D M Bass, H B Greenberg
Endothelial cell derived relaxing factor (EDRF) mediated relaxation of blood vessels is impaired in vessels exposed to lipoproteins in vitro and in arteries of hyperlipidemic humans and animals. To investigate the mechanism by which lipoproteins impair the effects of EDRF, which is likely nitric oxide (NO) or a related molecule, we have bioassayed EDRF/NO activity by measuring its ability to increase cGMP accumulation in rat fetal lung cultured fibroblasts (RFL-6 cells). Low density lipoprotein modified by oxidation (ox-LDL) induced a concentration-dependent inhibition of EDRF activity that had been released from bovine aortic endothelial cells (BAEC) stimulated with bradykinin or the calcium ionophore A23187. In addition, lipoproteins directly impaired authentic NO-induced stimulation of cGMP accumulation in the detector cells; stimulation by sodium nitroprusside was unaffected. Ox-LDL or oxidized HDL3 were highly potent in blocking NO-stimulated cGMP accumulation with EC50's of approximately 1 microgram/ml. Lipid extracted from ox-LDL blocked NO-stimulated cGMP accumulation to about the same extent as intact ox-LDL, while the protein component of ox-LDL did not inhibit the cGMP response. These results suggest that the lipid component of oxidized lipoproteins inactivate EDRF after its release from endothelial cells.
J H Chin, S Azhar, B B Hoffman
Rat liver perisinusoidal lipocytes (PL) cultured on uncoated plastic transform spontaneously within 6-10 d to myofibroblastlike cells (MFBlC). Parallel to the transformation the TGF alpha- and TGF beta 1-mRNA expression increased and was highest in MFBlC. Competitive radioligand binding assays demonstrated that in contrast to untransformed PL the MFBlC synthesize and secrete transforming growth factor (TGF)-alpha (15 fmol/cell per 24 h) and predominantly the latent form of TGF beta 1 (0.2 fmol/cell per 24 h). Medium conditioned by MFBlC (MFBcM) significantly stimulated PL proliferation with little effect on PL proteoglycan synthesis. By transient acidification of the MFBcM, known to activate the latent form of TGF beta 1, the stimulatory effect on PL proteoglycan synthesis was enhanced and furthermore PL transformation (measured by expression of iso-alpha smooth muscle actin and loss of retinylpalmitate) was accelerated. Preincubation of this medium with neutralizing antibodies to TGF beta resulted in (a) the growth inhibitory effect was converted to a growth stimulation and (b) the stimulatory effect on proteoglycan synthesis was abolished. In summary our data indicate that progressive activation of PL on plastic (transformation to MFBlC) leads to an enhanced expression of the TGF alpha- and TGF beta 1-mRNAs and secretion of the corresponding proteins. Medium conditioned by MFBIC stimulates proliferation, transformation, and PG synthesis of untransformed PL. These mechanisms are suggested to be relevant in self perpetuation of liver fibrogenesis.
M G Bachem, D Meyer, R Melchior, K M Sell, A M Gressner
We describe a novel experimental system in mice for the study of ovarian autoimmune disease, a condition encountered in women with premature ovarian failure. The ovarian autoimmune disease is induced in B6AF1 mice by a 15-amino acid peptide (Cys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg) from mouse ZP3, the sperm-binding component of the zona pellucida that surrounds growing and mature oocytes. Whereas the peptide induces both T cell and antibody responses, adoptive transfer of CD4+ T cell lines derived from affected animals causes oophoritis without observable antibodies to the zona pellucida peptide. The primacy of the T cell response in the pathogenesis of disease is further substantiated by defining oophoritogenic peptides as small as eight amino acids (Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln) that do not elicit an antibody response to the full-length ZP3 peptide. The identification of a well characterized peptide as a causative agent of autoimmune oophoritis should facilitate understanding of the pathogenesis of this T cell-mediated autoimmune disease. Because the proteins of the zona pellucida are conserved among mammals (the mouse and human ZP3 proteins are 67% identical), this murine model may lead to better understanding of the pathogenesis of human autoimmune oophoritis.
S H Rhim, S E Millar, F Robey, A M Luo, Y H Lou, T Yule, P Allen, J Dean, K S Tung
Liver glycogen formation can occur via the direct (glucose----glucose-6-phosphate----glycogen) or indirect (glucose----C3 compounds----glucose-6-phosphate----glycogen) pathways. In the present study we have examined the effect of hyperglycemia on the pathways of hepatic glycogenesis, estimated from liver uridine diphosphoglucose (UDPglucose) specific activities, and on peripheral (muscle) glucose metabolism in awake, unstressed control and 90% pancreatectomized, diabetic rats. Under identical conditions of hyperinsulinemia (approximately 550 microU/ml), 2-h euglycemic (6 mM) and hyperglycemic (+5.5 mM and +11 mM) clamp studies were performed in combination with [3-3H,U-14C]glucose, [6-3H,U-14C]glucose, or [3-3H]glucose and [U-14C]lactate infusions under postabsorptive conditions. Total body glucose uptake and muscle glycogen synthesis were decreased in diabetic vs. control rats during all the clamp studies, whereas glycolytic rates were similar. By contrast, hyperglycemia determined similar rates of liver glycogen synthesis in both groups. Nevertheless, in diabetic rats, the contribution of the direct pathway to hepatic glycogen repletion was severely decreased, whereas the indirect pathway was markedly increased. After hyperglycemia, hepatic glucose-6-phosphate concentrations were increased in both groups, whereas UDPglucose concentrations were reduced only in the control group. These results indicate that in the diabetic state, under hyperinsulinemic conditions, hyperglycemia normally stimulates liver glycogen synthesis through a marked increase in the indirect pathway, which in turn may compensate for the reduction in the direct pathway. The increase in the hepatic concentrations of both glucose-6-phosphate and UDPglucose suggests the presence, in this diabetic rat model, of a compensatory "push" mechanism for liver glycogen repletion.
A Giaccari, L Rossetti
Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteoclasts produce an autocrine or paracrine factor that enhances osteoclast formation. Conditioned media from long-term bone marrow cultures from patients with Paget's disease stimulated osteoclast-like MNC formation in normal marrow cultures. At least part of this activity could be ascribed to interleukin 6 (IL-6). In contrast, conditioned media from normal marrow cultures contained lower levels of IL-6 and did not stimulate formation of osteoclast-like MNC. 7 of 8 bone marrow plasma samples taken from involved bones and 18 of 27 peripheral blood serum samples from Paget's patients had high levels of IL-6. Normal marrow plasma and peripheral blood serum had no or very low levels of IL-6. These results suggest that IL-6 produced by marrow and/or bone cells in patients with Paget's disease may be an autocrine/paracrine factor for pagetic osteoclasts.
G D Roodman, N Kurihara, Y Ohsaki, A Kukita, D Hosking, A Demulder, J F Smith, F R Singer
Cytotoxic T lymphocytes (CTL) specific for human immunodeficiency virus (HIV) proteins have been analyzed in lymphoid organs from seropositive patients. Indeed, an active HIV replication coexists with a major CD8+ lymphocytic infiltration in these organs. We have shown in a previous report that HIV-seropositive patients lungs were infiltrated by HIV specific CD8+ lymphocytes. In the present report, we show that HIV-specific CTL responses can also be detected in lymph nodes and spleens, and were mainly directed against the ENV, GAG, and NEF HIV-1 proteins. The primary NEF-specific CTL responses were further characterized by epitope mapping. Determination of epitope-specific CTL frequencies were performed by limiting dilution analysis. Our results indicated that, in addition to the central region of NEF (AA66-148), a new immunodominant region is recognized by CTL. This region corresponds to the carboxyl-terminal domain of NEF (amino acids 182-206). AA182-206 is recognized in association with at least two common human histocompatibility leukocyte antigen (HLA) molecules (HLA-A1 and B8), with clonal frequencies of one CTL per 10(-5) to 10(-6) splenic lymphocytes. Our data indicate that lymphoid organs may represent a major reservoir for in vivo activated HIV-specific CTL. Furthermore, the carboxyl-terminal domain of NEF was found to be conserved among several HIV strains. Therefore, our finding is of interest for further HIV vaccines development.
F Hadida, A Parrot, M P Kieny, B Sadat-Sowti, C Mayaud, P Debre, B Autran
PBMC express cell surface receptors for extracellular matrix components known as integrins. We have recently shown that ligand binding to one PBMC integrin, the collagen receptor alpha 2 beta 1, stimulates the secretion of interleukin 1 (IL-1). We have now investigated the role of fibronectin (Fn), an adherence protein that has binding sites for both PBMC and collagen, in the generation of the IL-1 response to collagen. In contrast to collagen, Fn did not stimulate IL-1 release but Fn-depleted serum decreased the release of IL-1 induced by collagen. A polyclonal antiserum directed against Fn also decreased the collagen-induced IL-1 secretion. The IL-1 response to collagen from cells incubated in Fn-depleted serum was restored by the addition of either purified Fn or the 120-kD cell-binding fragment of Fn, which contains the cell-binding site but not the collagen-binding domain. Smaller Arg-Gly-Asp (RGD) peptides failed to enhance the PBMC response to collagen but inhibited in a concentration-dependent fashion the potentiating effect Fn. As expected, a MAb against the alpha 2 beta 1 collagen receptor decreased collagen-induced IL-1 release. However collagen-induced IL-1 release was also inhibited by a MAb against the alpha 5 beta 1 Fn receptor. The effect of the two MAbs was not additive, suggesting that the occupancy of both receptors by ligands is required in order for collagen to induce an maximal response from PBMC. The mechanism by which Fn exerts its effect remains unknown. However, flow-cytometric analysis revealed that Fn does not alter expression of the alpha2beta1 receptor on PBMC. These data demonstrate a potentiating effect of Fn on the collagen-induced secretion of IL-1 from human PBMC and suggest that this effect is mediated via the integrin alpha5beta1. These findings indicate a complex interactive role for specific integrin receptors in the regulation of the mononuclear cell immune response.
R Pacifici, C Basilico, J Roman, M M Zutter, S A Santoro, R McCracken
Cardiac work is a major determinant of heart size and growth. Heterotopic cardiac isografts are hemodynamically unloaded and undergo atrophy. To determine the molecular changes that occur as a result of hemodynamic unloading, we have studied the rate of synthesis of total cardiac proteins and myosin heavy chain (MHC) and the expression of the myosin heavy chain gene as reflected in the messenger RNA levels for alpha- and beta-MHC isoforms. 72 h after transplantation there is a significant decrease in left ventricular size accompanied by a 27% decrease in the rate of total cardiac protein synthesis and a 53% decrease in the rate of myosin heavy chain synthesis. In contrast to isografts 14 d after transplantation which have a decrease in protein synthetic capacity, simultaneous measurements of 18S ribosomal RNA and myosin messenger RNA suggest that after 3 d the decrease in synthesis is due to a change in the efficiency of protein translation. While the working in situ heart expresses primarily alpha-MHC mRNA (97%) hemodynamic unloading leads to a 43% decrease in alpha-MHC mRNA concentration and the de novo expression of the beta-MHC mRNA. Total MHC mRNA (alpha plus beta) concentration analyzed by a quantitative S1 nuclease protection assay was similar in the two groups of hearts. Thus, in association with hemodynamic unloading there are changes in cardiac myosin heavy chain content as a result of both gene transcription and protein translation mechanisms.
I Klein, K Ojamaa, A M Samarel, R Welikson, C Hong
Estrogen is generally considered to maintain bone mass through suppression of bone resorption. We have previously demonstrated that administration of pharmacologic doses of estrogen increases bone formation in ovary-intact rats. To assess the effects of physiological concentrations of estrogen on bone formation, estrogen was administered to ovariectomized rats in which bone resorption was suppressed by the bisphosphonate 3-amino-1-hydroxypropylidene-1-bisphosphonate (AHPrBP). Animals receiving exogenous 17 beta-estradiol (E2) (1, 10, and 100 micrograms/kg daily for 17 d) showed a dose-dependent increase in trabecular bone volume of 1.9, 25.8, and 43.6%, respectively, compared with those rats treated with AHPrBP alone. The increase in bone volume was associated with an increase in bone formation in E2-treated animals, in which bone resorption had been almost completely suppressed by AHPrBP. Neither ovariectomy, AHPrBP, nor E2 treatment had a significant effect on the volume or rate of formation of cortical bone. Thus, the increased bone resorption, which is a consequence of estrogen-deficiency, entrains increased bone formation, which masks a simultaneous reduction in estrogen-dependent bone formation. Therefore, in addition to the nonspecific effect of estrogen to depress formation via coupling, we have identified a specific effect of estrogen to increase formation independent of coupling. Thus it appears that estrogen maintains bone volume not only through inhibition of bone resorption, but also through stimulation of bone formation.
J Chow, J H Tobias, K W Colston, T J Chambers
We studied the synthesis, secretion, and aggregation into the extracellular matrix of fibrillin by dermal fibroblasts from 26 probands with the Marfan syndrome. Cells from seven probands synthesized approximately half the normal amount of fibrillin when compared with intrafamilial or unrelated controls. Cells from an additional seven probands synthesized a normal amount of fibrillin but secreted the protein less efficiently than control cells. Cells from a further eight probands synthesized and secreted normal amounts of fibrillin but the protein was poorly incorporated into extracellular matrix. Cells from the remaining four probands were indistinguishable from control cells in their synthesis and processing of fibrillin. Cells from 18 family members of 10 of the probands were also studied. Cells from affected individuals in the same family had the same biochemical defect and those from unaffected family members were indistinguishable from controls. These results indicate that mutations in the gene that encodes fibrillin are responsible for the Marfan syndrome in the majority of individuals (confirming recent immunohistochemical and genetic linkage studies) and that a variety of mutations can produce the phenotype associated with the syndrome.
D M Milewicz, R E Pyeritz, E S Crawford, P H Byers
T cell proliferative responses to hepatitis B virus-encoded envelope antigen (S + preS2 + preS1), recombinant core antigen (HBcAg), and natural hepatitis B e antigen (HBeAg) were examined in 22 HBeAg-positive patients with chronic type B hepatitis and 17 healthy hepatitis B surface antigen (HBsAg) carriers. The results showed that HBeAg-positive patients had (a) higher levels of T cell responses to HBcAg/HBeAg than those of healthy HBsAg carriers (P less than 0.001 and P less than 0.01, respectively); (b) a further increase in these T cell responses during acute exacerbations (P less than 0.05 and P less than 0.05, respectively); (c) subsidence in the T cell responses to HBcAg/HBeAg after recovery from acute exacerbations and HBeAg seroconversion, whereas the responses would persist at high levels if the patients did not enter a clinical remission; and (d) low levels of T cell responses to S + preS2 + preS1 either before or after HBeAg seroconversion. The appearance of increasing T cell responses to HBcAg/HBeAg usually occurred in the early phase of acute exacerbations. These findings imply that HBcAg/HBeAg-specific T cells play an important role in the exacerbations of chronic hepatitis B and in HBeAg seroconversion. HBcAg/HBeAg-specific precursor T cell frequencies were serially studied in selected cases by limiting dilution assay. Elevation (two- to fourfold) of HBcAg/HBeAg-specific precursor T cell frequencies contributed to the increase of HBcAg/HBeAg-specific T cell proliferation during acute exacerbations.
S L Tsai, P J Chen, M Y Lai, P M Yang, J L Sung, J H Huang, L H Hwang, T H Chang, D S Chen
Cyclooxygenase (COX), or prostaglandin (PG) H synthase, plays a role in inflammatory diseases, but very limited data exist on the regulation of COX in vivo. We, therefore, studied the in vivo expression of COX in synovia from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), as well as joints of rats with streptococcal cell wall (SCW) and adjuvant arthritis. Extensive and intense intracellular COX immunostaining, which correlated with the extent and intensity of mononuclear cell infiltration, was observed in cells throughout RA synovia. Significantly less or equivocal staining was noted in OA and normal human synovia. Similarly, COX immunostaining was equivocal in the joints of normal and arthritis-resistant F344/N rats. In contrast, high level expression developed rapidly in euthymic female Lewis (LEW/N) rats throughout the hindlimb joints and overlying tissues including skin, preceding or paralleling clinically apparent experimental arthritis. COX was expressed in the joints of athymic LEW.rnu/rnu rats 2-4 d after injection of SCW or adjuvant but was not sustained. Physiological doses of antiinflammatory glucocorticoids, but not progesterone, suppressed both arthritis and COX expression in LEW/N rats. These observations suggest that, in vivo, (a) COX expression is upregulated in inflammatory joint diseases, (b) the level of expression is genetically controlled and is a biochemical correlate of disease severity, (c) sustained high level up-regulation is T cell dependent, and (d) expression is down-regulated by antiinflammatory glucocorticoids.
H Sano, T Hla, J A Maier, L J Crofford, J P Case, T Maciag, R L Wilder
Studies in animal models suggest that oxygen radicals may be important in the pathogenesis of acute pancreatitis. Because glutathione is an essential component of the defense against radical-mediated cellular injury, we investigated whether pancreatic glutathione content is influenced by inducing acute pancreatitis and whether augmenting the intracellular supply of glutathione would alter the course of pancreatitis. Caerulein, a decapeptide cholecystokinin analogue, induces acute necrotizing pancreatitis in mice when given in high doses (50 micrograms/kg per h) over a period of 6 h. The pancreatic glutathione content (total, GSH + GSSG) in mice treated with high-dose caerulein fell to 17% of normal within 4 h of beginning caerulein and recovered toward normal after discontinuing caerulein treatment. Mice treated with glutathione monoethyl ester (20 mmol/kg 1 h before caerulein, 10 mmol/kg 3 and 7 h after starting caerulein) were found to have blunted depletion of pancreatic glutathione, diminished histologic evidence of pancreatitis (necrosis, inflammation, and vacuolization), and lower serum amylase values compared with mice treated with caerulein alone. These findings suggest that the profound depletion of pancreatic glutathione caused by hyperstimulation of the pancreas with caerulein is critically important in the pathogenesis of acute caerulein-induced pancreatitis.
B A Neuschwander-Tetri, L D Ferrell, R J Sukhabote, J H Grendell
In vitro incubated rat islet B cells differ in their individual rates of protein synthesis. The number of cells in biosynthetic activity increases with the glucose concentration. Flow cytometric monitoring of the cellular redox states indicated that islet B cells differ in their individual metabolic responsiveness to glucose. A shift from basal to increased NAD(P)H fluorescence occurred for 18% of the cells at 1 mM glucose, for 43% at 5 mM, and for 70% at 20 mM. The functional significance of this metabolic heterogeneity was assessed by comparing protein synthesis in metabolically responsive and unresponsive subpopulations, shortly after their separation by autofluorescence-activated cell sorting. The glucose-sensitive subpopulation exhibited four- to fivefold higher rates of insulin synthesis during 60-min incubations at 2.5-10 mM glucose. Its higher biosynthetic activity was mainly caused by recruitment of cells into active synthesis and, to a lesser extent, by higher biosynthetic activity per recruited cell. Cells from the glucose-sensitive subpopulation were larger, and presented a threefold higher density of a pale secretory vesicle subtype, which is thought to contain unprocessed proinsulin. It is concluded that intercellular differences in metabolic responsiveness result in functional heterogeneity of the pancreatic B cell population.
R Kiekens, P In 't Veld, T Mahler, F Schuit, M Van De Winkel, D Pipeleers
The aim of this experiment was to demonstrate whether histamine and histidine decarboxylase (HDC) contribute to mucosal repair in small intestine subjected to ischemia-reperfusion (I/R). The superior mesenteric artery was occluded for 15 min followed by reperfusion. In jejunal mucosa, histamine content and HDC activity increased after I/R. Histamine output in mesenteric lymph was also elevated after I/R. These increases in HDC activity, and mucosal and lymph histamine levels were suppressed by pretreatment of alpha-fluoromethylhistidine (alpha-FMH), a suicide inhibitor of HDC. alpha-FMH also attenuated the increase of ornithine decarboxylase (ODC) activity normally observed after I/R. Transport of dietary lipid into lymph markedly decreased at 24 h after I/R, yet it was restored to normal at 48 h after I/R. alpha-FMH inhibitor led to a sustained deficit in lipid transport at 48 h after I/R. This sustained functional impairment in alpha-FMH treated animals was associated with blunted responses of HDC activity and histamine content to I/R. Our results suggest that histamine and HDC contribute to the restoration in mucosal function observed at 48 h after I/R. This response may be related, at least in part, to stimulation of ODC activity by histamine.
K Fujimoto, I Imamura, D N Granger, H Wada, T Sakata, P Tso
Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Understanding the mechanisms which prevent maternal immunological rejection of the fetus remains a fundamental unsolved problem in immunology. We have previously reported that macrophages are inhibited by maternal decidual stromal cells residing at the maternal-fetal interface. In view of the central role of macrophages in cell-mediated immunity, this inhibition may contribute to preventing maternal antifetal responses. We now report that it was the solid phase signals embedded in the extracellular matrix (ECM) made by decidual cells which are responsible for inhibiting macrophage-mediated lysis of TNF-alpha-resistant P815 mastocytoma cells. The latter macrophage function is acquired after stimulation by interferon gamma and endotoxin. All these macrophage functions were also inhibited by ECM isolated from the Engelberth-Holm-Swarme (EHS) tumor. This tumor ECM has a similar biochemical composition to decidual ECM. This ECM inhibited the effector, as opposed to the stimulator, phase of macrophage-mediated tumor lysis. Laminin, type IV collagen, and heparan sulfate proteoglycans, the major known components of decidual and EHS ECMs, did not inhibit the above macrophage functions. Altogether these data indicate that macrophages were inhibited by solid phase signals embedded in decidual and EHS ECMs. Whether the solid phase signals in these two ECMs are biochemically identical remains to be determined. To our knowledge, such signals are a novel pathway of inhibiting macrophage functions which may be important in understanding the maternal-fetal immunologic relationship, and the pathogenesis of perinatal infections. Furthermore, the ability of EHS tumor ECM to inhibit macrophage functions may indicate that some tumors may defend themselves against host macrophage responses using solid phase signals. This may be important in understanding some host-tumor relationships.
D B McKay, M A Vazquez, R W Redline, C Y Lu
Pneumocystis carinii is the most common cause of life-threatening pneumonia in immunocompromised patients. In the current study, surfactant protein A (SP-A), the major nonserum protein constituent of pulmonary surfactant, is demonstrated to bind P. carinii in a specific and saturable manner. SP-A is surface bound and does not appear to be internalized or degraded by the P. carinii organism. Furthermore, SP-A binding to P. carinii is time- and calcium-dependent and is competitively inhibited by mannosyl albumin. In the absence of calcium or the presence of excess mannosyl albumin, SP-A binding to P. carinii is reduced by 95 and 71%, respectively. SP-A avidly binds P. carinii with a Kd of 8 x 10(-9) M and an estimated 8.4 x 10(6) SP-A binding sites per P. carinii organism, as determined from Scatchard plots. SP-A is shown to bind P. carinii in vivo, and a putative binding site for SP-A on P. carinii is demonstrated to be the mannoserich surface membrane glycoprotein gp120. These findings suggest that P. carinii can interact with the phospholipid-rich material in the alveolar spaces by specifically binding a major protein constituent of pulmonary surfactant.
P E Zimmerman, D R Voelker, F X McCormack, J R Paulsrud, W J Martin 2nd
Studies were performed on monolayers of cultured A6 cells, grown on permeable filters, to determine the second messenger system involved in the aldosterone-induced increase in electrogenic sodium transport. Addition of aldosterone (1 microM) to the solution bathing the basal surface of cells caused both an increase in Isc and threefold transient rise in intracellular calcium Cai2+ after a delay of approximately 60 min. Because both events were inhibited by actinomycin D and cyclohexamide, they appeared to require transcriptional and translational processes. Addition of BAPTA to the bathing media to chelate Cai2+ reduced Isc and the delayed Cai2+ transient; 50 microM BAPTA inhibited Isc and the rise in Cai2+ by greater than 80%. Further studies suggested that the action of aldosterone to increase Isc may be dependent on a calcium/calmodulin-dependent protein kinase, because W-7 and trifluoperazine reduced the aldosterone-induced Isc in a dose-dependent manner. Taken together, these observations suggest that calcium is a second messenger for the action of aldosterone on sodium transport, and suggest, for the first time, that agonists which bind to intracellular receptors can utilize, via delayed processes dependent on de novo transcription and translation, intracellular second messenger systems to regulate target cell function.
D Petzel, M B Ganz, E J Nestler, J J Lewis, J Goldenring, F Akcicek, J P Hayslett
We have investigated the T cell receptor V alpha and V beta gene family usage by T lymphocytes infiltrating affected thyroids in patients with autoimmune thyroid disease. We show that the intrathyroidal T lymphocytes from patients (n = 6) with autoimmune thyroid disease display a widespread usage of V beta gene families with an average of 14.4/19 V beta gene families similar to the peripheral T lymphocytes of the same patients. Because we recently reported that the utilization of V alpha gene families is markedly reduced within these mitogen-stimulated intrathyroidal T cell populations, as well as within intact tissue from similar patients (n = 4) (overall mean of 4.0/18 families detected), these results indicate that in thyroids of patients with autoimmune thyroid disease the lymphocytes are selectively accumulating based on their V alpha rather than V beta elements. This preferential hTcR V alpha and widespread V beta gene usage was not mimicked in most 7-d autologous mixed lymphocyte reactions using non-T cell stimulators (n = 6) or EB-virus immortalized autologous B cell lines (n = 3). Hence, the selective V gene utilization by intrathyroidal T cells is likely to be secondary to multiepitopic thyroidal autoantigens activating thyroid infiltrating T cells or to the presence of a superantigenlike thyroidal self-antigen, capable of determining a selective infiltration or activation of a variety of T lymphocytes on the basis of their V alpha gene usage.
T F Davies, A Martin, E S Concepcion, P Graves, N Lahat, W L Cohen, A Ben-Nun
Type VII collagen, a genetically distinct member of the collagen family, is present in the cutaneous basement membrane zone as an integral component of the anchoring fibrils. We have recently isolated several cDNAs that correspond to human type VII collagen sequences. One of these cDNAs (clone K-131) was utilized to examine type VII collagen gene expression in cultures of human cells by Northern analyses, in situ hybridizations and indirect immunofluorescence. Northern hybridizations revealed the presence of an approximately 9-kb mRNA transcript, and indicated a high level of expression in epidermal keratinocytes as well as in an oral epidermoid carcinoma cell line (KB), while the expression was considerably lower in skin fibroblasts and in several virally or spontaneously transformed epithelial cell lines. In situ hybridizations of cultured keratinocytes supported the notion of a high level of gene expression. Indirect immunofluorescence of skin from a 19-wk fetus revealed type VII collagen gene expression at the dermal-epidermal basement membrane zone. These results indicate that several different cell types including epidermal keratinocytes and dermal fibroblasts express the type VII collagen gene, but epidermal keratinocytes may be the primary cell source of type VII collagen in developing human skin.
J Ryynänen, S Sollberg, M G Parente, L C Chung, A M Christiano, J Uitto
The present studies were undertaken to determine whether lipolysis was increased in non-insulin-dependent diabetes mellitus (NIDDM) and, if so, to assess the influence of increased glycerol availability on its conversion to glucose and its contribution to the increased gluconeogenesis found in this condition. For this purpose, we infused nine subjects with NIDDM and 16 age-, weight-matched nondiabetic volunteers with [2-3H] glucose and [U-14C] glycerol and measured their rates of glucose and glycerol appearance in plasma and their rates of glycerol incorporation into plasma glucose. The rate of glycerol appearance, an index of lipolysis, was increased 1.5-fold in NIDDM subjects (2.85 +/- 0.16 vs. 1.62 +/- 0.08 mumol/kg per min, P less than 0.001). Glycerol incorporation into plasma glucose was increased threefold in NIDDM subjects (1.13 +/- 1.10 vs. 0.36 +/- 0.02 mumol/kg per min, P less than 0.01) and accounted for twice as much of hepatic glucose output (6.0 +/- 0.5 vs. 3.0 +/- 0.2%, P less than 0.001). Moreover, the percent of glycerol turnover used for gluconeogenesis (77 +/- 6 vs. 44 +/- 2, P less than 0.001) was increased in NIDDM subjects and, for a given plasma glycerol concentration, glycerol gluconeogenesis was increased more than two-fold. The only experimental variable significantly correlated with the increased glycerol gluconeogenesis after taking glycerol availability into consideration was the plasma free fatty acid concentration (r = 0.80, P less than 0.01). We, therefore, conclude that lipolysis is increased in NIDDM and, although more glycerol is thus available, increased activity of the intrahepatic pathway for conversion of glycerol into glucose, due at least in part to increased plasma free fatty acids, is the predominant mechanism responsible for enhanced glycerol gluconeogenesis. Finally, although gluconeogenesis from glycerol in NIDDM is comparable to that of alanine and about one-fourth that of lactate is terms of overall flux into glucose, glycerol is probably the most important gluconeogenic precursor in NIDDM in terms of adding new carbons to the glucose pool.
N Nurjhan, A Consoli, J Gerich
Differences in susceptibility to infection of most mononuclear phagocytes with HIV-1 are not known. We investigated the relative susceptibility of autologous freshly isolated blood monocytes (MN), MN cultured in vitro to allow differentiation (CM), and alveolar macrophages (AM) from healthy subjects to productive infection with HIV-1. Cells were infected with the macrophage tropic strain HIV-1JR-FL and p24 gag antigen levels measured in supernatants by ELISA. Freshly isolated MN had negligible levels of p24 in supernatants. In contrast AM had peak p24 levels of 4145 +/- 1456 pg/ml, mean +/- SE, and CM 9216 +/- 3118. As a measure of entry and extent of reverse transcription, levels of viral DNA in infected mononuclear phagocytes were analyzed by quantitative polymerase chain reaction (PCR). The data using primers that amplify all transcripts including incompletely formed reverse transcripts indicated that differences in entry of the virus may contribute to differences in virus production observed with MN, AM, and CM. Other primer pairs that detect intermediate and full-length double-stranded DNA showed that the ability to complete reverse transcription was similar among these mononuclear phagocytes. Since the lung is a major site of opportunistic infection and noninfectious complications in HIV-1-infected individuals, this increase in productive infection with HIV-1 in AM compared with MN could contribute to the immunopathogenesis of the lung disorders seen in the acquired immunodeficiency syndrome.
E A Rich, I S Chen, J A Zack, M L Leonard, W A O'Brien
Previously we demonstrated that arginine vasopressin (AVP) directly inhibits bicarbonate absorption (JHCO3, pmol/min per mm) in the medullary thick ascending limb (MTAL) of the rat. To determine whether changes in osmolality also may affect bicarbonate absorption, MTAL were studied in vitro with 25 mM HCO3- solutions. Control osmolality was 290 mosmol/kg H2O. In the absence of AVP, increasing osmolality to 560 in perfusate and bath by addition of 150 mM NaCl reduced JHCO3 from 13.7 to 4.5. With 2 x 10(-10) M AVP in the bath, adding 150 mM NaCl to perfusate and bath reduced JHCO3 from 6.9 to 0.6, while adding NaCl to the bath alone reduced JHCO3 from 7.1 to 0.5. Adding 150 mM NaCl to perfusate and bath caused a similar inhibition of JHCO3 in MTAL perfused with furosemide to inhibit net NaCl absorption. In the presence of AVP, adding 600 mM urea to perfusate and bath inhibited JHCO3 by 55%; adding 300 or 600 mM mannitol to perfusate and bath inhibited JHCO3 by 75%. The effects on JHCO3 were reversible and dissociable from changes in transepithelial voltage. Conclusions: (1) osmolality is a factor capable of regulating renal tubule bicarbonate absorption; (2) hypertonicity produced with NaCl, urea, or mannitol markedly inhibits bicarbonate absorption in the MTAL; (3) this inhibition occurs independent of, and is additive to, inhibition by vasopressin. Hypertonicity may shift TAL HCO3- absorption from medulla to cortex, thereby limiting delivery of bicarbonate to the medullary interstitium during antidiuresis.
D W Good
To develop a new approach to the treatment of advanced, hormone-refractory prostate cancer, the signal transductions regulating the growth of human androgen-independent prostate carcinoma cell lines were studied. Agonist-stimulated Ca2+ mobilization, a critical regulatory event in other secretory cell types, was studied as a means of identifying previously undescribed plasma membrane receptors that may transduce a growth inhibitory signal. In all of the cell lines tested, P2-purinergic receptor agonists, including ATP and certain hydrolysis-resistant adenine nucleotides, induced a rapid, transient increase in cytoplasmic free Ca2+ that was detectable at 50 to 100 nM ATP, was maximal at 100 microM ATP, and was inhibited approximately 50% by chelation of extracellular Ca2+. Within 8 s after addition, ATP stimulated accumulation of the polyphosphatidylinositol products inositol (1, 4, 5) trisphosphate, inositol (1, 3, 4) trisphosphate, and inositol tetrakisphosphate. In addition to stimulating phosphatidylinositol turnover and Ca2+ mobilization, ATP and hydrolysis-resistant ATP analogues induced greater than 90% inhibition of the growth of all lines tested. These data demonstrate that human androgen-independent prostate carcinoma cells express functional P2-purinergic receptors linked to phospholipase C, and that agonists of this receptor are markedly growth inhibitory, suggesting a novel therapeutic approach to this common adult neoplasm.
W G Fang, F Pirnia, Y J Bang, C E Myers, J B Trepel
The central importance of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in the pathobiochemistry of a number of clinical disorders underscores the need for a comprehensive understanding of the regulation of their expression. This study was undertaken to examine the effects of cytokines on XDH/XO activity and gene expression in pulmonary endothelial cells. The results indicate that IFN-gamma is a potent inducer of XDH/XO activity in rat lung endothelial cells derived from both the microvasculature (LMVC) and the pulmonary artery. In contrast, interferon-alpha/beta, tumor necrosis factor-alpha, interleukin-1 or -6, lipopolysaccharide and phorbol myristate acetate have no demonstrable effect. The increase in XDH/XO activity requires new protein synthesis. By Northern analysis, IFN-gamma markedly increases the level of the 5.0-kb XDH/XO mRNA in LMVC. The increase is due, in part, to increased transcription rate of the XDH/XO gene. Transcriptional activation does not require new protein synthesis. The physiologic relevance of these observations was evaluated by administering IFN-gamma to rats. Intraperitoneal administration leads to an increased XDH/XO activity and XDH/XO mRNA level in rat lungs. In sum, IFN-gamma is a potent and biologically relevant inducer of XDH/XO expression; the major site of upregulation occurs at the transcriptional level.
G P Dupont, T P Huecksteadt, B C Marshall, U S Ryan, J R Michael, J R Hoidal
One method to improve the immunogenicity of polysaccharide antigens is the covalent coupling of the native polysaccharide or a derivative oligosaccharide to a carrier protein. In general, T cell-dependent properties are enhanced in conjugates of smaller saccharides, but a conformational epitope of the native polysaccharide may be better expressed in conjugates of larger saccharides. We have reported previously the synthesis and immunogenicity in animals of an oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. In this study, we sought to determine the optimal size of group B Streptococcus type III oligosaccharide for use in a conjugate vaccine by evaluating the relative immunogenicity of conjugate vaccines containing oligosaccharides that were twofold smaller (7,000 Mr) or larger (27,000 Mr) than that reported previously (14,500 Mr). All three type III oligosaccharide conjugate vaccines were immunogenic in rabbits, in contrast to native, uncoupled group B Streptococcus type III polysaccharide. However, with respect to eliciting specific antibodies that were protective in vivo, the vaccine containing the intermediate-size oligosaccharide was superior to the smaller or larger conjugate vaccine. Analysis of opsonic activity of vaccine-induced antibodies demonstrated a predominance of IgG antibodies, thought to reflect T cell dependence, in response to shorter chain length conjugates, while the conformational epitope of the native polysaccharide was maximally expressed on longer chain length conjugates. These opposing trends may account for the optimal immunogenicity of an intermediate-size group B Streptococcus type III oligosaccharide conjugate vaccine.
L C Paoletti, D L Kasper, F Michon, J DiFabio, H J Jennings, T D Tosteson, M R Wessels
Development of the human embryo depends on the ability of first trimester cytotrophoblastic stem cells to differentiate and invade the uterus. In this process, transient expression of an invasive phenotype is part of normal cytotrophoblast differentiation. Morphologically, this process begins when polarized chorionic villus cytotrophoblasts form multilayered columns of nonpolarized cells, and invade the uterus. Using immunocytochemistry, we compared the presence of adhesion receptors and extracellular matrix ligands on cytotrophoblasts in villi, cell columns, and the uterine wall. Villus cytotrophoblasts, anchored to basement membrane, stained for alpha 6 and beta 4 integrin subunits and both merosin and A-chain-containing laminin. Nonpolarized cytotrophoblasts in columns expressed primarily alpha 5 and beta 1 integrin subunits and a fibronectin-rich matrix. Cytotrophoblast clusters in the uterine wall stained for alpha 1, alpha 5, and beta 1 integrins, but not for most extracellular matrix antigens, suggesting that they interact primarily with maternal cells and matrices. Tenascin staining was restricted to stroma at sites of transition in cytotrophoblast morphology, suggesting that tenascin influences cytotrophoblast differentiation. Our results suggest that regulation of adhesion molecule expression contributes to acquisition of an invasive phenotype by cytotrophoblasts and provide a foundation for studying pathological conditions in which insufficient or excessive trophoblast invasion occurs, such as preeclampsia or choriocarcinoma.
C H Damsky, M L Fitzgerald, S J Fisher
Tumor necrosis factor (TNF)-treated 3T3-L1 adipocytes were used as a model for studying the effects of systemic inflammation on adipose tissue. Lipopolysaccharide-treated monocyte-conditioned medium or recombinant human TNF alpha induced morphological dedifferentiation of the adipocytes and led to loss of adipocyte specific gene expression. Gel shift, Southwestern and Western immunoblot analysis demonstrated that dedifferentiation was preceded by a decrease in the DNA binding activity and protein level of the transcription factor CCAAT/enhancer binding protein (C/EBP). Liver activating protein, a related protein that binds identical DNA sequences, increased during cytokine treatment. Both proteins activate specific enhancer elements located in the promoter region of many genes whose transcription is altered during systemic inflammation. Pulse-chase labeling followed by immunoprecipitation demonstrated that C/EBP is a rapidly turning over protein in adipocytes and that cytokine treatment led to a specific, time dependent decrease in its rate of synthesis. Because C/EBP binding sites have been shown to play an important role in regulating the expression of genes involved in adipocyte metabolism, we propose that the TNF-induced changes in the complement of transcription factors binding those sites may be important in the pathogenesis of inflammation-induced atrophy of adipose tissue.
D Ron, A R Brasier, R E McGehee Jr, J F Habener
The effect of taxol, which is a microtubule stabilizer, was examined in a model of acute edematous pancreatitis induced in rat by the administration of caerulein. Prophylactic administration of taxol ameliorated inhibition of pancreatic secretion, increased level of serum amylase, pancreatic edema, and histological alterations in this model. Immunofluorescence studies revealed that taxol stabilized the arrangement of microtubules by the action of promoting tubulin polymerization and prevented inhibition of pancreatic digestive enzyme secretion. In isolated rat pancreatic acini, taxol reversed the inhibition of amylase secretion induced by supramaximal concentrations of cholecystokinin octapeptide and did not affect the binding of cholecystokinin octapeptide to its receptor. The results obtained in this study suggest that microtubule disorganization is the initiating event in caerulein-induced pancreatitis and that the inhibition of pancreatic digestive enzyme secretion by interfering with intracellular vesicular transport due to microtubule disorganization causes caerulein-induced pancreatitis.
T Ueda, Y Takeyama, K Kaneda, M Adachi, H Ohyanagi, Y Saitoh
Vascular endothelial growth factor (VEGF) is a secreted heparin-binding mitogen; its growth-promoting activity is limited to vascular endothelial cells in vitro and VEGF also stimulates angiogenesis in vivo. To identify target cells for VEGF and investigate the potential physiological role of this factor, iodinated recombinant human VEGF (125I-rhVEGF) was used for in vitro ligand autoradiography on tissue sections from adult rats. 125I-rhVEGF exhibited saturable, displaceable binding to a single class of sites with high affinity and low capacity in all tissues and organs examined. Colocalization of 125I-rhVEGF binding with Factor VIII-like immunoreactivity demonstrated binding sites associated with vascular endothelial cells of both fenestrated and nonfenestrated microvessels and the endothelium of large vessels, while no displaceable binding was evident on nonendothelial cells. Specific binding was associated with quiescent as well as proliferating vessels. These findings support the hypothesis that VEGF plays a specific role in both the maintenance and in the induction of growth of vascular endothelial cells.
L B Jakeman, J Winer, G L Bennett, C A Altar, N Ferrara
Gold-specific T lymphocyte clones were isolated from a patient with rheumatoid arthritis who developed delayed type hypersensitivity reactions to gold. All of the isolated T cell clones required histocompatible antigen presenting cells as well as gold for induction of proliferation. Using a panel of HLA-homozygous Epstein Barr virus-transformed B (EBV-B) cells and anti-HLA antibodies, the clones were shown to recognize gold in the context of DR1 molecules. Gold recognition did not require active antigen processing since specific proliferation was not affected by glutaraldehyde fixation of the DR1 homozygous antigen presenting cells. Furthermore, we could show that gold salts inhibited peptide-induced responses of a peptide-specific T cell clone. In addition to providing evidence for gold-specific T cells in gold-treated RA patients exhibiting delayed type hypersensitivity responses, these data suggest that gold can alter MHC-peptide complexes. The latter observation may in part explain the mechanism/s responsible for both the therapeutic and the toxic effects of gold.
P Romagnoli, G A Spinas, F Sinigaglia
We have investigated the role of leukocyte-endothelial cell interactions in a rabbit model of hemorrhagic vasculitis. Microvascular injury was produced in the skin by intradermal injection of Salmonella typhosa endotoxin followed 20 h later by intravenous zymosan, which activates complement. Hemorrhagic necrosis develops in the "prepared" skin sites which is characterized by microthrombi, neutrophil aggregation, platelet and fibrin deposition, and massive extravasation of erythrocytes. Hemorrhage in these Shwartzman-like lesions was quantitated by 99mTc-labeled autologous erythrocytes. Inhibition of the hemorrhagic response was obtained with mAb reactive with ICAM-1 as well as mAb against the leukocyte CD18 when either was administered intravenously just before intravenous zymosan challenge. This observation suggests that an intravascular event occurring in response to complement activation is required for the development of hemorrhagic vasculitis. We hypothesize that agents which successfully prepare the skin for the Shwartzman response after their intradermal injection do so by promoting increased intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Activation of complement then induces CD11/CD18 expression on circulating leukocytes thus producing an intravascular CD11/CD18-ICAM-1 (leukocyte-endothelium) adhesion event. Inhibition of intravascular leukocyte-leukocyte aggregation with mAb against CD11b (Mac-1) showed partial inhibition of hemorrhage, while mAb against CD11a (LFA-1) showed no inhibitory activity. This type of cytokine-primed, neutrophil-dependent vascular damage may be a model of human vasculitic processes where microvascular damage is produced in the absence of immune-complex deposition.
L W Argenbright, R W Barton
No gene for a hematopoietic cell carboxypeptidase has previously been characterized. Mast cell carboxypeptidase A (MC-CPA) is a prominent secretory granule marker of mast cell differentiation and phenotype. The 32-kb human MC-CPA gene was isolated, localized to chromosome 3, and found to contain 11 exons. No significant homology was found between the 5' flanking region of the MC-CPA gene and those of three rat pancreatic carboxypeptidase genes (carboxypeptidase A1 and A2, and carboxypeptidase B [CPB]). In contrast, the intron/exon organization of the MC-CPA gene was conserved, most closely resembling the CPB gene. MC-CPA is unique among carboxypeptidases in having a CPA-like substrate-binding pocket and enzymatic activity despite overall protein and gene structures more similar to CPB. Evolutionary tree analysis of the carboxypeptidase gene family showed that, before the mammalian species radiation, a common MC-CPA/CPB ancestor diverged by gene duplication from the lineage leading to CPA, and then underwent another gene duplication to form separate but similar gene structures for MC-CPA and CPB. MC-CPA mRNA was prominent in dispersed lung cells enriched for mast cells but was undetectable in other nontransformed populations of several lineages, demonstrating that transcription of MC-CPA, a novel carboxypeptidase gene, provides a specific molecular marker for mast cells among normal hematopoietic cell populations.
D S Reynolds, D S Gurley, K F Austen
Insulin-dependent diabetes mellitus (IDDM) is thought to result from the autoimmune destruction of the insulin-producing beta cells of the pancreas. Years before IDDM symptoms appear, we can detect autoantibodies to one or both forms of glutamate decarboxylase (GAD65 and GAD67), synthesized from their respective cDNAs in a bacterial expression system. Individual IDDM sera show distinctive profiles of epitope recognition, suggesting different humoral immune responses. Although the level of GAD autoantibodies generally decline after IDDM onset, patients with IDDM-associated neuropathies have high levels of antibodies to GAD, years after the appearance of clinical IDDM. We note a striking sequence similarity between the two GADs and Coxsackievirus, a virus that has been associated with IDDM both in humans and in experimental animals. This similarity suggests that molecular mimicry may play a role in the pathogenesis of IDDM.
D L Kaufman, M G Erlander, M Clare-Salzler, M A Atkinson, N K Maclaren, A J Tobin
The enzyme steroid 5 alpha-reductase catalyzes the conversion of testosterone into the more potent androgen, dihydrotestosterone, and impairment of this reaction causes a form of male pseudohermaphroditism in which genetic males differentiate predominantly as phenotypic females. We previously isolated cDNA clones that encode a human steroid 5 alpha-reductase enzyme. Here, we report molecular and genetic studies demonstrating that the gene encoding this cDNA is normal in subjects with the genetic disease steroid 5 alpha-reductase deficiency. We further show that in contrast to the major steroid 5 alpha-reductase in the prostate and cultured skin fibroblasts, the cDNA-encoded enzyme exhibits a neutral to basic pH optima and is much less sensitive to inhibition by the 4-aza steroid, finasteride (MK-906). The results provide genetic, biochemical, and pharmacological support for the existence of at least two steroid 5 alpha-reductase isozymes in man.
E P Jenkins, S Andersson, J Imperato-McGinley, J D Wilson, D W Russell
Monochloramine (NH2Cl), a granulocyte-derived reactive oxygen metabolite (ROM), increases short-circuit current (Isc) in cultured T84 monolayers in a concentration-dependent manner up to nonlethal concentrations of 75 microM. Isc increases slowly after NH2Cl, reaching a peak value of 18 +/- 2 microA/cm2 20 min after addition. The Isc changes are persistent (lasting over 20-30 min), depend on medium Cl, and are inhibitable with bumetanide. 36Cl flux studies demonstrated that NH2Cl increases serosa-to-mucosa flux of Cl without changing mucosa-to-serosa flux, consistent with stimulation of electrogenic Cl secretion. Isc responses to NH2Cl, but not PGE2, are dependent on medium calcium. As demonstrated in fura-2-loaded T84 cells, NH2Cl increases free cytosolic calcium by influx of extracellular Ca2+ and by release of Ca2+ from endogenous stores. However, NH2Cl had no effect on phosphatidylinositol metabolism or cyclic nucleotide levels. We conclude that ROM directly stimulate electrolyte secretion, an effect in part mediated by increases in cytosolic Ca2+, possibly through increasing Ca2+ permeability of cellular membranes.
H Tamai, T S Gaginella, J F Kachur, M W Musch, E B Chang
The function of gamma delta T cells is still elusive. The nature of the antigens that they recognize and the mode of presentation of these antigens are largely unknown. The majority of human peripheral gamma delta T cells bear a V gamma 9/V delta 2 T cell receptor, and display nonclonal reactivity to mycobacteria, without restriction by MHC. It is unknown whether these cells have clonal antigenic specificity as well. Here we describe rheumatoid arthritis-derived V gamma 9/V delta 2 T cell clones, displaying dual antigenic recognition: a nonclonal, MHC-unrestricted recognition of mycobacteria, and a clonal recognition of a short tetanus toxin peptide presented by HLA-DRw53, a nonpolymorphic class II MHC molecule associated with susceptibility to rheumatoid arthritis. This is the first evidence that V gamma 9/V delta 2 T cells can recognize nominal antigenic peptides presented by class II MHC molecules. These results suggest that much like alpha beta T cells, V gamma 9/V delta 2 cells may contribute to the immune response against foreign antigens in an antigen-specific and MHC-restricted manner. The reactivity of these gamma delta T cells to mycobacteria may represent a superantigen-like phenomenon.
J Holoshitz, L M Vila, B J Keroack, D R McKinley, N K Bayne
Rapid endothelial cell migration and inhibition of thrombosis are critical for the resolution of denudation injuries to the vessel wall. Inhibition of the endothelial cell autocrine angiotensin system, with either the angiotensin-converting enzyme inhibitor lisinopril or the angiotensin II receptor antagonist sar1, ile8-angiotensin II, leads to increased endothelial cell migration and urokinase-like plasminogen activator (u-PA) activity (Bell, L., and J. A. Madri. 1990. Am. J. Pathol. 137:7-12). Inhibition of the autocrine angiotensin system with the converting-enzyme inhibitor or the receptor antagonist also leads to increased expression of the proto-oncogene c-src: pp60c-src mRNA increased 7-11-fold, c-src protein 3-fold, and c-src kinase activity 2-3-fold. Endothelial cell expression of c-src was constitutively elevated after stable infection with a retroviral vector containing the c-src coding sequence. Constitutively increased c-src kinase activity reconstituted the increases in migration and u-PA observed with angiotensin system interruption. Antisera to bovine u-PA blocked the increase in migration associated with increased c-src expression. These data suggest that increases in endothelial cell migration and plasminogen activator after angiotensin system inhibition are at least partially pp60c-src mediated. Elevated c-src expression with angiotensin system inhibition may act to enhance intimal wound closure and to reduce luminal thrombogenicity in vivo.
L Bell, D J Luthringer, J A Madri, S L Warren
Investigation of the in vitro ability of plasma from pregnant women to inhibit exogenous thrombin (25 nM) demonstrated that heparin cofactor II inhibited more thrombin (3.0 +/- 0.7 nM, mean +/- SD) than plasma from women 3-5 d postpartum (1.9 +/- 0.5 nM) or plasma from nonpregnant adults (1.5 +/- 0.4 nM). Levels of heparin cofactor II were only slightly increased over normal in both pregnant and postpartum women and did not account for the observed increase in thrombin bound to heparin cofactor II. Assay of pregnancy plasma for dermatan sulfate anticoagulant activity demonstrated the presence of activity equivalent to 0.23 +/- 0.02 micrograms/ml of porcine mucosal dermatan sulfate. This activity could not be demonstrated in normal adult plasma or plasma from women on the contraceptive pill. The mass of dermatan sulfate in pregnancy and umbilical cord plasmas was increased over adult control plasma by 0.20 micrograms/ml (53%) and 0.29 micrograms/ml (76%), respectively. The glycosaminoglycan-containing fraction of plasma was isolated and an assay for anticoagulant dermatan sulfate confirmed its presence in both pregnancy and cord plasmas but minimal activity in adult plasma. Gel chromatography of isolated fractions from both pregnancy and cord plasmas revealed a polydisperse, active species with apparent Mr 150,000 D. Reductive elimination decreased the apparent Mr of the active species on gel chromatography to 31,000 D for cord and 21,000 D for pregnancy products. This confirmed the presence of an anticoagulant active dermatan sulfate proteoglycan circulating in the plasmas of pregnant women at term and fetuses at delivery.
M Andrew, L Mitchell, L Berry, B Paes, M Delorme, F Ofosu, R Burrows, B Khambalia
Immunological responses to bacterial heat shock proteins have been implicated in the pathogenesis of arthritis in animals and humans. The predicted amino acid sequence of dnaJ, a heat shock protein from Escherichia coli, contains an 11-amino acid segment that is homologous to the third hypervariable region of the human histocompatibility antigen (HLA) DRB10401 (formerly known as HLA Dw4), the part of the molecule that carries susceptibility to rheumatoid arthritis. To test the biological significance of this finding, we expressed and purified recombinant dnaJ (rdnaJ), and determined its immunologic cross-reactivity with HLA DRB10401. A rabbit antipeptide antiserum raised against the sequence of the third hypervariable region of HLA DRB10401 specifically bound to 'dnaJ, thus confirming that a similar sequence is expressed on the bacterial protein. Of greater consequence, an antiserum to the 'dnaJ protein recognized not only a peptide from the third hypervariable region of HLA DRB10401, but also the intact HLA DRB10401 polypeptide. Furthermore, the antibody to 'dnaJ reacted with HLA DRB10401 homozygous B lymphoblasts, but not with HLA DRB11501, DRB10101, DRB10301, and DRB10701 (formerly known as HLA Dw2, DR 1, DR 3, and DR 7, in the same order) homozygous cells. These results demonstrate that exposure to a bacterial heat shock protein can elicit antibodies against the rheumatoid arthritis susceptibility sequence in the third hypervariable region of HLA DRB10401.
S Albani, J E Tuckwell, L Esparza, D A Carson, J Roudier
Individuals with chronic hepatitis B virus (HBV) infection are generally divided into asymptomatic healthy carriers and patients with chronic liver disease. Several studies have suggested that the hepatitis B core antigen could be an immunological target of cytotoxic T lymphocytes (CTL). To investigate the possible pressure site from CTL, the entire core region of HBV DNA was sequenced in 30 subjects (10 asymptomatic healthy carriers and 20 patients with chronic liver disease). No significant changes in the nucleotide sequence and deduced amino acid residue were noted in the 10 healthy carriers. In contrast, a cluster of changes in a small segment of 18 amino acids (codons 84-101 from the start of the core gene) was found in 15 of the 20 chronic liver disease patients. All these 15 patients had advanced liver diseases (chronic active hepatitis and cirrhosis), whereas only mild liver disease (chronic persistent hepatitis) was found in the five patients without mutations. These data suggest that the region with mutation clustering is the major target of CTL, and that the mutations evolve under the pressure of immune selection.
T Ehata, M Omata, O Yokosuka, K Hosoda, M Ohto
Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.
G M Denning, L S Ostedgaard, S H Cheng, A E Smith, M J Welsh