Semiquantitative classification of ductus venosus blood flow patterns.

OBJECTIVES: To identify the range of waveform abnormalities in the ductus venosus (DV) characterized by their timing in the cardiac cycle and to evaluate if they can be categorized into distinct patterns. METHODS: DV velocity ratios were calculated from peak velocities during ventricular systole (S), end-systolic ventricular relaxation (v), early diastole (D) and atrial systole (a) (S/v, S/D, v/D, S/a, v/a and D/a ratios). The ratios were converted to their Z-scores and elevation > 2 SD was assigned as abnormal. Combinations of ratio abnormalities were grouped to define distinct waveform patterns and their distribution was related to the clinical presentation. RESULTS: Five-hundred and forty-two abnormal DV waveforms fell into three principal patterns. In Pattern 1 only the a-wave-related ratios were abnormal (180, 33.2%), in Pattern 2 the v/D ratio was abnormal (143, 26.3%) and in Pattern 3 combinations of a-wave abnormalities in the presence of a normal v/D ratio were normal (94, 17.3%). CONCLUSIONS: Interpretation of venous waveform patterns is complex because the multiphasic waveforms reflect events in the cardiac cycle that may be differentially affected by clinical pathology. We sought to present a classification for the DV flow profile that characterizes abnormal flow confined to atrial systole and occurs during ventricular relaxation or during holodiastole. Further research is warranted to determine the significance of these patterns in specific fetal conditions.

Inhibition of collagen synthesis by mononuclear cell supernates.

Mononuclear cell infiltration and alteration in the connective tissues are prominent features of the inflammatory response in a number of diseases. To determine whether mononuclear cell products can modulate collagen synthesis, human peripheral mononuclear cells from normal donors were isolated by Ficoll-Hypaque gradient centrifugation and then incubated for 48 h with or without phytohemagglutinin. Confluent cultures of normal, human skin fibroblasts were incubated with [14C]proline and various amounts of dialyzed supernates from the mononuclear cell cultures. Labeled, newly synthesized collagen was estimated by [14C]hydroxyproline analysis, collagenase digestion, and chromatography on Agarose A-5m in sodium dodecyl sulfate. The total incorporation of [14C]proline was not significantly affected by addition of the mononuclear cell supernates, but as much as 90% decrease in the synthesis by the fibroblasts of labeled collagen was found relative to controls. Supernates from the phytohemagglutinin-stimulated cultures were more active than those from nonstimulated cells. These results suggest that mononuclear cells can synthesize a factor(s) which can selectively inhibit collagen synthesis.

Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development.

During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located approximately 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.

Intracellular pool of unhydroxylated polypeptide precursors of collagen.

The hydroxyproline and hydroxylysine in collagen are synthesized by an apparently unique pathway in which proline and lysine are hydroxylated after they are incorporated into a large polypeptide precursor of collagen called protocollagen. When the hydroxylation of protocollagen in isolated tissues is intermittently interrupted, hydroxylation can occur after complete polypeptides are released from ribosomal complexes. Cartilage from chick embryos was incubated with the iron chelator alpha,alpha'-dipyridyl for 2 hours to inhibit protocollagen hydroxylase, and then the inhibition was reversed by transferring the tissues to medium containing ferrous iron and no alpha,alpha'-dipyridyl. "Pulse labeling" of the tissues during these two periods indicated that both the accumulated protocollagen and the polypeptides synthesized after reversal of the inhibition were hydroxylated at the same rate. Even when no measures are taken to inhibit the hydroxylation of protocollagen, most of the hydroxyproline in collagen is probably synthesized after complete protocollagen polypeptides are released from ribosomes.

Selective inhibition of human diploid fibroblast collagen synthesis by interferons.

The effects of alpha- and gamma-interferons (IFNs) on collagen production by confluent human diploid fibroblasts in culture were examined. It was found that partially purified alpha-IFNs and affinity purified gamma-IFNs caused greater than 50% inhibition of collagen synthesis by these cells independently of their effect on cell proliferation. Recombinant alpha-IFNs showed a similar effect (38.8% inhibition), indicating that collagen synthesis inhibition was a constitutive property of IFNs. Collagen synthesis inhibition by IFNs was concentration dependent. Gel filtration chromatography of the newly synthesized proteins from the media of fibroblasts incubated with partially purified alpha-IFNs demonstrated a selective depression of molecules eluting in the region of procollagen. No detectable increase in collagen degradation products or underhydroxylation of procollagen was observed. Short-term kinetic studies further demonstrated that the major effect of IFNs was due to a net decrease in fibroblast collagen production rather than to impairment of secretion or increased extracellular degradation of the newly synthesized molecules. These results indicate that alpha- and gamma-IFNs are potent inhibitors of human fibroblast collagen production and suggest that they may play an important role in the regulation of normal and pathologic fibrogenesis.

Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts.

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides -804 to -675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease.

Immunolocalization of elastase in human emphysematous lungs.

The current working hypothesis concerning the pathogenesis of human pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes into the interstitium. These enzymes, in particular elastase, can bind to and degrade interstitial elastin. This report describes an immunohistochemical, ultrastructural technique that utilizes polyclonal antibodies to localize neutrophil elastase in human lungs. Using both the immunoperoxidase and the immunogold methods on thin, embedded sections of surgically resected human emphysematous lung tissue, elastase was localized in neutrophils in the lung interstitium and extracellularly in association with interstitial elastic fibers in human lungs that showed local emphysema of varying severity. Quantitative morphometric data were obtained from the lungs of eight patients undergoing lobectomy for removal of pulmonary carcinomas. Patients had preoperative forced expiratory volume (FEV1)% levels ranging from 55 to 77. There was a correlation between a quantitative measure of the local distribution of neutrophil elastase in contact with alveolar interstitial elastin and the local presence of emphysematous change as determined by mean linear intercept of the various histologic sections. These data support the validity of the "protease-protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysema.

Developmental expression of latent transforming growth factor beta binding protein 2 and its requirement early in mouse development.

Latent transforming growth factor beta (TGF-beta) binding protein 2 (LTBP-2) is an integral component of elastin-containing microfibrils. We studied the expression of LTBP-2 in the developing mouse and rat by in situ hybridization, using tropoelastin expression as a marker of tissues participating in elastic fiber formation. LTBP-2 colocalized with tropoelastin within the perichondrium, lung, dermis, large arterial vessels, epicardium, pericardium, and heart valves at various stages of rodent embryonic development. Both LTBP-2 and tropoelastin expression were seen throughout the lung parenchyma and within the cortex of the spleen in the young adult mouse. In the testes, LTBP-2 expression was seen within lumenal cells of the epididymis in the absence of tropoelastin. Collectively, these results imply that LTBP-2 plays a structural role within elastic fibers in most cases. To investigate its importance in development, mice with a targeted disruption of the Ltbp2 gene were generated. Ltbp2(-/-) mice die between embryonic day 3.5 (E3.5) and E6.5. LTBP-2 expression was not detected by in situ hybridization in E6.5 embryos but was detected in E3.5 blastocysts by reverse transcription-PCR. These results are not consistent with the phenotypes of TGF-beta knockout mice or mice with knockouts of other elastic fiber proteins, implying that LTBP-2 performs a yet undiscovered function in early development, perhaps in implantation.

Control of elastin synthesis.

mRNA was isolated from the thoracic aortas of 16-day chick embryos and used to synthesize blunt-ended heteroduplex molecules consisting of one strand of mRNA and one of cDNA using AMV reverse transcriptase and S1 nuclease. The duplexes were tailed with dCTP and hybridized to the plasmid pBR322 which had been restricted with Pst I and tailed with dGTP. Recombinant plasmids were used to transform E. coli C600 and colonies containing elastin cDNA were selected by in situ hybridization with 32P labeled elastin mRNA and by hybrid selected translation using the nuclease-treated reticulocyte lysate system. mRNA recovered from hybridization to DNA of one clone, pWB1, markedly stimulated incorporation of [3H]valine into a protein which was immunoprecipitable with elastin-specific antibody and had a molecular weight of 72,000, characteristic of tropoelastin. The 230 bp insert of pWB1 was sequenced by the technique of Maxam and Gilbert and found to be derived from a nontranslated region of the 3' end of the mRNA. Nick-translated pWB1 was used to identify and to estimate the relative amounts of elastin mRNA in the developing chick embryo aorta by blot hybridization. A single mRNA species of 3.5 kb hybridized to the pWB1 probe and this species increased greatly in amount between day 7 and day 14. This increase was paralleled by an increase in translatable elastin mRNA and by the rate of elastin synthesis of aortas from various age embryos incubated in vivo. The injection of 150 microgram of hydrocortisone 21-phosphate into 8-day eggs produced a significant increase in both the relative rate of tropoelastin synthesized by the isolated aortas and the relative amount of elastin mRNA. These results suggest that the observed changes in elastin synthesis during development and after hydrocortisone administration are governed by the elastin mRNA content of the aortas.

Regulation of elastin gene expression: evidence for functional promoter activity in the 5'-flanking region of the human gene.

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.

UVB irradiation alters cellular responses to cytokines: role in extracellular matrix gene expression.

Solar radiation causes cutaneous photodamage characterized by alterations in the quantity and structure of the extracellular matrix. We determined the direct and cytokine-mediated effects of UV irradiation on mRNA levels for two matrix elements, tropoelastin and fibrillin 1. (i) Comparison of normal versus end-stage photodamaged skin failed to reveal differences in these message levels. (ii) Acutely irradiated skin showed suppression of both tropoelastin and fibrillin mRNAs. (iii) UVB irradiation (50 mJ) of cultured skin fibroblasts suppressed fibrillin mRNA by 50%, consistent with a direct effect of radiation. Addition to the cultured fibroblasts of several cytokines upregulated by UVB showed that IL-1alpha had no effect on fibrillin mRNA in unirradiated cells, but in irradiated cells, this cytokine enhanced the suppression of fibrillin mRNA. There were no changes in the message stability, suggesting altered gene transcription. In contrast, UVB had no effect on tropoelastin mRNA levels in cultured fibroblasts, indicating the absence of a direct effect of radiation. IL-1alpha stimulated tropoelastin mRNA 2.8-fold in unirradiated cells, and this stimulation was entirely blocked by UVB. Overall, our results indicate acute suppression of matrix genes by UVB in vivo. The suppression of fibrillin message was a direct effect of UVB on fibroblasts and was augmented by IL-1alpha. Suppression of tropoelastin message by UVB occurred in vitro only in IL-1alpha-stimulated cells. We conclude that UVB substantially alters the pattern of cellular response to cytokines. The interplay between UVB and cytokines is essential to explain the acute responses of matrix genes to UVB in vivo.

Cloning of full-length elastin cDNAs from a human skin fibroblast recombinant cDNA library: further elucidation of alternative splicing utilizing exon-specific oligonucleotides.

A human cDNA library was constructed utilizing RNA isolated from cultured skin fibroblasts. Recombinant clones containing elastin sequences were identified by plaque hybridizations with previously characterized human placental elastin cDNAs. Seven positive recombinant clones with inserts of approximately 3.2-2.2 kb were isolated. Characterization of the clones by restriction endonuclease analysis and dot-blot hybridizations with exon-specific synthetic oligonucleotides demonstrated considerable variability in the primary nucleotide sequence. Dideoxy nucleotide sequencing confirmed this finding. The variability is most likely a result of alternative splicing of exons from the primary elastin transcripts. The two largest clones contained approximately 1 kb of 3' untranslated sequence and approximately 2.2 kb of translated sequence encoding 730 amino acids. Six amino acids, encoded by exon 12A, have not been previously noted in human elastin cDNAs. In addition, these human skin fibroblast clones contained a 49 bp 5' untranslated sequence. These results demonstrate that there is considerable variability in the processed nucleotide sequence of the elastin mRNAs. These transcripts may code for isoforms of tropoelastin with different biologic properties.

Analysis of the human gene encoding latent transforming growth factor-beta-binding protein-2.

Transforming growth factor (TGF)-beta is secreted as an inactive complex, which frequently contains a large molecular weight binding protein designated latent TGF-beta-binding protein (LTBP). Recently, the LTBPs have been shown to be a gene family that contains three known members and exhibits a multidomain structure containing cysteine-rich motifs that are also found in the fibrillin gene family. The present work seeks to characterize the gene encoding LTBP-2 and to compare its features to that of the other LTBPs and to the fibrillins. Human fibroblast libraries were used to isolate cDNA encoding LTBP-2 which was then used to identify LTBP-2 transcripts and to isolate the corresponding LTBP-2 gene. The cloned cDNA encodes a 195 kDa protein containing 20 epidermal growth factor (EGF)-like repeats, three repeats containing eight cysteines, and one segment that appears to be a hybrid of the two. Single exons encode EGF repeats while the eight-cysteine repeats are encoded in two exons. Northern analysis identified two transcripts of 7.5 and 9.0 kb, with the presently analyzed cDNA probably corresponding to the 7.5 transcript. Phylogenetic sequence comparisons demonstrated that LTBP-3 is more similar to LTBP-1 than LTBP-2, while LTBP-2 shows the most similarity to the fibrillins. These analyses suggest that LTBP-1 diverged from LTBP-3, and that LTBP-2 diverged from LTBP-1. Within the fibrillin family, fibrillin-1 is nearest to the LTBPs. While the domain structure of LTBP-2 is similar to that of the other LTBPs, LTBP-2 possesses unique regions that make it the largest member of the LTBP family. LTBP-2 may have dual functions as a member of the TGF-beta latent complex and as a structural component of microfibrils.

Rat somatotroph insulin-like growth factor-II (IGF-II) signaling: role of the IGF-I receptor.

The anterior pituitary contains insulin-like growth factor-II (IGF-II) and expresses abundant IGF-II-binding sites, but the IGF-II signaling pathway in the pituitary has not been defined. We, therefore, tested the effects of recombinant human IGF-II on pituitary function by assessing the GH responsiveness of the primary rat somatotroph to IGF-II. IGF-II (3.3 nM) suppressed GH secretion by 50%, similar to the effect elicited by equimolar doses of IGF-I. In contrast, a low concentration of IGF-II (0.2 nM) did not attenuate GH secretion, while a similar IGF-I dose was sufficient to produce 50% inhibition of basal GH secretion. Fifty percent competition for [125I]IGF-I binding by IGF-I and IGF-II in GC rat pituitary cells demonstrated a 14-fold lesser affinity of the IGF-II ligand for the IGF-I receptor compared to IGF-I; therefore, the binding affinity of IGF-II for the IGF-I receptor correlates with the concentration of IGF-II required for 50% GH inhibition. Transfected GH-secreting cell lines derived from GC cells overexpressing intact human IGF-I receptors exhibited enhanced responsiveness to IGF-II. In contrast, cells transfected with a truncated IGF-I receptor cDNA lacking the cytoplasmic receptor beta-subunit (952STOP) failed to transduce the IGF-II signal, indicating that functional IGF-I receptors are required for IGF-II signaling. In addition, a mutant IGF ligand, [Leu27]IGF-II, which selectively exhibits high affinity for the type II receptor, but only minimal binding to the IGF-I receptor, did not attenuate GH secretion. However, the analog [Arg54,Arg55]IGF-II, which exhibits high affinity to the IGF-I receptor, but no binding to the type II receptor, appropriately suppressed GH secretion. This unique model of somatotroph signaling provides evidence for IGF-II regulation of polypeptide hormone secretion mediated by the IGF-I receptor.

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.

Production of collagen synthesis inhibitory lymphokine by human leukemic T-lymphocyte cell lines.

It has been previously shown that normal human peripheral blood mononuclear cells produce a soluble factor(s) that causes selective inhibition of collagen production by cultured human diploid fibroblasts, and that stimulation of the mononuclear cells by appropriate mitogens greatly enhances its production. Because of the difficulties inherent in obtaining sufficient peripheral blood lymphocytes to purify the collagen synthesis inhibitory factor (CSIF) further and to study in detail its mechanisms of action, we have examined CSIF production by several leukemic human T- and B-lymphocyte cell lines. From the 10 lines tested (4 T- and 6 B-lymphocyte cell lines), we found 2 T-lymphocyte cell lines which produced CSIF constitutively. When the effects of various lymphocyte mitogens on CSIF production by these cell lines were examined, it was found that only phorbol myristic acid was stimulatory. Further studies demonstrated that optimal CSIF production by the phorbol myristic acid-stimulated leukemic T-lymphocytes could be achieved in a chemically defined, serum-free medium. The CSIF in the supernatants from such cultures was further purified by ammonium sulfate precipitation and gel filtration chromatography and it was shown that it had similar properties to those of CSIF produced by normal human peripheral blood lymphocytes. These findings indicate that certain leukemic T-lymphocyte cell lines are capable of CSIF production in continuous cultures. These results will permit the large scale production of CSIF and a better understanding of its mechanisms of action.

Structure of the elastin gene and alternative splicing of elastin mRNA: implications for human disease.

The protein elastin is largely responsible for the elastic properties of vertebrate lungs, large blood vessels, and skin. The structure of the human, bovine, and chick elastin gene and protein monomer, tropoelastin, has recently been elucidated by using techniques of molecular biology. Extensive homology of amino acid sequence exists among the mammalian species and there is in addition strong conservation of nucleotide sequences in the 3' untranslated region of the gene. The translated exons are small and embedded in large expanses of introns. Sequences coding for the hydrophobic regions, responsible for the elastic properties of the molecule, and the alanine-lysine rich regions, responsible for crosslink formation between molecules, reside in separate exons and alternate for the most part in the elastin gene. S1 analyses and sequence analysis of cDNA and genomic clones have indicated that there is substantial alternative splicing of the primary elastin transcript. Variations in the structure of mRNAs resulting from alternative splicing could explain the existence of the multiple forms of tropoelastin observed electrophoretically in several species. Different kinds of splicing patterns could occur in human populations and may contribute to aging and pathological situations in the cardiovascular and pulmonary systems.

Cytokine networks in the regulation of inflammation and fibrosis in the lung.

To understand the processes regulating inflammation and fibrosis in the human lung, we characterized the effects of recombinant interleukin-1, tumor necrosis factor, and gamma interferon on fibroblast proliferation, collagen production, interleukin-1-alpha production, interleukin-1-beta production, and interleukin-6-production. These studies demonstrated the existence of complex cytokine networks by which inflammatory cells regulate fibroblast function and fibroblasts, in turn, feed back to regulate inflammatory cell function. They also demonstrated that, in this complex network, the effect of an individual cytokine varies with the state of activation of the target cell, the presence of other cytokines in the local microenvironment, and the ability of the target cell to produce bioactive autocoids such as prostaglandins. Aspects of this cytokine network are discussed and a testable hypothesis for granuloma and abscess formation is detailed.

Synthesis of elastin. A rapid formation of lysine-derived crosslinks by chick embryo aorta.

Aortas of 13-day-old chick embryo were labeled for 0.5 hr with [14C]lysine and subjected to a serial extraction after chase for 1-24 hr with [12C]lysine. Substantial radioactivity was found in insoluble elastin after 3 hr chase. The effect of beta-amino-propionitrile on labeling with [14C]lysine was also examined. Each fraction was hydrolyzed and applied to a short column on an amino acid analyzer. Radioactivity was found in desmosine and isodesmosine of insoluble elastin as early as 1 hr after the beginning of chase. The radioactivity increased rapidly at 2 hr and very slowly thereafter. A large count, which was separated into five peaks on a long column, was observed in other lysine derivatives at 2 hr and increased steadily up to 24 hr, while the lysine count decreased from 1 : 0.5 to 1 : 6 against lysine derivatives and from 1 : 0.04 to 1 : 0.9 against quarter-desmosine after 24 hr. The oxidation of lysine residues incorporated during the 0.5 hr pulse was almost completed during the first 1 hr of chase, and these oxidized residues were incorporated into crosslinks during the following 1 hr. It is suggested that poorly crosslinked elastin accumulated in the soluble fractions. The presence of crosslinking derived from lysine residues was also indicated in the microfibril fraction.

Regulation of human lung fibroblast collagen production by recombinant interleukin-1, tumor necrosis factor, and interferon-gamma.

Several cytokines have been shown to modulate the synthesis of matrix molecules, but few reports have defined how the cytokines interact with one another in mediating these regulatory effects. To define the cytokine network regulating collagen production, we have studied the effects of interferon-gamma, interleukin-1, and tumor necrosis factor on collagen synthesis by cultured human lung fibroblasts. Confluent cells were treated with cytokines for 24 h in the absence of serum or in media containing 1% serum. In the absence of serum, IL-1 and TNF each dose-dependently stimulated types I and III collagen production, with a maximal 2-4-fold increase in collagen accumulation being observed. Cells treated with IL-1 and TNF in combination showed less stimulation than with either cytokine alone. IFN-gamma also diminished the stimulatory effects of both IL-1 and TNF. In contrast, in 1% serum IL-1 and TNF individually had relatively minor effects, while IFN-gamma inhibited collagen production. However, the combination of IL-1 and TNF inhibited collagen production. Generally, these effects were achieved through control of mRNA levels. These studies demonstrate the existence of a cytokine network regulating fibroblast collagen production and suggest that cytokine effects in vivo may vary depending upon the constellation of factors present in the tissue.