Inflammatory cues enhance TGFß activation by distinct subsets of human intestinal dendritic cells via integrin αvß8.

Regulation of intestinal T-cell responses is crucial for immune homeostasis and prevention of inflammatory bowel disease (IBD). A vital cytokine in regulating intestinal T cells is transforming growth factor-ß (TGFß), which is secreted by cells as a latent complex that requires activation to function. However, how TGFß activation is regulated in the human intestine, and how such pathways are altered in IBD is completely unknown. Here we show that a key activator of TGFß, integrin αvß8, is highly expressed on human intestinal dendritic cells (DCs), specifically on the CD1c+ but not the CD141+ intestinal DC subset. Expression was significantly upregulated on intestinal DC from IBD patients, indicating that inflammatory signals may upregulate expression of this key TGFß-activating molecule. Indeed, we found that the Toll-like receptor 4 ligand lipopolysaccharide upregulates integrin αvß8 expression and TGFß activation by human DC. We also show that DC expression of integrin αvß8 enhanced induction of FOXP3 in CD4+ T cells, suggesting functional importance of integrin αvß8 expression by human DC. These results show that microbial signals enhance the TGFß-activating ability of human DC via regulation of integrin αvß8 expression, and that intestinal inflammation may drive this pathway in patients with IBD.

A role for the integrin alphavbeta8 in the negative regulation of epithelial cell growth.

The control of cell growth is regulated through coordinated responses to growth factors and cell-extracellular matrix (ECM) interactions. Integrins, the major family of cell-ECM receptors, are vital to these coordinated responses. Although much is known of the role of integrins in growth promotion, specific examples of integrin-mediated cell growth inhibition are few. On the basis of our findings that the integrin beta8 subunit is expressed in airway epithelial cells and is absent in lung cancers, we investigated the role and mechanism of the integrin alphavbeta8 in mediating growth inhibition. When introduced into either a lung or colon carcinoma cell line, beta8 inhibited cell growth without inducing apoptosis. Ligation of alphavbeta8 also induced cell rounding, inhibited focal contact formation, and initiated an inhibitory signaling pathway as demonstrated by increased expression of the cyclin-dependent kinase inhibitor p21Cip1. The cytoplasmic domain of beta8 was capable of both growth inhibition and causing cell shape changes as shown by the use of a chimeric integrin construct consisting of the beta8-cytoplasmic domain coupled to the beta6-extracellular domain. Finally, when tested in vivo, beta8 potently inhibited tumor growth in nude mice. Together, these results implicate alphavbeta8 as a novel growth-regulatory molecule of epithelial cells.

Pulmonary and mediastinal glomus tumors--report of five cases including a pulmonary glomangiosarcoma: a clinicopathologic study with literature review.

Pulmonary and mediastinal glomus tumors are rare lesions, with four previously reported primary pulmonary cases and three mediastinal cases. The authors report one mediastinal glomus tumor, a locally infiltrative type, and four pulmonary glomus tumors, including the first case of primary pulmonary glomangiosarcoma. These tumors show a variety of clinical and pathologic differences from the more common cutaneous variety, including later age at presentation, larger size, and more frequent atypical/malignant features. Mediastinal and pulmonary glomus tumors both have an average patient age at presentation of 45 years. However, compared with their pulmonary counterparts, mediastinal glomus tumors are less common, more often symptomatic, and are larger (average size, 5.4 cm). Additionally, mediastinal glomus tumors more often demonstrate malignant or atypical features. Pulmonary glomus tumors average 3.3 cm in greatest dimension, with the majority measuring less than 2.5 cm. The pulmonary glomangiosarcoma presented was large, measuring 9.5 cm, and showed increased mitotic count (9 mitoses/10 high-power fields), necrosis, cytologic atypia, and was associated with disseminated disease. Regardless of clinical symptoms, histologic features, and even metastases, the vast majority of all benign and malignant glomus tumors are indolent and cured surgically, with adjuvant therapy needed only for occasional patients with more advanced disease. The four patients with glomus tumors reported are currently alive and free of disease as of last follow up. The patient with the glomangiosarcoma developed widespread metastases and died of disease 68 weeks after initial therapy.

Synaptic and glial localization of the integrin alphavbeta8 in mouse and rat brain.

Integrins are a large family of cell adhesion receptors mediating cell-extracellular matrix (ECM) interactions and are widely distributed in tissues. The beta8 integrin subunit mRNA has been shown to be expressed at higher levels in the central nervous system (CNS) than in other organs [M. Moyle, M.A. Napier, J.W. McLean, Cloning and expression of a divergent integrin subunit beta8, J. Biol. Chem. 266 (29) (1991) 19650-19658] but its cellular and subcellular localization in the CNS are unknown. In this report, we demonstrate that beta8 pairs exclusively with the alphav subunit in the CNS to form the alphavbeta8 heterodimer. Immunohistochemical analysis of the distribution of beta8 in adult mouse and rat brains revealed that the protein is expressed in several regions of the hippocampal formation and in the molecular layer and glomeruli of the granular cell layer of the cerebellum. Punctate and diffuse immunolabeling was observed occasionally surrounding neuronal pericarya and extensively throughout dendritic fields suggesting both pre- and post-synaptic localization and/or expression in non-neuronal cells. By immunoelectron microscopy, beta8 immunoreactivity was detected in dendritic spines where it was often localized at post-synaptic densities, occasionally in axon terminals and in glial processes. Association of beta8 with synaptic membranes was further supported by its enrichment in synaptosomal preparations as detected by immunoblotting. These results demonstrate that alphavbeta8 is present in mature synapses and therefore may play a role in synaptic function.

Asbestos-induced pleural disease.

Asbestos-induced pleural disease has become the most common manifestation of asbestos exposure. Asbestos has an unusual affinity for the pleural space and leads to plaques, benign effusions, fibrosis, and malignant mesothelioma. The explanation for its affinity for the pleura may lie in part with new evidence showing that asbestos fibers can accumulate in certain regions of the parietal pleura at higher concentrations than in the lung. With the control of industrial exposures to asbestos, the incidence of this disease should decrease, with the incidence of mesothelioma peaking in the years 2000 to 2020. Nonetheless, the toxic features of asbestos including shape, chemical composition, and surface characteristics should be understood to avoid toxicity in fibers used to replace asbestos and to know the risks of low level exposures from asbestos currently in our environment.

Biochemical characterization of high-affinity 3H-opioid binding. Further evidence for Mu1 sites.

In saturation studies with [3H]dihydromorphine, unlabeled D-Ala2-D-Leu5-enkephalin (1 nM) inhibited the high-affinity binding component far more potently than the lower-affinity one. Similarly, morphine (1 nM) inhibited the higher-affinity binding of 3H-D-Ala2-D-Leu5-enkephalin to a greater extent than its lower-affinity binding component, consistent with a common high-affinity binding site for opiates and enkephalins. Treatment of tissue with either trypsin (1 microgram/ml) or N-ethylmaleimide (25 microM) effectively eliminated the high-affinity binding component of a series of 3H-opiates and opioid peptides. Competition studies following both treatments were consistent with a common high-affinity binding site. Both treatments also eliminated the ability of low morphine concentrations (less than 1 nM) to inhibit 3H-D-Ala2-D-Leu5-enkephalin binding and of low D-Ala2-D-Leu5-enkephalin concentrations (less than 1 nM) to inhibit [3H]dihydromorphine binding. Protection experiments examining N-ethylmaleimide (25 microM) inhibition of [3H]dihydromorphine binding showed significant protection (p less than 0.002) by both unlabeled D-Ala2-D-Leu5-enkephalin and morphine (both at 1 nM). When studied together, both naloxonazine and N-ethylmaleimide inhibited [3H]dihydromorphine binding to a similar extent. Equally important, tissue previously treated with naloxonazine was far less sensitive to N-ethylmaleimide than was untreated control tissue, consistent with the possibility that both treatments affected the same site. Together, these results support the concept of a common high-affinity binding site for opiates and opioid peptides.

Integrin alpha v beta 8. Interaction with vitronectin and functional divergence of the beta 8 cytoplasmic domain.

The integrin beta 8 subunit was identified by cloning and sequencing of the cDNA and has been shown to associate with the alpha v subunit (Moyle, M., Napier, M. A., and McLean, J. W. (1991) J. Biol. Chem. 266, 19650-19658). We now present initial data on its functional properties. We produced a recombinant secreted form of alpha v beta 8 and used it to raise monoclonal antibodies that recognize the alpha v beta 8 complex or the beta 8 subunit alone on the surface of melanoma cells and on beta 8-transfected human embryonic kidney (293) cells. Affinity chromatography experiments showed that secreted alpha v beta 8 bound to vitronectin but not to fibronectin, collagen, or fibrinogen. Supporting evidence that intact full-length alpha v beta 8 could also bind to vitronectin-Sepharose was provided by performing affinity chromatography with the melanoma cell line MeWo, which normally expresses the intact beta 8 subunit. By studying the adhesive properties of melanoma cells and beta 8-transfected 293 cells, we found that alpha v beta 8 by itself does not promote cell adhesion on a vitronectin-coated substrate. To test the respective functional activities of the beta 8 extracellular and cytoplasmic domains, we analyzed chimeric beta 8/beta 3 subunit constructs. The beta 3 subunit was chosen because full-length beta 3, when transfected into 293 cells, strongly supports cell adhesion. We found that a chimeric integrin containing the beta 3 extracellular domain combined with the beta 8 transmembrane and cytoplasmic domains did not promote 293 cell adhesion. Conversely, a chimeric integrin construct combining the beta 8 extracellular domain with the beta 3 transmembrane and cytoplasmic domains did promote adhesion of transfected 293 cells. This suggests that the beta 8 cytoplasmic domain does not interact with the cytoskeleton and with cytoplasmic signaling pathways in an adhesion-promoting fashion. We conclude that the beta 8 cytoplasmic domain, which is structurally unrelated to the conserved cytoplasmic domains of other beta subunits, is functionally distinct.

Stromal fibroblasts influence oral squamous-cell carcinoma cell interactions with tenascin-C.

In this study we identified tenascin-C (TN-C) and one of its integrin receptors, alpha(v)beta6, in oral squamous-cell carcinoma (SCC) specimens. Neither TN-C nor alpha(v)beta6 are expressed in normal oral mucosa. We also studied 2 human oral squamous-cell carcinoma cell lines: the highly invasive HSC-3 cells, and the poorly invasive SCC-25 cells. We determined that adhesion of these cells to TN-C involves both alpha2 and alpha(v) integrins. Migration on TN-C by oral SCC cells required fibroblast-conditioned medium and did not occur in its absence. This migration was blocked by anti-alpha2 and anti-alpha(v) antibodies and was partially inhibited by antibodies to hepatocyte growth factor, epidermal growth factor and transforming growth factor-beta1. When seeded on TN-C, the poorly invasive SCC-25 cells formed alpha(v)beta6-positive focal contacts; the HSC-3 cells did not. HSC-3, SCC-25 and PTF cells secrete TN-C into the culture medium, as determined by Western blot. However, when HSC-3 cells were inoculated into the floor of the mouth of nude mice, only murine TN-C could be identified in the reactive stroma adjacent to the resulting tumor nests, demonstrating that in vivo, HSC-3 cells do not secrete TN-C. Our results demonstrate that alpha(v)beta6 and tenascin-C are neo-expressed in oral squamous-cell carcinoma, and that the tumor stromal environment is influential in oral SCC behavior.

Evaluation of an enzyme immunoassay for detection of cryptococcal capsular polysaccharide antigen in serum and cerebrospinal fluid.

The Premier enzyme immunoassay (Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared with a latex agglutination assay (CALAS; Meridian) for the ability to detect cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid (CSF). A total of 594 specimens (471 serum samples and 123 CSF samples) obtained from 430 patients, most of whom were at risk for or had AIDS, were tested in parallel by both systems. Both tests were independently evaluated for their ability to (i) detect CrAg when used as a screening test and (ii) quantitate the CrAg present when used as a titration assay. Chart review to assess clinical outcome after the time of specimen collection was conducted for all patients. When both assays were used as screening assays, 103 serum samples and 18 CSF samples were positive and 356 serum samples and 104 CSF specimens were negative by both assays (97.8% concordance). Thirteen specimens (12 serum samples, 1 CSF sample) gave discrepant screening results. When the tests were used as semiquantitative assays for titer determinations, the CrAg titers determined by the enzyme immunoassay were generally higher than those obtained with the latex agglutination assay. In summary, results obtained with the enzyme immunoassay correlated well with those obtained with the latex agglutination test for screening for the presence of CrAg and for determining the titer of CrAg in serum or CSF.

Clinical and pathological features of bacillary peliosis hepatis in association with human immunodeficiency virus infection.

BACKGROUND: Peliosis hepatis is characterized by cystic, blood-filled spaces in the liver and is seen in patients with chronic infections or advanced cancer and as a consequence of therapy with anabolic steroids. Cutaneous bacillary angiomatosis is a bacterial infection that occurs in patients with human immunodeficiency virus (HIV) infection; its histologic appearance is that of a pseudoneoplastic vascular proliferation. METHODS: We studied liver tissue from eight HIV-infected patients with peliosis hepatis, two of whom also had cutaneous bacillary angiomatosis. For comparison we examined tissue from four patients who had peliosis hepatis without HIV infection. Tissues were examined histologically on routine sections and with special stains and electron microscopy. RESULTS: The histologic features seen in peliosis hepatis associated with HIV infection, but not in the four cases unrelated to HIV infection, were myxoid stroma and clumps of a granular purple material that on Warthin-Starry staining and electron microscopy proved to be bacilli. The bacilli, which could not be cultured, were morphologically identical to those found in the skin lesions of cutaneous bacillary angiomatosis. The clinical courses of two of the patients with this "bacillary peliosis hepatis" indicate that it responds to antibiotic treatment. CONCLUSIONS: HIV-associated bacillary peliosis hepatis is an unusual, treatable opportunistic infection, probably caused by the same organism that causes cutaneous bacillary angiomatosis. Our failure to find bacilli in non-HIV-associated cases implies that other pathogenetic mechanisms may also be responsible for peliosis hepatis.

Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling.

The alpha v beta 6 integrin was identified in cultured epithelial cells and functions as a fibronectin receptor. We have now used monoclonal antibodies to determine in vivo expression patterns of the beta 6 subunit in normal and pathological human or primate tissues, and during experimental wound healing or induced lung injury. The results indicate that beta 6 expression is restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumorigenesis, and epithelial repair. During development of the kidney, lung, and skin, we found that beta 6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin. In contrast, we detected high-level expression in several types of carcinoma. For example, beta 6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, often focally localized at the infiltrating edges of tumor islands. Expression of beta 6 is also upregulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing. Similarly, beta 6 expression is induced in type II alveolar epithelial cells during lung injury caused by injection of live bacteria. We also observed beta 6 expression in adult lungs and kidneys at focal sites of subclinical inflammation, as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys. From these findings and earlier results, we hypothesize that alpha v beta 6 affects cell spreading, migration and growth during reorganization of epithelia in development, tissue repair, and neoplasia.