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Previous reports from our laboratories showed that type IV collagen from anterior lens capsule (ALC) inhibited stimulated neutrophil function. This property was shown to reside in the region comprising residues 185-203 of the non-collagenous domain (NC1) of the alpha 3(IV) chain. We also reported that ALC-type IV collagen or the synthetic alpha 3(IV) 185-203 peptide, induced a rise in intracellular cAMP which persisted for up to 60 minutes. In the present work we extend our previous studies on signal transduction by alpha 3(IV) 185-203 and we provide new data showing the involvement of cAMP-dependent PKA and protein phosphatases. The data also show that the alpha 3(IV) peptide triggered a rise in intracellular calcium that was dependent on phospholipase C activation. Inhibitors of the Ca(2+)/calmodulin system suppressed both the alpha 3(IV) 185-203 peptide-induced cAMP increase and the inhibitory activity of the peptide on f-Met-Leu-Phe triggered O(2)(-) generation. When alpha 3(IV) 185-203 peptide-induced calcium mobilization was blocked by U-73122, an inhibitor of phospholipase C activation, or by BAPTA/AM, a chelator of intracellular calcium, the inhibitory effect of the peptide on PMA-triggered O(2)(-) production was also abolished. These findings provide evidence that signal transduction by the alpha 3(IV) peptide occurs via pathways which involve calcium. Indeed, the cAMP increase was shown to be mediated by adenosine and adenosine A2 receptors and required calcium elevation, since adenosine deaminase, theophilline, dimethylpropargylxanthine, trifluoperazine or autocamtide-2 related inhibitory peptide, suppressed the activity of the alpha 3(IV) peptide. The inhibitory effect of the peptide on f-Met-Leu-Phe-induced O(2)(-) generation was slightly affected by 1 microM KT5720 or H89, two inhibitors of cAMP-dependent PKA, but was completely suppressed by 10 nM calyculin A or 10 microM okadaic acid, two inhibitors of ser/thr phosphatases. These results suggest that Ser/Thr protein phosphatases and/or cAMP-dependent PKA are involved in signal transduction by the alpha 3(IV) 185-203 peptide and is consistent with the concept that adenosine receptor occupancy modulates neutrophil function.

Studies from our laboratory (Shahan, T. A., Sorenson, W. G., and Lewis, D. M. (1994) Environ. Res. 67, 98-104) demonstrated that spores from different fungal species differentially activate rat alveolar macrophages as detected by the measurement of superoxide anion and cytokine production (Shahan, T. A., Siegel, P. D., Sorenson, W. G., Kuschner, W. G., and Lewis, D. M. (1998) Am. J. Respir. Cell Mol. Biol. 18, 435-441). Spores from Aspergillus candidus stimulated production of the highest levels of superoxide anion (5.2 nmol/1.0 x 10(6) alveolar macrophages (AMs)/30 min), followed by those from Aspergillus niger (2.4 nmol/1.0 x 10(6) AMs/30 min) and Eurotium amstelodami (0.4 nmol/1.0 x 10(6) AMs/30 min). The mechanism of this differential activation was studied. Our data demonstrate that the tyrosine kinases p56(Hck), p72(Syk), p77(Btk), p62(Yes), p56(Lck), and p59(Fyn) were specifically activated in response to spores from A. candidus, whereas spores from either A. niger or E. amstelodami activated p56(Hck), p72(Syk), and p77(Btk). Kinetic analysis of specific tyrosine kinases demonstrated that p56(Hck), p72(Syk), and p77(Btk) were activated faster and to a greater extent by spores from A. candidus as compared with spores from E. amstelodami. These data suggest a relationship between reactive oxygen species and tyrosine kinase activation. Treatment of AMs with H(2)O(2) (1 mM) caused the activation of p72(Syk) only, whereas treatment with superoxide dismutase and catalase before treatment with the spores had no effect on tyrosine kinase activation. Incubation with NADPH oxidase inhibitors inhibited both superoxide anion production and the activation of p56(Hck), p72(Syk), and p77(Btk) in response to fungal spores. These data indicate that endogenous reactive oxygen species are necessary for the activation of p56(Hck), p72(Syk), and p77(Btk) by spores; they also indicate that some species of spores are capable of activating tyrosine kinases independent of superoxide anion.

Studies from our laboratories demonstrated that synthetic peptides from the non-collagenous (NC-1) domain of the alpha3 (IV) chain of type IV collagen (COL IV) enhanced tumor cell adhesion (Han, J., Ohno, N., Monboisse, J. C., Pasco, S., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). We have isolated the receptors for the alpha3(IV)185-203 peptide from melanoma and prostate tumor cells and identified them as CD47/integrin-associated protein and the integrin alpha(V)beta(3) (Shahan, T. A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584-4590). In the present study we have examined the effect of CD47 and the integrin alpha(V)beta(3) on in vitro tumor cell chemotaxis and Ca(2+)(i) modulation in response to COL IV, from the anterior lens capsule (ALC-COL IV) and peptides from its NC-1 domain. COL IV as well as the alpha3(IV) peptide promoted tumor cell chemotaxis with an immediate increase in intracellular [Ca(2+)]. Treating tumor cells with CD47 and integrin alpha(V)beta(3)-reactive antibodies reduced chemotaxis as well as the rise in [Ca(2+)](i) in response to ALC-COL IV or the alpha3(IV)185-203 peptide but not to Engelbreth-Holm-Swarm-COL IV or fibronectin. The alpha3(IV)185-203 synthetic peptide stimulated an increase in calcium from intracellular stores exclusively, whereas ALC-COL IV, Engelbreth-Holm-Swarm-COL IV, and fibronectin stimulated Ca(2+) flux from both internal and external stores. Furthermore, treatment of the cells with Ca(2+) chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraaceticacid- acetomethoxy ester inhibited chemotaxis toward both ALC-COL IV and the alpha3(IV)185-203 peptide. These data indicate that CD47 and integrin alpha(V)beta(3) regulate tumor cell chemotaxis in response to COL IV and the alpha3(IV)185-203 peptide through a Ca(2+)-dependent mechanism.

The invasive properties of melanoma cells correlate with the expression of matrix metalloproteinases (MMPs) and their physiological modulators (tissue inhibitors of metalloproteinase and membrane-type MMPs) and with that of the alphaVbeta3 integrin. We investigated the effect of anterior lens capsule type IV collagen and of the alpha3(IV) collagen chain on the invasive properties of various tumor cell lines (HT-144 melanoma cells, HT-1080 fibrosarcoma cells). We demonstrated that anterior lens capsule type IV collagen or specifically the synthetic peptide alpha3(IV) 185-203 inhibited both the migration of melanoma or fibrosarcoma cells as well as the activation of membrane-bound MMP-2 by decreasing the expressions of MT1-MMP and the beta3 integrin subunit.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189-191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475-25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395-20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221-232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the alpha3(IV)179-208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin beta3 subunit, and the alpha(v)beta3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand alpha3(IV)187-191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin beta3 subunit, and the alpha(v)beta3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the alpha(v)beta3 integrin-reactive antibodies prevented the alpha3(IV)185-203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the alpha3(IV)187-191 (-YYSNS-) peptide also inhibited their adhesion to the alpha3(IV)185-203 peptide substrate, whereas the inactive alpha1(IV)185-203 peptide, from the same region of the alpha1 chain as the alpha3(IV)185-203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or alpha(v)beta3 integrin-reactive antibodies inhibited their adhesion to the alpha3(IV)185-203 peptide, whereas antibodies to the beta1 and beta2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the alpha(v)beta3 integrin.

We have shown that basement membrane (BM) collagen (type IV), and specifically the peptide CNYYSNSYSFWLASLNPER (a.a. 185-203), from the non-collagenous domain of the alpha3 chain inhibits PMN. We examined the role of this peptide on PMN damage to BM in a vessel wall model. The presence of the endothelial monolayer as well as treatment of PMN with the alpha3(IV) 185-203 peptide reduced damage to BM by non-activated but not by activated PMN. The damage inhibition is unique to the alpha3(IV) peptide and not exhibited by comparable alpha1(IV) and alpha2(IV) chain peptides. A shorter peptide alpha3(IV) 185-191, containing the -SNS- triplet, reduced damage, whereas the one lacking the triplet, residues 194-203, was not effective. The CD47-alphavbeta3 integrin complex is the receptor for the alpha3(IV) peptide. Incubation of PMN with CD47 reactive mAb followed by the alpha3(IV) peptide abolished its protective effect on BM damage., (Copyright 1999 Academic Press.)

Previously we had shown that basement membrane collagen (COL IV), and specifically residues 185-203 of the non-collagenous domain of the alpha3(IV) chain, inhibits PMN activation. Since acetylcholinesterase (AchE) possesses collagenous and non-collagenous domains, we tested its effect on PMN activation. Whole AchE and the AchE recombinant catalytic subunit inhibited PMN superoxide anion (O2-) generation. AchE synthetic peptides, residues 139-154 and 252-266 of the catalytic subunit, with sequence homology to that of the alpha3(IV) peptide also inhibited O2- production by PMN. Reactive pAb and mAb to the alpha3(IV) 185-203 peptide abolished the inhibitory effect of the AchE. The data show that the non-collagenous domain of the AchE down-regulates O2- production by PMN. We suggest that this inhibitory activity may serve as a protective mechanism against PMN-mediated injury at the level of vessel wall and the neuromuscular junction.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation; this property requires the presence of the triplet -SNS- in residues 189-191 (Monboisse et al., J. Biol. Chem., 269, 25475, 1994; Han et al., J. Biol. Chem., 272, 20395, 1997). In the present study, we demonstrate that whole native COL IV and -SNS- containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells, but also breast-, pancreas- and stomach-tumor cells up to 67%, and prostate tumor cells by 15%. ALC-COL IV at 5 microg/ml was shown to inhibit melanoma cell proliferation maximally at 69% and the alpha3(IV)185-203 peptide inhibited proliferation (62%) maximally at 10 microg/ml. Treatment of the alpha3(IV)185-203 peptide with either a specific mAb or a polyclonal antibody, prepared against the sequence alpha3(IV)179-208, decreased the ability of the peptide to inhibit cell proliferation by 97%, while treatment of ALC-COL IV with the same antibodies inhibited proliferation by 44%. Exposure of the above tumor cells to COL IV or the peptides resulted in an increase of intracellular cAMP that was inhibited by prior treatment of the protein with the above antibodies. To investigate the role of cAMP in the inhibition of cell proliferation, cAMP analogs and inhibitors were used. cAMP analogs mimicked the inhibitory effect of the peptide. Rp-cAMPS, a cAMP competitive inhibitor, suppressed the inhibitory effect of ALC-COL IV and of the cAMP analogs. The protein kinase-A inhibitor H-89 blocked the ability of ALC-COL IV and of the alpha3(IV)185-203 peptide to inhibit tumor cell proliferation. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation utilizing a signal transduction pathway which includes cAMP and cAMP-dependent protein kinase(s).

Previous studies have shown that tumor cells with metastatic propensity secrete more of the laminin alpha2 chain than non-metastatic tumor cells do, and that laminin-2, which contains the alpha2 chain, promotes cell adhesion better than laminin-1 (Jenq et al. (1994). Differentiation, 58, 29-36). The current studies were designed to determine whether a correlation exists between the expression of the laminin-2 isoform and the metastatic phenotype in melanoma cells. We found that expression of the laminin-2 isoform was upregulated in the metastatic melanoma cell lines tested. Cell attachment studies showed that metastatic melanoma cells attached more efficiently to laminin-2 substrates. Studies on integrin expression revealed that the presence of alpha2beta1 integrin correlated with expression of the laminin-2 isoform in metastatic melanoma cells; anti-integrin alpha2 antibody prevented cell attachment to laminin-2 substrates. The data suggest that the alpha2beta1 integrin is the receptor mediating cell attachment to the laminin-2 isoform. This interaction, mediated by the alpha2beta1 integrin, stimulates secretion of the 72 kD type IV collagenase and induces a specific 185 kD protein tyrosine phosphorylation. The 185 kD tyrosine-phosphorylated protein was identified as the p185/C-erb B2 oncoprotein by immunoprecipitation. These studies suggest that upregulation of expression of the laminin-2 chain correlates with the metastatic phenotype of melanoma cells and provides evidence that the specific p185/C-erb B2 tyrosine phosphorylation may be involved in integrin-mediated signaling during tumor cell invasion and metastasis.

Our previous studies have shown that a peptide corresponding to the residue sequence 185-203 of the NC1 domain of the alpha3 chain of basement membrane collagen (type IV) inhibits the activation of polymorphonuclear leukocytes. Peptides from the same region of the alpha1, alpha2, alpha4, and alpha5(IV) chains did not exhibit this property. Because of the intimate relationship between metastasizing neoplastic cells and vascular as well as epithelial basement membranes, we measured the cell adhesion-promoting activity of peptides from the NC1 domain of type IV collagen and their effect on proliferation of human melanoma cells. We found that peptide alpha3(IV)185-203 (CNYYSNSYSFWLASLNPER) not only promotes adhesion of human melanoma cells but also inhibits their proliferation. Adhesion increased by 50-60% over control. Melanoma cell proliferation was inhibited by 40% when cells were grown in a medium containing 5 microg/ml peptide for 5 days. Studies showed that replacement of serine in position 189 or 191 by alanine resulted in significantly reduced adhesion. Similarly, serine replacement resulted in reduced ability to inhibit proliferation. Our data suggest that a region of the NC1 domain of the alpha3(IV) chain, contained within the sequence 185-203, not only specifically promotes adhesion but also inhibits proliferation of melanoma cells. These properties appear to be dependent on the presence of the triplet sequence -SNS- (residues 189-191), which is unique to the alpha3 chain and may represent an important functional epitope.

Previous studies have indicated that laminin from neoplastic cells of high tumorigenicity is less active in promoting cell adhesion than aminin from normal cells or tissues. In the present study, we tested the hypothesis that laminin of metastatic tumor cells differs from that of nonmetastatic cells. Accordingly, we determined the subunit composition of laminin in highly metastatic, ras-transformed cells (4R) and compared it with laminin produced by nonmetastatic cells transformed with ras plus E1a (RE4). Metastatic 4R cells produced three to four times more of the alpha 2-subunit of laminin than RE4 cells did. Furthermore, the highly metastatic human melanoma cells (1205 and A2058) made and secreted into the medium, laminin containing significantly more of the alpha 2-subunit than laminin from the highly tumorigenic but nonmetastatic melanoma WM793 or HT1080 fibrosarcoma cells. Using HT1080 cells, laminin (250 ng/well) from 4R cells showed more adhesion promoting activity (68%) than laminin from RE4 cells (39%). Similarly, laminin isolated from human placenta, which expresses both the alpha 1 beta 1 gamma 1 and alpha 2 beta 1 gamma 1 isoforms, promoted cell adhesion better (63%) than EHS laminin (26%), which contains only the former isoform, at 250 ng/well. In addition, both 4R and RE4 cells attached more efficiently to 4R laminin-coated substratum than to RE4 laminin at 0.3 and 0.6 microgram/well.(ABSTRACT TRUNCATED AT 250 WORDS)

Our initial observation that type I collagen activates polymorphonuclear leukocytes (PMN) prompted the testing of the activating potential of type IV collagen. It was noted, however, that type IV collagen isolated from bovine lens capsule did not activate PMN but rather prevented their stimulation by N-formylmethionyl-leucyl-phenylalanine, phorbol myristate acetate, or type I collagen. This observation led to the present study, which demonstrates that the inhibitory effect of lens capsule type IV collagen resides in the noncollagenous (NC1) domain of the alpha 3 chain and specifically in the region comprising residues 185-203 of the NC1 domain of both the human and bovine molecules. Synthetic peptides from the same region of the NC1 domains of the alpha 1, alpha 2, alpha 4, and alpha 5 chains did not possess the inhibitory effect seen with the alpha 3 chain. The sequence S-N-S (residues 189-191) is unique to the peptide of the alpha 3 chain, and substitution of either serine with alanine abolishes the inhibition. Type IV collagen isolated from the mouse Engelbreth-Holm-Swarm (EHS) tumor, a molecule that lacks the alpha 3 chain, did not prevent PMN activation but instead stimulated the secretion of elastase and type IV collagenase. Incubation of PMN with intact lens capsule type IV collagen or a peptide comprising residues 185-203 of the alpha 3 (IV) chain resulted in a 3-fold increase of intracellular cAMP, whereas, Ca2+ levels remained unchanged. Incubating PMN with forskolin or with dibutyryl-cAMP resulted in the inhibition of O2- production and degranulation by PMN, thus mimicking the effects of type IV collagen and the alpha 3 (IV) 185-203 peptide. The data suggest that type IV collagen, through its alpha 3 chain, down-regulates PMN activation and thus decreases the potential for damage as these cells traverse the capillary wall. Our in vitro experiments suggest that the higher the content of the alpha 3 (IV) chain is in a basement membrane, the wider would be its capacity for self-protection.

To investigate the role of cell-cell interactions between keratinocytes and fibroblasts at the dermal-epidermal junction in the regulation of extracellular matrix protein expression, we have developed a cell coculture model that allows both cell types to interact on a reciprocal basis and yet be isolated as pure populations for quantitative analysis. Using porous membrane inserts as a substrate for keratinocytes grown in coculture over fibroblast monolayers, we report that coculture stimulates cell growth and total protein synthesis in both cell types when compared to monocultured controls. Both keratinocytes and fibroblasts synthesize laminin B1 and B2 chains with an additional subunit comigrating with laminin M chain detected in keratinocytes but only faintly visible in fibroblasts. Laminin A chain synthesis could not be detected. Although laminin subunit composition did not change in either cell type, fractional laminin synthesis increases by 41.0 +/- 2.3% in cocultured keratinocytes and decreases by 73.8 +/- 8.1% in cocultured fibroblasts when compared to monocultured controls. In cocultured keratinocytes, steady-state mRNA levels for laminin B1, B2, and M chains increased by 69.4, 63.5, and 136.8%, respectively, when compared to monocultured controls. However, in cocultured fibroblasts, laminin B1 and B2 chain mRNA decreased by 74.2 and 72.7%, respectively. Laminin M chain mRNA could not be detected in fibroblasts. These results suggest that synthesis and expression of laminin is regulated through reciprocal cell-cell interactions in fibroblasts and keratinocytes grown in coculture.

Background: Patients treated with lithium salts for manic depression had a lower incidence of herpes simplex infections. Initial studies in our laboratory demonstrated that addition of LiCl in cultures of human endothelial cells infected with herpes simplex virus suppressed viral replication and allowed synthesis of host proteins., Experimental Design: Based on the above observations, we decided to study the optimal condition for the lithium effect and determine the process of inhibition of viral replication. Endothelial cell cultures infected with herpes simplex virus-1 were exposed to LiCl at various times postinfection. The levels of host and viral mRNAs were measured by Northern and slot blot hybridization. The pattern of protein synthesis was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot and replication was assessed by plaque assay., Results: LiCl inhibited virus replication in a dose- and time-dependent manner as was reflected in the sharp decrease or absence of infectious virus production. The condition for optimal effects of LiCl were the addition of the salt between 0-3 hours postinfection, and at a concentration of 30 mM. LiCl suppressed the synthesis of viral polypeptides, whereas the synthesis of host proteins was maintained. Similar results were observed with phosphonoacetic acid, an inhibitor of viral DNA polymerase. NaCl, at the same concentration as LiCl, did not prevent the virus-induced inhibition of host cell protein synthesis. The level of host mRNA for fibronectin, thrombospondin, collagen type IV, actin, and plasminogen activator inhibitor-1 were maintained in the presence of LiCl. mRNAs for viral proteins, ICP-4 and DNA polymerase were nearly undetectable when LiCl was added with the virus (0 time postinfection)., Conclusions: The data indicate that LiCl treatment results in suppression of herpes virus mRNAs, i.e., mRNAs for ICP-4 and DNA polymerase, thereby inhibiting replication. On the other hand, the levels of host mRNAs are maintained to varying degrees depending on the message. The data suggest that a very early step in the process of viral replication is affected by LiCl, since the drug is maximally effective when added with the virus.

The cell adhesion promoting activity of laminin isolated from normal human placenta was compared with that isolated from mouse EHS tumor and from the cultures of a mouse epithelial cell line B82 and its tumorigenic derivative, B82HT. The adhesion promoting properties of commercial merosin isolated from placenta was also compared with the above preparations using the human fibrosarcoma HT1080 cells. Percent attachment was defined as (radioactivity extracted from attached cells)/(radioactivity in cells added to assay) x 100. HT1080 cells adhered more efficiently on laminin (0.5 micrograms/well), isolated from the B82 rather than B82HT cell conditioned medium, (82% vs 64%). Percent attachment of HT1080 cells on isolated native placental laminin or commercial merosin was significantly higher compared to laminin from the EHS tumor (at 0.75 micrograms/well, 69%, 73% and 20% respectively). In parallel experiments the steady-state levels of mRNAs for subunits A, M, B1 and B2 in cultures of B82 and B82HT cells were determined. The ratio of mRNA for the laminin subunits in B82 and B82HT cells was 1:0.9 for the A chain, 1:0.6 for the M chain, 1:0.4 for the B1 chain, and 1:0.3 for the B2 chain. Protein studies indicated that the M subunit is absent in laminin preparations from the EHS tumor whereas it is abundant in the laminin from placenta and in commercial merosin. Laminin isolated from B82 cells contains a higher proportion of the M subunit compared to that from B82HT cells. The data suggest that there are functional differences between the laminin found in normal tissue and that present in a solid tumor. Functional differences were noted between the laminins synthesized by the B82 cell line and its tumorigenic counterpart, B82HT. These differences may result from the lack of gene expression for the laminin subunit M by the EHS tumor and by the lower degree of gene expression for this subunit by B82HT cells. The possibility that the laminin synthesized by the tumorigenic cell line may be structurally different from that synthesized by the B82 cells should also be considered.

Sera from patients with antiglomerular basement membrane (anti-GBM) antibodies associated with Goodpasture syndrome (GP) or glomerulonephritis were tested by ELISA and electroimmunoblot against whole basement membrane collagen (type IV) isolated from bovine anterior lens capsule (ALC) and bacterial collagenase resistant domains of the collagen molecule, that is, the NC-1 and 7-S domains isolated from either ALC or bovine and human glomerular basement membrane (GBM). Reactivity was high with the NC-1 domain by both the ELISA and the electroimmunoblot techniques. Some of the anti-GBM sera reacted with both the NC-1 and 7-S domains of both human and bovine type IV collagen. At a time when the patients' sera reacted weakly with a collagenase digest of human GBM using a radioimmunoassay, the reactivity with the NC-1 domain was also low, but some of the sera continued to react with the 7-S domain. The data suggest that there may be heterogeneity in the nature of autoantibodies with respect to collagen type IV domain reactivity in the sera of patients with anti-GBM antibody disease.

Peptides representing potential antigenic regions of the NC-1 and 7-S domains of the human alpha 1 and alpha 2, and bovine alpha 3 chains of type IV collagen were synthesized either chemically or by the recombinant DNA technique and tested by ELISA using antibodies raised in rabbits against the whole type IV collagen or the NC-1 domain. Sera from patients with Goodpasture syndrome (GP) or with acute poststreptococcal glomerulonephritis (APSGN) were also tested. The location of antigenic determinants was predicted from the primary and secondary structure of the chains, that is, aromaticity, hydrophilicity and presence of beta-turns. All synthetic peptides reacted with the antiserum to type IV collagen (anti-Col IV). Whereas all peptides arising from the NC-1 domain reacted with anti-NC-1, intact 7-S or peptides of the alpha 1 or alpha 2 chain of the 7-S domain did not react. However intact 7-S reacted with anti-Col IV. Two synthetic peptides from the NC-1 domain of alpha 1, (a.a. 71-90 and a.a. 176-190), one from the alpha 2 (a.a. 70-83) and four from the alpha 3 chain (a.a. 72-89, a.a. 104-117, a.a. 133-145, a.a. 185-203) reacted with anti-NC-1 and anti-COL IV. The above peptides, except alpha 3 (72-89) and alpha 3 (185-203), were tested and found to be reactive with sera from patients with GP.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.

The biosynthesis of interstitial collagens (types I and III) and proteoglycans was studied in fibroblasts isolated from the parietal layer of bovine pericardium. Confluent cultures were labeled with Na2 35SO4 for proteoglycans or 14C-proline for collagens. The proteoglycans synthesized by pericardial fibroblasts were purified by DEAE-Sephacel chromatography and further fractionated into three components by gelfilitration. Two minor high molecular weight proteoglycans were shown by SDS-PAGE to be resistant to chondroitinase ABC and AC, and partially degraded by nitrous acid. The major, low molecular weight proteoglycan had a core protein of 45 kDa and is considered to be a dermatan sulfate/chondroitin sulfate proteoglycan since it was resistant to nitrous acid, but digested partially by chondroitinase AC and completely by ABC. The pericardial fibroblasts synthesized predominantly type I collagen and low amounts (about 10%) of type III collagen which was detected by delayed reduction on SDS-PAGE. The data show that pericardial fibroblasts synthesize the same macromolecules that can be extracted from the intact tissue and suggest that the proteoglycan may play a structural as well as physiological role.

Earlier studies from this laboratory have shown that infection of vascular cells with herpes simplex virus 1 or 2 (HSV-1, HSV-2) results in the differential suppression of extracellular matrix proteins including fibronectin (FN), type IV collagen, thrombospondin (TSP) and Factor VIII von Willebrand protein. The present study was designed to determine whether a correlation exists between suppression of synthesis of specific proteins and their mRNA levels. We have measured the steady-state levels of mRNAs for several extracellular matrix proteins (type IV collagen, FN and TSP) and two intracellular proteins (actin and tubulin) in human endothelial cells (EC) following HSV-1 infection. The results show that during the first 5 h post-infection, when there is a rapid decrease in the synthesis of extracellular matrix proteins, the steady-state levels of the corresponding mRNAs remain relatively high, but progressively decline to levels of less than 20% by 13 h post-infection. These findings suggest that in the early hours post-infection there is an alteration in the translatability of the hybridizable message followed by degradation in the later hours.

Intact human laminin and laminin type IV collagen complexes were extracted from term placental membranes and their structures were examined by electroimmunoblot and by rotary shadowing electron microscopy. Rotary shadowing electron microscopy revealed a structure of human laminin which is essentially similar to the cruciform structure of the mouse tumor (EHS) laminin, but with some notable differences. The observed lengths of the short arms in the human laminin are different. One short arm has an average length of 34 nm and another short arm a length of 42 nm. In the mouse laminin all three short arms are of equal length. The average length of the long arm is 97 nm, which is longer than that of mouse tumor laminin (77 nm). The distal portion of the long arm has two clearly separated globular domains instead of the single one observed with tumor laminin. Human laminin was found in various states of aggregation including dimers, trimers and higher aggregates. Laminin molecules appeared to attach to a point in type IV collagen located 87 nm from C-terminus, or at 174 nm from C-terminus and one with less frequency, at 251 nm from C-terminus. A small number of molecules appeared to bind at the N-terminus of the collagen. These laminin-collagen interactions occurred via the distal globular domains of both the short and long arms of laminin. The data suggest that human laminin extracted from placenta is structurally different from that isolated from the mouse EHS tumor.

We compared the structure and subunit composition of laminin in less tumorigenic mouse A9 and highly tumorigenic mouse A9HT cells by pulse-chase studies. During a 15' pulse, the ratio of laminin B1 to B2 subunits in cell lysates is 1:1 in both the A9 and A9HT cells; however, after a 3 hr chase, this ratio changes to 6:1 and 2:1 in the A9 and A9HT cells, respectively. Analysis of mature laminin subunits in culture media after a 3 hr chase also showed a similar higher ratio of B1 to B2 in the A9 cells as compared to the A9HT cells. The higher ratio of B1 to B2 subunits in A9 cells was evident as early as after a 30' pulse. A comparative analysis of steady-state levels of mRNAs for the laminin subunits B1 and B2 between A9 and A9HT cells showed a ratio of 1:1 for B1 and a ratio of 1:1.65 for B2. The ratio of B1 to B2 mRNAs in A9 cells was 1:1.3 whereas in A9HT cells it was 1:2.5, suggesting changes in the processing of mRNA in the highly tumorigenic A9HT cells. These observations suggest that the processing of laminin B subunits is altered during the process of increased tumorigenicity, thus resulting in the synthesis and secretion of structurally different laminin in tumorigenic A9HT cells as compared to the parent and less tumorigenic A9 cells.

In the present study, the ability of a 43 K fragment, originating from the long arm of the 400 K laminin "A" chain (Rao and Kefalides, manuscript in press), to promote the attachment and de novo neurite outgrowth by the pheochromocytoma cell line, (PC12) was studied. Unprimed PC12 cells (not treated with nerve growth factor, NGF), just as the primed cells (treated with NGF), rapidly and efficiently attach to the laminin fragment but less efficiently to the intact laminin coated bacteriological plastic surfaces. The neurite outgrowth was continuous from primed cells attached to both laminin and laminin fragment substrates, when cultured for four days in the presence of NGF. On the other hand, no significant neurite outgrowth was observed from unprimed PC12 cells attached to the laminin fragment coated bacteriological dishes, in the absence of NGF. These data suggest that the 43 K laminin "A" chain fragment contains the PC12 cell attachment promoting site.

A recently described procedure of reduction and carboxymethylation followed by heparin-Sepharose chromatography [Arumugham et al. (1988) Connect. Tissue Res. 18, 135-147] was used to characterize high-affinity heparin binding fragments of the laminin "A" chain. Two laminin fragments of Mr 53K and 43K selectively bound to the heparin-Sepharose column from the chymotrypsin digest of laminin, indicating that these fragments originate from the "A" chain. Without reduction and carboxymethylation but in the presence of 2.0 M urea, the heparin-Sepharose-bound material from the chymotrypsin laminin digest contains all the attachment-promoting activity for normal mouse mammary epithelial cells. The reduced 200-kDa intact three short arm fragment, fragments of Mr 70K-160K obtained either from laminin or from the reduced 200-kDa three short arm fragment, and the 53-kDa heparin binding fragment were all inactive in promoting the adhesion of mouse mammary epithelial cells. The mammary epithelial cell adhesion and spreading properties of laminin are associated with the high-affinity heparin binding 43-kDa fragment. The mammary epithelial cells attach to the 43-kDa fragment substrate and synthesize laminin, collagen type IV, and desmoplankins I and II as are the cells attached to laminin substrate and to the cells grown on tissue culture dishes. The biologically active 43-kDa fragment is generated from laminin, but not from the three short arm fragment. These results suggest that normal mouse mammary epithelial cells interact with laminin through a single site which is present in the 43-kDa heparin binding fragment located on the long arm of the "A" chain.

The effects of herpes simplex virus type 1 (HSV-1) infection on proteoglycan synthesis by human endothelial cells were studied as a model of endothelial cell injury. Confluent cultures of early passage endothelial cells from human umbilical vein were infected with HSV-1 at multiplicities of infection from 0.001 to 1.0. HSV-1 infection produced a dose-dependent inhibition of total proteoglycan synthesis of up to 85%. Although there was a 2- to 3-fold increase in the quantity of virus necessary to cause 50% inhibition of heparan sulfate compared to chondroitin/dermatan sulfate proteoglycan, the inhibition was relatively parallel, even up to high virus doses. There was no inhibition of an undersulfated heparan sulfate proteoglycan that contained glycosaminoglycan chains shorter than the predominant species. The results indicate that HSV-1 infection of human endothelial cells produces complex effects on host-cell metabolism. The viral-induced changes in proteoglycan metabolism may influence cell-matrix interactions and lead to altered vessel wall function.

Herpes simplex virus type 1 produces an early decrease in protein synthesis in cultured smooth muscle cells isolated from human umbilical artery. To confirm that suppression of host protein synthesis occurs before virus protein expression, cultures were treated with actinomycin D (5 micrograms/ml) continuously from the beginning of infection. In the presence of actinomycin D, by 3.5 hours postinfection, virus-infected cultures showed much less incorporation of [35S]methionine into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than did identically treated mock-infected cultures. In the absence of actinomycin D, there was incorporation of radiolabel into viral proteins as early as 4.5 hours postinfection. Hybridization of slot-blotted total RNA demonstrated that virus-infected cultures treated with and without actinomycin D showed a marked decrease in hybridizable RNA by 2 hours PI when compared with mock-infected controls. This decrease was seen with cDNA probes for the mRNA for secreted extracellular matrix macromolecules (fibronectin, thrombospondin and collagen chains alpha 2(I), alpha 1(III] as well as with cDNA probes for RNA of intracellular macromolecules actin and beta-tubulin. In vitro translation of total RNA isolated from infected human smooth muscle cells at 2 and 5 hours postinfection resulted in far less [35S]methionine incorporation into sodium dodecyl sulfate-polyacrylamide electrophoresis-separated products than would be predicted from the amount of hybridizable RNA available. The results suggest that host message is rapidly rendered nonfunctional as well as degraded by means of a virion-competent mechanism(s).

Studies to eludicate the effect of heparin on the synthesis of extracellular matrix components by cultured human umbilical vein endothelial cells (EC) were conducted. Using pulse-labeling and ELISA techniques, we found that EC grown in the presence of heparin (90 micrograms/ml) and endothelial cell growth factor (ECGF) synthesized 50% less fibronectin (FN) than did ECGF-treated control cultures. No change in the synthesis of thrombospondin (TSP) was induced by heparin. The effect of heparin on EC FN synthesis was independent of whether the cells were cultivated on plastic or gelatin substrates. However, ECGF modulates the effect of heparin on EC synthesis of FN. RNA slot-blot analysis demonstrated that heparin treatment specifically decreased the steady-state mRNA levels for both FN and TSP in the cells. Steady-state levels of mRNA for two intracellular proteins, actin and tubulin, were unchanged. These data suggest that heparin decreases EC expression of FN at least in part by decreasing the amount of FN mRNA available for translation. The failure of heparin to inhibit TSP expression, although it reduces TSP mRNA levels, points to the possibility that the rate of EC synthesis of TSP is translationally or post-translationally regulated.

In previous studies we have shown that herpes simplex virus type 1 (HSV-1) infection suppresses host-cell protein synthesis in human endothelial cells (EC). It has been demonstrated that lithium salts prevent viral replication in HSV-1 infected cells. In the present study, we have measured host-cell protein synthesis in HSV-1 infected EC in the presence or absence of 20 and 30 mM LiCl. Although LiCl restored synthesis of almost all host-cell proteins, [35S]methionine incorporation was most pronounced in thrombospondin and plasminogen activator inhibitor 1 and least in fibronectin and type IV collagen. LiCl was more effective at the higher concentration (30 mM) and when the compound was added to the EC culture at the time of infection rather than after adsorption of HSV-1. Synthesis of virus proteins continued in LiCl-treated EC but at a reduced rate. The data suggest that LiCl not only interferes with virus replication, but may also, to some extent, interfere with the virion-associated inhibition of host protein synthesis.

[14C]Proline and [14C]lysine were incorporated into collagen by cultures of endothelial cells derived from calf aortae. The isomer 3-hydroxy[14C]proline accounted for 10% of the total hydroxy[14C]proline in the collagen isolated from the medium. Approximately 81% of the hydroxy[14C]lysine isolated from the medium was glycosylated, and 91% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosylgalactose. Gel filtration chromatography or acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the initially synthesized peptide chain of [14C]collagen had a molecular weight of about 135,000; after pepsin digestion this was converted to 115,000. The ratio of hydroxy[14C]proline to total [14C]proline x 100 in the pesin-resistant fraction was 59. When examined by immunofluorescence microscopy, the endothelial cultures stained positively with antiserum to (Type IV) collagen from basement membrane of bovine anterior lens capsule. The data indicate that cultured endothelial cells derived from calf aortae synthesize collagen that resembles that of basement membrane collagen.

Monoclonal antibodies (mAbs) (4F2 and 7E12) were prepared against heparan sulfate proteoglycan (HSPG) isolated from bovine glomeruli. Enzyme linked immunosorbent assays (ELISA) and immunoblotting demonstrated that the mABs reacted with HSPG. Indirect immunofluorescence (IF) showed that the mAbs stained renal basement membranes (BMs) and BMs in other organs of normal bovine and human tissues in patterns typical of HSPG. Immunoinhibition studies, and immunoblotting of heparan lyase digested HSPG, indicated that the mAbs recognize HSPG core protein. In kidney biopsies from patients with acute poststreptococcal GN, intact linear glomerular BM (GBM) staining for HSPG was noted despite markedly widened capillary loops. In membranous and in diffuse proliferative lupus GN, loss of HSPG staining was demonstrated at sites of immunodeposition of IgG or C3, while increased staining for HSPG was noted in areas of newly formed GBM. Extensive loss of HSPG was seen in areas of glomerular sclerosis and necrosis. In biopsies from patients with minimal change glomerulonephritis (GN) and mesangioproliferative lupus GN, a normal linear GBM distribution of HSPG was noted. The findings are discussed in the context of current knowledge regarding the pathogenesis of glomerular injury. MAbs to BM HSPG should prove useful for future immunochemical studies, and for the study of diseases of the basement membrane.

Adherence of granulocytes to tissue culture monolayers of endothelium averaged 26.2 +/- 1.3% SEM, which was similar to their adherence on 50-mg nylon fiber columns (27.7 +/- 3.6%). In contrast, adherence to epithelial cells, fibroblasts, kidney cells, and plastic Petri dishes without monolayers was only 12.4, 9.9, 11.1, and 4.3%, respectively. Cyclic nucleotides and adherence-modifying plasma factors induced changes of adherence to endothelium similar to those in nylon fiber columns. Adherence of granulocytes in whole blood was the same as for purified granulocytes in Hank's balanced salt solution. Exposure of endothelial monolayers to 0.18% trypsin for 10 min reduced subsequent granulocyte adherence to 25.2% of control values. Incubation of trypsin-treated monolayers with nutrient medium for 4 h did not improve adherence, but values returned to normal or above by 24 h, with or without serum proteins present in the nutrient medium. The similarity of granulocyte adherence to nylon fiber and to endothelial monolayers in vitro suggests that results with the nylon fiber assay reflect in vivo granulocyte-endothelium interaction. Furthermore, the endothelial monolayer offers a new model for studying this cell-cell relationship in vitro.

The mechanism by which immune complexes deposit in vascular tissue is uncertain. Several human viruses, including herpes simplex virus, have recently been demonstrated to replicate in human endothelial cells. Such viruses may injure vascular tissue and could play a role in the pathogenesis of immune complex deposition. Therefore, we studied the expression of receptors for immune complexes containing IgG and C3 on endothelial cells after infection with herpes simplex virus type I.Human umbilical vein endothelial cells were incubated with (51)Cr-labeled sheep erythrocytes sensitized with IgG, IgM, or IgM plus complement. Preferential binding of IgG or complement-coated erythrocytes to uninfected endothelial monolayers was not observed. In contrast, significant binding of erythrocytes coated with IgG or IgM plus complement was observed after viral infection. Phase-contrast and scanning electron microscopy demonstrated erythrocyte adherence around the infected endothelial cells in a rosette pattern. Binding of IgG-coated erythrocytes was fully inhibited by Fc (0.31 mg/ml) but not Fab' fragments of nonimmune IgG. Binding of complement-coated cells was unaffected by the presence of IgG (1 mg/ml). With purified individual components, binding of complement-coated erythrocytes depended on the presence and was proportional to the concentration of C3. Binding of IgG-or C3-coated cells was detected beginning 4 h after infection. These studies indicate that herpes simplex virus type I infection can induce IgG and C3 receptors on human endothelial cells. These receptors may promote the deposition of immune complexes in vascular tissue after certain viral infections.

The synthesis of matrix proteins was investigated in cultures of human umbilical vein endothelial cells (EC) infected with herpes simplex virus type 1 (HSV-1). EC cultures were either mock-infected or infected for 1 hour at a multiplicity of infection (MOI) of 5 or 20 infectious particles per cell. Synthesis was followed by determining [14C]-proline or [35S]-methionine incorporation into non-dialyzable proteins. Using SDS-PAGE we observed that synthesis of fibronectin (FN), type IV procollagen and thrombospondin (TSP) was inhibited in infected cultures as early as 2 hours becoming almost complete by 15 hours post-infection. The degree of inhibition of matrix protein synthesis was dependent on the dose of the virus inoculum (MOI 20 greater than MOI 5) and varied with the particular matrix protein, i.e. shut-off of type IV collagen occurred first followed by that of FN and then TSP. Pulse-chase experiments suggest that the absence of labeled matrix protein in the medium of infected cultures is not due to accumulation of protein within the infected cells since there was an equal and parallel reduction in the cell layer. Suppression of matrix proteins in infected cultures was confirmed by measuring the level of FN and TSP in uninfected and infected cultures in an enzyme-linked immunosorbent assay (ELISA). The three proteins were identified by ELISA, electroimmunoblot, immunoprecipitation and ion-exchange chromatography. The data suggest that HSV-1 infection of human EC suppresses matrix protein synthesis; the degree and time of complete shut-off varies with the protein and the virus dose.

Adherence of human granulocytes was measured on endothelial monolayers of human and bovine origin, grown in 35-mm Diam petri dishes and in cluster wells. Adherence to human endothelium in petri dishes using 1.0 ml of whole blood averaged 17.9+/-3.7%, and to bovine endothelium was 20.3+/-3.7%. Cluster wells required only 1/5 the endothelial cells needed for petri dishes, and 0.25 ml of whole blood yielded average adherence of 26.2+/-3.4 to human cells and 28.0+/-3.7 to bovine in the wells. The impact of infection of the endothelium by different viruses on subsequent granulocyte adherence was measured. Polio virus produced an acute lytic infection of human endothelial cells, with associated increased adherence to 185.4% of control 24 h after inoculation. Significantly increased adherence was noted at 6 h, before detectable cytopathic effect. Herpes simplex type I caused a similar rapidly lytic infection of bovine endothelium associated with increased adherence to 213.7% of control 6 h after inoculation. This augmented adherence could be demonstrated when granulocytes were suspended in physiologic saline solution, showing that antibody and complement need not be present. Trypsin treatment of infected monolayers did not prevent the augmentation, and supernate from infected monolayers increased the adherence of polymorphonuclear leukocytes to normal, uninfected monolayers. Chronic, slowly lytic infections, lasting 7 d or more, were induced with adenovirus in human endothelium and with measles virus in bovine cells. Adherence increased as virus was noted in the cell cultures on day 4, several days before cytotoxicity was seen. Thus, chronic viral infection of the endothelium appears possible, and results in increased granulocyte adherence. In naturally occurring disease, such an infection may act synergistically with adherent granulocytes to damage the endothelium, and may represent an in vitro model of vasculitis.

Laminin components were solubilized from basement membranes of amnion, chorion and chorionic microvessels of human placenta without prior protease digestion. These structures, after isolation, were initially processed in a sonicator bath containing a solution of Triton X-100, EDTA and 2M NaCl and the laminins extracted sequentially with 0.5M NaCl, 8 M urea, and 8 M urea + 2% 2-mercaptoethanol + 2% SDS. A high molecular weight (appr. 1 x 10(6)) complex containing laminins was purified by gel filtration on a Sepharose CL-2B column. This complex migrated as a single band on gel electrophoresis before reduction but resolved, after reduction, into four major laminin components, laminin A (350,000 M.W.), laminin M (240,000 M.W.) and laminins B1 and B2 (195,000 and 185,000 M.W.). Laminin M is a new molecular species of this protein.

Cultures of human diploid fibroblasts were shown to accumulate elevated levels of intracellular triglyceride when grown in medium supplemented with sera from patients with endogenous hypertriglyceridemia (Type IV hyperlipoproteinemia). Time course studies indicated cell triglyceride levels reached maxium in 6 to 12 hours. Isotopic studies indicated that the source of the accumulated cell triglyceride was serum triglyceride, and that most of the triglyceride was taken up without hydrolysis. Triglyceride was not significantly removed by trypsinization or extensive washing. The accumulated triglyceride was also demonostrated to be metabolized, as indicated by conversion to phospholipids and free fatty acids. Increasing concentrations of hypertriglyceridemic serum in the medium resulted in increasing cell triglyceride levels. However, increasing amounts of normal serum did not result in comparable cell triglyceride accumulation. Intracellular triglyceride accumulation seemed to correlate with levels of hypertriglyceridemic serum triglyceride, whether this serum was present at equivalent concentrations or normalized to triglyceride levels equivalent to normal serum. It is concluded that elevated serum triglyceride levels can result in intracellular triglyceride accumulation even in the absence of lipolytic activity.

The major proteoglycan in bovine parietal pericardium is a low molecular weight dermatan-sulfate proteoglycan. It possesses structural and immunologic characteristics similar to those of the small proteoglycan found in tendon. We demonstrate that digestion of purified pericardial proteoglycan with low levels of V8 protease results in the liberation of the glycosaminoglycan chain and of a 40 kDa resistant fragment. A similar 40 kDa fragment can be obtained by V8 protease digestion of the proteoglycan deglycosylated by chondroitinase ABC. Although the protein core size of the pericardial proteoglycan is similar to that of tendon PG II, the size of the glycosaminoglycan chain liberated from the former is smaller. The pericardial proteoglycan and its V8 protease products reacted with an anti-PG II antiserum by immunoblotting. The anti-PG-II antibody localized in the pericardial tissue by the immunoperoxidase technique. The presence of intrachain disulfide bonds in the structure of pericardial proteoglycan core protein and V8 resistant fragment was demonstrated by their decreased electrophoretic mobility after disulfide reduction. Digestion of pericardial proteoglycan with Cathepsin C resulted in a rapid liberation of the glycosaminoglycan chain from the core protein, indicating that its attachment site was very close to the amino terminus. Ultrastructural examination of pericardial tissue utilizing Cuprolinic Blue revealed a periodic association of the proteoglycan with the d/e band on the collagen fibrils. Electron microscopic immunohistochemical studies confirmed the perifibrillar association of pericardial proteoglycan. The present data demonstrate that, although the pericardial proteoglycan possesses some unique structural features, it shares structural and immunological characteristics to place it in the category of the small PG II family.

Basement membrane collagen from bovine anterior lens capsules (ALC) was isolated by a non-degradative procedure and characterized. The material was obtained by extracting the ALC with 0.5 M acetic acid, passing the extract through a DEAE-cellulose column, collecting and concentrating the unbound fraction. Amino acid and carbohydrate analysis, polyacrylamide gel electrophoresis and rotary shadowing electron microscopy showed the purified extract to have the characteristics of basement membrane procollagen. Examination by rotary shadowing revealed the material to consist of type IV procollagen-like tetramers in various degrees of aggregation. When this purified procollagen was injected into rabbits, antibody of high titer and specificity was obtained. The competitive ELISA was then used to demonstrate lack of reactivity of the antibody with collagen types I, II, III and V and fibronectin. The electroimmunoblot technique was used to demonstrate lack of reactivity with laminin. Competition with collagenase resistant fragments of type IV procollagen representing the carboxyl terminal domain (NCI) and the 7-S domain, as well as with a pepsin-resistant alpha 1 (IV)125K chain was markedly weaker as compared to the native type IV procollagen molecule.

Parietal yolk sacs isolated from 14.5-day rat embryos and incubated in vitro with either [14C]proline, [3H]mannose or 3H-labeled amino acid mixture synthesized and secreted basement membrane collagenous and noncollagenous glycoprotein components with relative molecular weights of 350,000 (350K), 220,000 (220K), 185,000 (185K), 175,000 (175K) and 150,000 (150K). The 185K and 175K components appeared to be similar to the pro-alpha 1 (IV) and pro-alpha 2(IV) chains, respectively, which have been isolated from other sources. These components were completely susceptible to bacterial collagenase, but were only partially susceptible to alpha-chymotrypsin digestion. The 350K and 220K components appeared to be similar to subunits of laminin (or PYS A and PYS B, respectively) which have been characterized by others, while the 150K component may be similar to entactin (or PYS C). These components were completely resistant to bacterial collagenase and completely susceptible to alpha-chymotrypsin digestion. In addition, the basement membrane of the parietal yolk sac (Reichert's membrane) stained intensely with antibodies directed against either rat laminin or mouse basement membrane procollagen. The results of these experiments suggest that the 14.5-day rat embryo parietal yolk sac is a useful system for studying the structure, biosynthesis and deposition of basement membrane components.

Endothelial injury is important in the pathogenesis of thrombosis, atherosclerosis, disseminated intravascular coagulation, and vasculitis. The ability of several common human viruses to infect cultures of endothelial cells obtained from human umbilical veins or bovine thoracic aorta was demonstrated. Indicators of infection included cytopathology, viral growth curves, and antigen detection by immunofluorescence. Herpes simplex virus type 1, adenovirus type 7, measles virus, and parainfluenza virus type 3 infected both human venous and bovine aorta endothelium. Mumps virus, poliovirus type 1, and echovirus type 9 grew only in human venous cells; coxsackievirus B4 infected only bovine arterial cultures; and cytomegalovirus, influenza A/Victoria/75 (H3N2) virus, and respiratory syncytial virus failed to grow in either cell culture. During replication some viruses caused acute lytic changes; some produced chronic, less destructive alterations; and other induced no apparent cytopathology. The results suggest that viral replication within endothelium may be important in the pathogenesis of viral disease of initiation of vessel-wall injury.

Screening of a human fibroblast cDNA library with a mouse type IV procollagen clone resulted in one 1.05-kilobase isolate that was used to identify a 1.7-kilobase clone overlapping the former by less than 150 nucleotides. EcoRII digestion revealed that the larger clone exhibited the pattern characteristic of DNA coding for a collagenous sequence. Blot hybridization to RNA from mouse F9 stem cells and from these cells treated with retinoic acid and N6, O2'-dibutyryl-cAMP showed induction of type IV mRNA. DNA sequencing and comparison of the derived amino acids with the reported protein data demonstrated that the clones encode part of the alpha 1 chain of human type IV procollagen. Alignment of alpha 1 (IV) with other human procollagens showed minimal but detectable homology. A small cluster of charged residues in the alpha chain is partially shared by type IV. In close proximity is an interruption in the alpha 1 (IV) Gly-Xaa-Yaa region corresponding to the 3' end of a unique proline-free, and therefore also less rigid, area in other collagen triple helices. Analysis of the carboxyl-terminal alpha 1 (IV) peptide showed a repeat symmetry possibly dictated by the six cysteines in each half of the structure. The position of five cysteines in addition to four tyrosine/tryptophan groups allowed a correlation to be drawn between the 3' noncollagenous type IV region and the larger, highly conserved carboxyl propeptides of other human procollagens. Such similarities in the different chains may define functional domains conserved throughout evolution.

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [14C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultres to change in exogenous lipid is dependent on passage level and state of growth.

Previous studies have shown that there is microscopic and biochemical evidence that rat parietal yolk sac synthesizes basement membrane (type IV) collagen; this study shows that a radioimmunoassay may be used for the detection of type IV collagen in such biosynthetic systems. Rat parietal yolk sacs incubated in medium containing (14C) proline either with or without alphaalpha-dipyridyl produced either unhydroxylated or hydroxylated (14C)collagen. The immunological reactivity of these two preparations was investigated using antibodies to bovine type IV collagen in a radioimmunoassay which demonstrated that the hydroxylated (14C)collagen preparation had a considerably higher level of antigenicity than the unhydroxylated (14C)collagen. Hydroxylated rat type IV (14C)collagen which had been reduced and alkylated was intermediate in antigenicity between hydroxylated and unhydroxylated material. These findings suggest that there are antigenic determinants which depend upon hydroxylation of the collagen molecule, and others dependent upon intact disulphide bonds. In addition, various levels of pepsin extracted unlabelled calf anterior lens capsule collagen caused inhibition of antibody binding to (14C)collagen. Rat type IV (14C)collagen which had been digested with collagenase was inactive in the radioimmunoassay, while pepsin digestion caused no reduction in antigenicity. These findings suggest that the antiserum is directed towards the collagenous part of the molecule and may be a useful tool in the detection of biosynthesized basement membrane collagen.

The synthesis of collagen has been demonstrated in endothelial cells of Descemet's membrane isolated from rabbit cornea. Incorporation of [14C]proline and [14C]lysine into nondialyzable protein was measured in the medium and cell fraction after incubating Descemet's membrane for up to 5 hours. In the [14C]collagen synthesized by the endothelium, 15% of the hydroxy[14C]proline was present as the 3-isomer. About 98% of the hydroxy[14C]lysine in the 14C-labeled-protein found in the medium was glycosylated; 95% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosyl-galactosyl-hydroxy[14C]lysine. Time course experiments with [14C]proline indicated that there was a delay of about 60 min before significant amounts of [14C]collagen were secreted into the medium. The initial polypeptides of [14C]collagen synthesized by the corneal endothelium had an apparent molecular weight of 155,000. The chemical and physical properties of the [14C]collagen synthesized by rabbit corneal endothelium are consistent with those of basement membrane collagen synthesized by other cell types.

Using a monoclonal anti-tubular basement membrane antibody (alpha TBM-Ab) affinity column, we isolated from collagenase-solubilized human renal tissue (HSRTA) a predominantly 48,000-mol-wt moiety (H3M-1) which is selectively recognized by antisera from two patients with alpha TBM-Ab-associated interstitial nephritis (alpha TBM disease). Whereas both antisera had alpha TBM-Ab titers of 1:64-1:128 by immunofluorescence on tissue sections, their reactivity with H3M-1 in a solid-phase radioimmunoassay was demonstrable at dilutions up to 1:10,000. While these sera displayed some reactivity with pre-column HSRTA, this was markedly less than with H3M-1. HSRTA depleted of H3M-1 by passage over the alpha TBM-Ab affinity column was almost completely depleted of reactivity. Neither pooled normal human sera nor sera from patients with a variety of renal lesions not associated with alpha TBM-Ab (including interstitial nephritis and antiglomerular basement membrane disease) were reactive with H3M-1. Both patient antisera containing alpha TBM-Ab were also highly reactive with R3M-1, the 48,000-mol-wt rabbit glycoprotein antigen of experimental alpha TBM disease. Furthermore, a competitive inhibition radioimmunoassay revealed that alpha TBM-Ab from rodents with experimental alpha TBM disease could inhibit 45-98% of the R3M-1 binding reactivity of patient antisera and 85% of the H3M-1 binding reactivity of patient antisera, thus suggesting paratypic cross-reactivity. We conclude, therefore, that tubular basement membrane target epitopes and their paratypic recognition are highly conserved among mammals.