [residues 915, 916 and 917 of the complete alpha 1(I) chain, located in the helical part] Asp-Gly-Gly-Arg-Tyr-Tyr (residues 1034-1039, located in the C-terminal non-helical telopeptide).

Human type VI collagen: isolation and characterization of alpha 1 and alpha 2 chains by two-dimensional preparative gel electrophoresis.

Two 140 kDa collagenous glycoproteins were isolated from 5 M guanidinium chloride extracts of human uterine leiomyoma by two-dimensional preparative gel electrophoresis. The glycoproteins represented the major concanavalin A binding fraction of the extract and were also present in adult human skin. On two-dimensional gel electrophoresis the glycoproteins appeared as elongated spots, indicating variations of their isoelectric points from 5 to 6. These glycoproteins were disulfide-bonded components of high molecular mass protein and, after reduction, became sensitive to collagenase treatment that generated peptides corresponding in size to those of the noncollagenous domains of type VI collagen. Antisera raised against these purified glycoproteins reacted with either pepsin-derived alpha 1(VI) or pepsin-derived alpha 2(VI) chains but not with alpha 3(VI) chain of human type VI collagen. Reciprocally, these glycoproteins reacted with monoclonal antibodies against type VI collagen. These results indicate that the glycoproteins represent the integral alpha 1 and alpha 2 chains of type VI collagen. The globular domains of alpha 1(VI) and alpha 2(VI) chains remaining after collagenase treatment appeared on two-dimensional gel electrophoresis as elongated spots, suggesting that the noncollagenous portions determine the well known microheterogeneity of the molecule. The differences in isoelectric points between and within alpha chains may facilitate the formation of microfibrillar network.

Nonisotopic evaluation of collagen in fibroblasts cultures.

A method for the evaluation of collagen concentrations in the medium of fibroblasts in culture was developed. Collagen was precipitated with other proteins by addition of ethanol and hydrolyzed by 6 M HCl. The primary amino acids of the hydrolyzate were reacted with o-phthalaldehyde (OPA) and secondary amino acids (Pro, Hyp) were derivatized with 9-fluorenylmethyl-chloroformate (FMOC-Cl). The mixture was separated by isocratic HPLC on a reverse-phase column. FMOC-derivatives were detected by fluorometry, whereas OPA-derivatives were not. This method is suitable for the monitoring of collagen metabolism in fibroblast cultures exposed to various effectors.

A parallel between two techniques of extraction of connective tissue macromolecules.

A method of extraction of the collagen and noncollagen proteins from deep dermis of young adult rabbits using a 0.1 M tartaric acid solution was set up. The tartaric acid extraction, together with the preliminary neutral salt extraction, solubilized 95% of the total collagen and 98% of the noncollagen proteins, far more than the 6 M guanidinium Cl solution used for comparison. Elastin was not extracted. Studies on the fibrillation of the extracted collagen in neutral solution at 25 degrees C or on the results of pepsin digestion in acidic solution at +4 degrees C showed that the tartaric acid-extracted collagen was in a nondenatured form, whereas that extracted by guanidinium Cl was largely denatured. Polyacrylamide gel electrophoresis (PAGE) indicated that most of the collagen was of type I and that many noncollagen proteins were present, mostly in the molecular weight range of 40 kDa. Bidimensional PAGE gave a reproducible pattern of these noncollagen proteins, showing that several additional proteins were present in tartaric acid extracts and not in guanidinium chloride extracts.

Collagen degradation by superoxide anion in pulse and gamma radiolysis.

Delipidated collagen fibrils reconstituted from acid-soluble calf skin collagen, suspended in 50 mM phosphate buffer, pH 7.4, containing 100 mM sodium formate, were submitted to pulse radiolysis in Febetron devices or to gamma radiolysis in a 60Co irradiator. A collagen degradation process was found. The kinetics of this degradation was followed by evaluation of the amount of 4-hydroxyproline present in the small peptides liberated during the irradiation period. The yield of 4-hydroxyproline small peptides was low (0.1 mol/100 eV for an initial collagen concentration 3.2 microM). It increased linearly with the dose of irradiation and the concentration of collagen in suspension. The kinetic competition between O2-. dismutation and O2-. reaction with collagen was studied by pulse radiolysis at several concentrations of collagen. A value of the kinetic constant of k(O2-. + collagen) = 4.8 . 10(6) mol-1.l.s-1 was determined.

Collagen activates superoxide anion production by human polymorphonuclear neutrophils.

Human polymorphonuclear neutrophils (PMNs), purified on Ficoll-Hypaque cushions, were incubated for 5 min with calf skin acid-soluble collagen and the released superoxide anions (O2-) measured spectrophotometrically by reduction of ferricytochrome c or by chemiluminescence analysis. This collagen stimulated the release of O2- unless it had been treated with pepsin. The stimulatory activity remained in denatured collagen, was contained only in the alpha 1(I) chain and was present in the alpha 1(I)-CB 6 (CNBr-cleaved) peptide, which is C-terminal. The activity was linearly dependent on the collagen concentration up to about 200 micrograms/ml. In addition, this collagen induced a release of beta-glucuronidase and N-acetyl-beta-glucosaminidase from PMNs.

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures.

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.

Effects of isaxonine phosphate and analogs on fibroblast metabolism in culture.

Human skin fibroblasts in confluent cultures were incubated for 24 h in the presence of isaxonine phosphate (Nerfactor) and several related factors. The incorporation of 14C-proline into secreted proteins and the release of collagen into the medium were inhibited. When the cells were incubated for an additional period of 24 h after thorough washing, protein and collagen syntheses were found to be identical to those of controls, demonstrating that the inhibition of protein synthesis was independent of any toxic effect. When cells were incubated in the presence of both isaxonine and colchicine, the secretion of collagen was more inhibited than by colchicine alone, and proteins accumulated in the cells.

A study of collagen metabolism in cell cultures by fluorometric determination of proline and hydroxyproline.

A technique of derivatization of proline (Pro) and 4-hydroxyproline (Hyp) by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole permitted the measurement of Pro and Hyp radioactivities, concentrations, and specific activities in the main fractions separated from cultures of fibroblast cells (extracellular collagen and non-collagen proteins, intracellular free Pro and Hyp, Pro- and Hyp-containing peptides, procollagen, and non-collagen proteins). The evaluation of collagen in the medium was obtained from as few as 10(4) cells. The method might advantageously replace [14C] Pro or [3H] Pro incorporation studies. It permits measurement of the size of the Pro pool and the amount of peptides formed by intracellular catabolism of collagen. It demonstrates that the time necessary for a full equilibration of intracellular Pro and intracellular collagen is longer than is generally believed. It avoids the uncertainties of protein labelling, which may vary with uncontrolled variations of the intracellular Pro specific activity.

Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis.

It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. [14C]Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of [14C]proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.

Modifications of collagens in the course of inflammatory tracheal stenoses.

A comparison was made between the biochemical and histological properties of collagens contained in samples of normal tracheas obtained at autopsy or of stenosed tracheas obtained during surgery. The amounts of total collagen solubilized by pepsin was increased seven times in the pathological samples, and the proportion of cartilage type II collagen decreased by about one half, being replaced by type I collagen, whose ratio was increased five times. Microscopic studies confirmed that cartilage underwent a degenerative process and was progressively infiltrated by fibrils of interstitial collagen.