Bryan R. Hewlett

Transfer of Tumor Necrosis Factor-α to Rat Lung Induces Severe Pulmonary Inflammation and Patchy Interstitial Fibrogenesis with Induction of Transforming Growth Factor-β1 and Myofibroblasts

Tumor necrosis factor-alpha is up-regulated in a variety of different human immune-inflammatory and fibrotic pulmonary pathologies. However, its precise role in these pathologies and, in particular, the mechanism(s) by which it may induce fibrogenesis are not yet elucidated. Using a replication-deficient adenovirus to transfer the cDNA of tumor necrosis factor-alpha to rat lung, we have been able to study the effect of transient but prolonged (7 to 10 days) overexpression of tumor necrosis factor-alpha in normal adult pulmonary tissue. We have demonstrated that local overexpression resulted in severe pulmonary inflammation with significant increases in neutrophils, macrophages, and lymphocytes and, to a lesser extent, eosinophils, with a peak at day 7. By day 14, the inflammatory cell accumulation had declined, and fibrogenesis became evident, with fibroblast accumulation and deposition of extracellular matrix proteins. Fibrotic changes were patchy but persisted to beyond day 64. To elucidate the mechanism underlying this fibrogenesis, we examined bronchoalveolar fluids for the presence of the fibrogenic cytokine transforming growth factor-beta1 and tissues for induction of alpha-smooth muscle actin-rich myofibroblasts. Transforming growth factor-beta1 was transiently elevated from day 7 (peak at day 14) immediately preceding the onset of fibrogenesis. Furthermore, there was a striking accumulation of myofibroblasts from day 7, with the most extensive and intense immunostaining at day 14, ie, coincident with the up-regulation of transforming growth factor-beta1 and onset of fibrogenesis. Thus, we have provided a model of tumor necrosis factor-alpha-mediated pulmonary inflammation and fibrosis in normal adult lung, and we suggest that the fibrogenesis may be mediated by the secondary up-regulation of transforming growth factor-beta1 and induction of pulmonary myofibroblasts.

Gastrointestinal stromal tumors may originate from a subset of CD34-positive interstitial cells of Cajal.

Most gastrointestinal stromal tumors (GISTs), a subgroup of mesenchymal neoplasms of the gut wall, express both Kit (CD117) and CD34 proteins. It has been suggested that GISTs originate from or differentiate into interstitial cells of Cajal (ICC), after several reports indicated that ICC are likely the only cells in the gut which express both Kit and CD34. ICC are among the few cell types resident in the gut which express Kit, together with mast cells. However, the question whether or not ICC express CD34 is currently disputed. Using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) on cultured murine intestinal cells, single ICC were selected by morphology and tested for the expression of c-kit and CD34 mRNA. Most ICC were only c-kit-positive, however a subset (7 out of 43) were double positive for both c-kit and CD34. In the human small intestine, sequential immunohistochemical staining for Kit and CD34 proteins on the same 3-microm sections showed that some of the ICC surrounding Auerbachs plexus and ICC within the circular muscle layer of the small intestine were positive for both Kit and CD34. In addition, CD34(+)Kit(-) cells were seen adjacent to ICC. These data from two different techniques indicate that ICC can be double positive for Kit and CD34. Thus, GISTs with the Kit(+)CD34(+) phenotype may arise from a subpopulation of CD34(+) Kit(+) ICC.

Immunohistochemical evidence for estrogen receptors in meibomian glands.

PURPOSE To look for sex hormone receptor distribution in three structures contributing to the normal human tear film: the conjunctiva, the accessory lacrimal glands, and the meibomian glands. DESIGN An immunohistochemical study. TISSUES AND CONTROLS: Forty-one upper eyelid specimens were collected from 15 male and 26 female patients (age range, 1.5-85 years) during blepharoptosis surgery via posterior tarsoconjunctival mullerectomy (Fasanella-Servat or Gavaris). In addition, control sections of histologically normal breast, prostate, and skin tissue were obtained. METHODS Immunohistochemical staining using mouse monoclonal antibodies against estrogen, progesterone, and androgen receptors was performed on all tissues and controls. Quantitation of the receptors was performed and expressed as percentage nuclear positivity. Specimens were divided into three groups based on the age of the patient: <12 years (n = 9); 18-55 years (n = 1); >55 years (n = 12). RESULTS Forty-one specimens contained conjunctiva. All were negative for estrogen, progesterone, and androgen receptors. Twenty-four specimens contained accessory lacrimal glands of Wolfring. All were negative for the three receptors. Twenty-two specimens contained meibomian glands. All were positive for estrogen receptors; one was positive for progesterone receptors and one for androgen receptors. Using Minitab statistical software (Minitab Inc. State College, PA), analysis of variation revealed no statistical difference between sexes or between age groups studied. The sebaceous glands of skin were uniformly positive for androgen receptors. Sebaceous glands of the face and scalp (3 of the 15 skin samples) were also positive for estrogen receptors. CONCLUSIONS Estrogen receptors are present in the meibomian glands of the upper eyelid. Unlike sebaceous glands elsewhere on the skin, the meibomian glands lack androgen receptors. Estrogen receptors may play a role in modulation of the lipid layer of the tear film, and their activity may be linked to meibomian gland dysfunction and dry eye syndrome.

Cytokeratin 18 Is Expressed on the Hepatocyte Plasma Membrane Surface and Interacts with Thrombin-Antithrombin Complexes

During experiments to identify putative hepatic receptors for thrombin-antithrombin (TAT) complexes, a 45-kDa protein was identified by ligand blotting. Following gel purification, amino acid sequencing revealed the 45-kDa TAT-binding polypeptide to be cytokeratin 18 (CK18). The presence of CK18 on the surface of intact rat hepatoma cells was demonstrated by binding of125I-anti-CK18 antibodies. Anti-CK18 antibodies reduced the binding and internalization of 125I-TAT by rat hepatoma cells. Immunocytochemical analysis, to determine the location of CK18 in vivo, revealed a periportal gradient of CK18 staining; with hepatocytes around the portal triads demonstrating striking pericellular staining. In addition, anti-CK18 IgG associated with perfused livers to a significantly greater extent than preimmune IgG. Taken together, these data provide evidence that CK18 is found on the extracellular surface of hepatocytes and could play a role in TAT removal. Finally, these data, in conjunction with recent reports of CK8 (Hembrough, T. A., Li, L., and Gonias, S. L. (1996)J. Biol. Chem. 271, 25684–25691) and CK1 cell membrane surface expression (Schmaier, A. H. (1997) Thromb. Hemostasis 78, 101–107), indicate a novel role for these proteins as putative cellular receptors or cofactors to cellular receptors.

T Cell-Mediated Exocrine Pancreatic Damage in Major Histocompatibility Complex Class II-Deficient Mice

BACKGROUND & AIMS Recent observations suggest a role for lymphocytes in human pancreatitis. However, existing animal models of pancreatitis are not immunologically based. In studies on major histocompatibility complex (MHC) II-deficient mice backcrossed five generations onto a C57BL/6 background, we discovered a progressive wasting disease due to pancreatic damage. The purpose of this study was to characterize this model of immune-based pancreatic injury. METHODS The pathology was characterized histologically and functionally by assaying for pancreatic enzymes and glucose. RESULTS By 6 months, a periductal lymphocytic infiltrate was observed that later developed into pancreatic lesions with extensive, but selective, destruction of acinar cells. Mice eventually lost weight, developed a hunched appearance, and began to pass large, pale pellets. Histology of affected mice revealed pancreatic atrophy with almost complete loss of acinar cells, although islets remained intact. Serum levels of amylase, lipase, and glucose confirmed the selective loss of the exocrine pancreas, with both amylase and lipase levels being significantly decreased in affected mice. However, glucose levels remained unaffected. Adoptive transfer of splenic mononuclear cells to athymic mice was found to transfer the disease. CONCLUSIONS Aged MHC II-deficient mice develop an immune-based pancreatitis with selective loss of exocrine cells and function.

Distribution of adrenergic receptor subtypes in the retractor muscles of the upper eyelid

PURPOSE To identify adrenergic receptor subtypes and their relative distribution in the retractor muscles of the upper eyelid, the levator palpebrae superioris, and the Müller muscle. The pattern of distribution of these receptors in the Müller muscle was further compared in patients with dysthyroid eyelid retraction and in normal subjects. METHODS Müller muscle specimens were collected from 19 patients undergoing ptosis correction and from 8 patients undergoing repair of dysthyroid eyelid retraction. Immunohistochemical staining for alpha 1-, alpha 2-, beta 1-, and beta 2-adrenergic receptors was performed using antihuman rabbit polyclonal antibodies. RESULTS alpha 2-Adrenergic receptors were the predominant subtype in the Müller muscle, and beta 1-adrenergic receptors were the predominant subtype in the levator muscle. There was no significant difference in the staining pattern between specimens collected from patients with dysthyroid eyelid retraction and those from normal subjects. CONCLUSIONS The interaction between the alpha 2 and beta 1 receptors in the upper eyelid retractor muscles may be important in the control of the upper eyelid position and may contribute to the development of dysthyroid eyelid retraction. Specific alpha 2 antagonists could be developed and may be effective pharmacologic agents for the treatment of eyelid retraction.

Characterization of adrenergic receptors in the accessory lacrimal glands of the upper eyelid.

PURPOSE To identify the histologic location as well as the exact subtype of adrenergic receptors in the accessory lacrimal glands of the upper eyelid. METHODS Upper eyelid specimens were collected from 19 patients undergoing routine blepharoptosis correction via a posterior tarsoconjunctival mullerectomy. Immunohistochemical staining using polyclonal antibodies against human alpha 1, alpha 2, beta 1, and beta 2 receptors was performed on all specimens. RESULTS beta 1 receptors were the predominant adrenergic receptor subtype in the glands of Wolfring. CONCLUSIONS The presence of beta 1 receptors in the accessory lacrimal glands of the upper eyelid may suggest a possible role for selective beta 1 agonists in the treatment of keratitis sicca.