PubMed Health⌕ Search

Biomedical subjects

R A Panettieri

Publications and source records attributed to R A Panettieri.

At least 19 recordsLinked to original sources
PubMed1997

Antigen receptor-stimulated peripheral blood and bronchoalveolar lavage-derived T cells induce MHC class II and ICAM-1 expression on human airway smooth muscle.

The current model of lymphocyte extravasation into areas of inflammation involves the sequential engagement of multiple cell adhesion molecules (CAMs) expressed on lymphocytes and endothelial cells. In addition, the expression of CAMs and the elaboration of matrix by subendothelial/submucosal cells may contribute to the retention and stimulation of infiltrating cells in an inflammatory lesion. We previously demonstrated that mitogen-activated T cells adhered to airway smooth muscle (ASM) in an integrin-dependent fashion. ASM are MHC class II-negative and expressed low basal levels of intercellular adhesion molecule-1 (ICAM-1). In this study, we demonstrate that anti-CD3-stimulated peripheral blood T cells also adhere to ASM and markedly upregulate ICAM-1 expression and induce the expression of MHC class II on ASM. The induction of HLA-DR was completely inhibited, and the induction of ICAM-1 partially inhibited, by neutralizing antibody against interferon-gamma. Furthermore, in studies with bronchoalveolar lavage-derived T cells isolated from atopic donors following local antigen challenge, we observed adhesion to ASM and upregulation of ASM expression of ICAM-1 and HLA-DR similar to that seen with in vitro-activated T cells. Finally, we found that despite expression of ICAM-1 and HLA-DR, ASM could not present alloantigen to CD4+ T cells. These findings suggest that the interaction of activated T cells with parenchymal cells of the lung such as airway smooth muscle affects the phenotype of myocytes and thus may have significant implications for inflammatory diseases such as asthma or transplant rejection.

Antigen Presentation↗
PubMed1997

TGF-beta 1 modulates human airway smooth-muscle cell proliferation induced by mitogens.

Asthma is a disease of airway inflammation and bronchoconstriction that results in airway smooth-muscle cell hypertrophy and hyperplasia. The underlying mechanisms that induce myocyte proliferation remain unknown. Evidence suggests that cytokines such as transforming growth factor (TGF)-beta 1 may play a role in modulating this process. In this study, we examined the effects of TGF-beta 1 on human airway smooth-muscle (HASM) cell proliferation. We found that treatment of HASM cells with TGF-beta 1 inhibited epidermal growth factor (EGF)- and thrombin-induced DNA synthesis. This inhibition was both dose and time dependent. We then investigated whether these effects are mediated through activation of mitogen-activated protein kinase (MAP kinase), an enzyme that is thought to play a central role in the regulation of cell proliferation. We found that MAP kinase activation induced by EGF was not modulated by TGF-beta 1, despite TGF-beta 1 inhibiting EGF-induced HASM cell growth. These data suggest that TGF-beta 1 inhibits mitogen-induced HASM cell proliferation, but does so downstream from MAP kinase activation, or via a parallel pathway that is independent of MAP kinase activation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon.

1. In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin-1 (ET-1), ET-3, and sarafotoxin S6c (S6c), the ETB receptor-selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2. In confluent, growth-arrested human airway smooth, neither ET-1 (0.01 nM-1 microM) nor ET-3 (0.001 nM-1 microM) or S6c (0.01 nM-1 microM) induced cell proliferation, as assessed by [3H]-thymidine incorporation. In contrast, EGF (1.6 pM-16 nM) produced concentration-dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16 nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16-16 nM) also produced a concentration-dependent increase in cell counts, whereas ET-1 (1-100 nM) was without effect on this index of mitogenesis. 3. ET-1 (1-100 nM) potentiated EGF-induced proliferation of human tracheal smooth muscle cells. For example, ET-1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET-1 on EGF-induced proliferation was antagonized by BQ-123 (3 microM), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ-788 (10 microM). 4. Neither ET-3 (1-100 nM) nor S6c (1-100 nM) influenced the mitogenic effects of EGF (0.16-1.6 nM). 5. [125I]-ET-1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 ( +/- 3; n = 4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6. These data indicate that ET-1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor-mediated mechanism. These findings suggest that ET-1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.

Amino Acid Sequence↗
PubMed1996

Pharmacological activation changes stiffness of cultured human airway smooth muscle cells.

Using magnetic twisting cytometry (MTC), we measured the cytoskeletal stiffness of adherent human airway smooth muscle (HASM) cells. We hypothesized that modulation of actin-myosin interactions by application of contractile agonists would induce changes in cytoskeletal stiffness. In cells plated on high-density collagen, bradykinin (10(-6) M) and histamine (10(-4) M) increased stiffness by 85 +/- 15 and 68 +/- 16%, respectively. Increases in cell stiffness were also consistently observed after acetylcholine, substance P, and KCl. The bronchodilator agonists isoproterenol, prostaglandin E2, forskolin, dibutryl adenosine 3', 5'-cyclic monophosphate, and 8-bromoguanosine 3', 5'-cyclic monophosphate each caused a dose-dependent decrease in cell stiffness in unstimulated as well as bradykinin-treated cells. HASM cells plated on high-density collagen were stiffer than cells plated on low-density collagen (126 +/- 16 vs. 43 +/- 3 dyn/cm2) and developed more pronounced increases in stiffness in response to bradykinin as well as more pronounced decreases in stiffness in response to isoproterenol. These results are consistent with the hypothesis that modulation of actin-myosin interactions by application of contractile agonists causes changes in cytoskeletal stiffness of HASM cells. MTC may be a valuable tool for evaluating the mechanisms of pharmacomechanical coupling in airway smooth muscle cells in culture.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Activation of the TNF alpha-p55 receptor induces myocyte proliferation and modulates agonist-evoked calcium transients in cultured human tracheal smooth muscle cells.

Evidence suggests that cytokines may modulate smooth muscle cell function in a variety of inflammatory diseases. In the present study, we characterized the specific receptor subtypes that mediate tumor necrosis factor alpha (TNF alpha) effects on myocyte proliferation and on agonist-induced calcium transients in cultured human tracheal smooth muscle cells (TSMC). Pretreatment of human TSMC with TNF alpha potentiated cytosolic calcium [(Ca2+)i] transients evoked by carbachol. In a similar manner, selective TNF alpha-p55 receptor agonists such as htr-9, an activating monoclonal antibody, or a recombinant TNF-p55 (rTNF-p55), which specifically activates the TNF alpha-p55 receptor but not the TNF alpha-p75 receptor, also augmented [Ca2+]i transients evoked by carbachol. In parallel experiments, TNF alpha, rTNF alpha-p55, and htr-9 induced human TSMC proliferation as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Interestingly, activation of the TNF alpha-p75 receptor with a selective agonist, recombinant TNF alpha-p75 (rTNF alpha-p75), or inhibition of the TNF alpha-p75 receptor with utr-1, an inhibitory anti-TNF alpha-p75 receptor antibody, had no effect on TNF alpha-augmented calcium transients or on myocyte growth. To further confirm the receptor specificity of these findings, immunocytochemical studies were performed using receptor-specific antibodies. These studies demonstrated marked cell-surface expression of the TNF alpha-p55 receptor compared with expression of the TNF alpha-p75 receptor on human TSMC. Taken together, our results suggest that TNF alpha modulates agonist-induced calcium transients and induces human TSMC proliferation by specific activation of the TNF alpha-p55 receptor. Further studies addressing the cellular and molecular mechanisms regulating cytokine modulation of airway smooth muscle function may provide new insight into mechanisms that induce airway hyperresponsiveness in asthma.

Antibody Specificity↗
PubMed1995

Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF-alpha-induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion.

Asthma is a disease of airway inflammation and hyper-reactivity associated with lymphocytic infiltration in the bronchial submucosa. We recently demonstrated that human airway smooth muscle (ASM) cells express the cell adhesion molecules ICAM-1 and VCAM-1, which are up-regulated by cytokines such as TNF-alpha, and which mediate binding of activated T lymphocytes. In this study, we examined whether an increase in [cAMP]i, presumably via activation of cAMP-dependent protein kinase, modulates TNF-alpha-induced ICAM-1 and VCAM-1 on ASM. We found that treatment of ASM with either forskolin, which directly activates adenylyl cyclase, or with cholera toxin, which activates the heterotrimeric GTP-binding protein, Gs, inhibited TNF-alpha-induced cell adhesion molecule expression. In addition, treatment with either isoproterenol or prostaglandin E2, which activates receptors coupled to Gs and increases [cAMP]i, also inhibited TNF-alpha-induced expression of ICAM-1 and VCAM-1 on ASM. Furthermore, adhesion of activated T cells to TNF-alpha-stimulated ASM was inhibited by treating the ASM cells with either forskolin or PGE2. These data suggest that cAMP-dependent protein kinase activation decreases cytokine-induced expression of cell adhesion molecules on ASM cells, modulates T cell binding to airway myocytes and, thus, suggests novel therapeutic approaches to airway inflammation.

Asthma↗
PubMed1995

Defective phosphorylation and hyaluronate binding of CD44 with point mutations in the cytoplasmic domain.

CD44 is a cell surface adhesion molecule that plays a role in leukocyte extravasation, leukopoiesis, T lymphocyte activation, and tumor metastasis. The principal known ligand for CD44 is the glycosaminoglycan hyaluronate, (HA), a major constituent of extracellular matrices. CD44 expression is required but is not sufficient to confer cellular adhesion to HA, suggesting that the adhesion function of the receptor is regulated. We recently demonstrated that CD44 in primary leukocytes is phosphorylated in a cell type- and activation state-dependent fashion. In this study we demonstrate that serines 325 and 327 within the cytoplasmic domain of CD44 are required for the constitutive phosphorylation of CD44 in T cells. Furthermore, we demonstrate that cells expressing mutated CD44 containing a serine to glycine substitution at position 325 or a serine to alanine substitution at amino acid 327 are defective in HA binding, CD44-mediated adhesion of T cells to smooth muscle cells, as well as ligand-induced receptor modulation. The effect of these mutations can be partially reversed by a monoclonal anti-CD44 antibody that enhances CD44-mediated HA binding.

Amino Acid Sequence↗
PubMed1995

alpha-Thrombin increases cytosolic calcium and induces human airway smooth muscle cell proliferation.

In a variety of diseases including asthma, inflammation causes microvascular leakage and activates thrombin. In addition to cleaving fibrinogen to fibrin, thrombin may have other important cellular effects. Because airway inflammation and vascular permeability are important determinants of airway hyperreactivity, we have studied the effects of thrombin on airway smooth muscle. Using cultured human airway smooth muscle cells, we have examined whether alpha-thrombin can evoke calcium responses, phosphoinositide turnover, or cell proliferation. We have demonstrated that alpha-thrombin does increase cytosolic calcium and phosphoinositide hydrolysis in a dose- and time-dependent manner that may be inhibited by pretreating cells with r-hirudin. In addition, we have shown that thrombin stimulates airway smooth muscle cell proliferation. By contrast, bradykinin, which evoked comparable increases in cytosolic calcium and phosphoinositide turnover, did not stimulate airway smooth muscle cell growth. We conclude that thrombin effectively increases cytosolic calcium and induces PI hydrolysis and, in addition, is capable of stimulating airway smooth muscle cell growth. However, the lack of an effect of bradykinin on cell growth suggests that increases in calcium and PI turnover alone will not induce airway smooth muscle cell proliferation. We suggest that alpha-thrombin may be important in the pathogenesis of both increased airway resistance as well as the structural changes seen as a consequence of chronic asthma.

Bradykinin↗
PubMed1994

T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis.

Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma.

Animals↗
PubMed1994

Expression of endothelial cell adhesion molecules in human bronchial xenografts.

The role of endothelial cell adhesion molecules (CAMs) in the selective recruitment of leukocyte subsets to the airway remains unclear. The goal of the present study was to examine the expression of human endothelial CAM in a cytokine-induced airway inflammatory response. To accomplish this, an in vivo model of human bronchus was developed by heterotopically transplanting intact sections of human airway into severe combined immunodeficient (SCID) mice. Three weeks after transplantation, the xenografts closely resembled normal bronchus with little evidence of rejection. Less than 15% of the submucosal vessels expressed E-selectin and vascular cell adhesion molecule-1 (VCAM-1), whereas intercellular adhesion molecule-1 (ICAM-1) was present constitutively on approximately 35% of bronchial microvessels. Intrabronchial instillation of tumor necrosis factor-alpha (TNF-alpha) significantly increased expression of microvascular E-selectin to 40%, ICAM-1 to 65%, and VCAM-1 to 41%, and was accompanied by an influx of murine leukocytes into the bronchial submucosa. These results demonstrate that the human bronchial microvasculature expresses cytokine-inducible adhesion molecules. Mast cells and other resident or migratory cells that secrete TNF-alpha may thus activate the bronchial microvasculature and thereby recruit leukocytes to the airway.

Animals↗
PubMed1994

Hyperoxia increases airway cell S-phase traversal in immature rats in vivo.

Exposure of 21-day-old Sprague-Dawley rats to hyperoxia (> 95% O2 for 8 days) causes thickening of the airway epithelial and smooth muscle layers. To test the hypothesis that hyperoxic exposure increases airway layer DNA synthesis, we labeled the nuclei of cells undergoing S-phase by administering the thymidine analog bromodeoxyuridine (BrdU). BrdU was administered on days 3 and 4, 5 and 6, or 7 and 8 of air or O2 exposure, and the lungs were harvested immediately thereafter. Histologic sections were stained with an avidin-biotin-immunoperoxidase stain that revealed BrdU incorporation into nuclei, and a hematoxylin counterstain. After 4 days of air or O2 exposure, there was no difference in BrdU fractional labeling between control and hyperoxic animals. Thereafter, fractional BrdU labeling of the small airway (circumference < 1,000 microns) epithelium and smooth muscle layer was significantly increased in O2-exposed animals (P < 0.01, unpaired t test). The fractional labeling of larger, central airway smooth muscle layer cells was also increased after 8 days of O2 exposure (P < 0.01). In another cohort of O2-exposed animals, measurements of airway layer dimensions demonstrated increases in small airway epithelial and smooth muscle layer thickness that paralleled the time course seen for BrdU incorporation. We conclude that O2 exposure of immature rats increases airway epithelial and smooth muscle layer cellular DNA synthesis. These data suggest that hyperplasia of airway epithelial and smooth muscle layer cells may contribute to hyperoxia-induced airway remodeling.

Animals↗
PubMed1993

Changes in levels of mRNA encoding myosin heavy chain in porcine trachealis during ontogenesis.

We determined the steady-state level of mRNA for myosin heavy chain (MHC) from airway smooth muscle during maturation in domestic swine. Tissues were excised, and airway smooth muscle was dissected from three neonatal (NEO), three 2-wk-old swine (2ws), three 10-wk-old swine (10ws), and three adult swine. Total RNA was isolated, fractionated, and transferred to a nitrocellulose membrane (Northern blot). A single-stranded oligonucleotide of 63-nt was synthesized corresponding to the 3' coding region of the chicken gizzard MHC cDNA. This region appeared to be highly conserved (92% nucleotide sequence homology with the corresponding portion of rabbit uterine smooth muscle MHC cDNA). Northern blots, which were loaded with equivalent quantities of total RNA, were probed with gamma 32P-labeled synthetic oligonucleotide, and, under stringent washing conditions, the 5' end-labeled DNA was hybridized to a single band of the expected molecular weight. The mRNA for total myosin was quantified using autoradiograms of blots, and signal intensity was measured as integrated areas expressed as arbitrary densitometric units x mm (AU). The content of mRNA for MHC was substantially greater in NEO than in more mature animals; maximal area was 1.33 +/- 0.15 AU for NEO, 0.33 +/- 0.05 AU for 2ws, 0.30 +/- 0.04 AU for 10ws, and 0.34 +/- 0.08 AU for adult swine (P < 0.05, NEO versus 2ws, 10ws, and adult). Rehybridization of each blot with a 28S ribosomal RNA probe confirmed comparable total RNA loadings for all tissue samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1991

The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.

Although murine X-linked muscular dystrophy (mdx) and Duchenne muscular dystrophy (DMD) are genetically homologous and both characterized by a complete absence of dystrophin, the limb muscles of adult mdx mice suffer neither the detectable weakness nor the progressive degeneration that are features of DMD. Here we show that the mdx mouse diaphragm exhibits a pattern of degeneration, fibrosis and severe functional deficit comparable to that of DMD limb muscle, although adult mice show no overt respiratory impairment. Progressive functional changes include reductions in strength (to 13.5% of control by two years of age), elasticity, twitch speed and fibre length. The collagen density rises to at least seven times that of control diaphragm and ten times that of mdx hind-limb muscle. By 1.5 years of age, similar but less severe histological changes emerge in the accessory muscles of respiration. On the basis of these findings, we propose that dystrophin deficiency alters the threshold for work-induced injury. Our data provide a quantitative framework for studying the pathogenesis of dystrophy and extend the application of the mdx mouse as an animal model.

Animals↗
PubMed1991

Metabolic and contractile protein expression in developing rat diaphragm muscle.

Progressive changes in myosin isozyme expression and in energy-generating enzyme activities were followed in the diaphragm and, for comparison, in axial and appendicular muscles of rats from 18 d gestation to maturity. Native myosins were characterized by pyrophosphate gel electrophoresis. Myosin heavy-chain (MHC) isozymes were measured with ELISA using monoclonal antibodies and were localized by immunocytochemistry. RNA transcripts for the MHCs were demonstrated on Northern blots and by RNase protection assays. Quantitative activities of malate dehydrogenase (MDH), beta-hydroxyacyl CoA dehydrogenase (beta OAC), 1-phosphofructokinase (PFK), lactate dehydrogenase (LDH), creatine kinase (CK), and adenylokinase (AK) were measured in muscle homogenates and in individual fibers by fluorometric pyridine nucleotide-dependent assays. Compared to limb muscles, expression of neonatal myosin in the diaphragm is precocious. Neonatal MHC mRNA is prominent in the diaphragm at 19 d gestation, and neonatal myosin is the major MHC isoform present at birth. Slow and fast IIa MHCs are also present at birth. Transcripts for IIa MHC are detectable in the diaphragm at 21 d gestation and are upregulated at birth. Comparable signal for IIa MHC mRNA is not found in the gastrocnemius until 10 d postpartum. Adult fast IIb MHC mRNA was detected only as a faint signal at 30-40 d in the diaphragm and then disappeared. Results indicate that a separate phenotype, the IIx type, matures late in diaphragmatic development. The activities of enzymes representing all of the major energy pathways are higher in the fetal diaphragm than in the fetal hindlimb muscles. For example, beta OAC had sixfold higher activity in the diaphragm than in the extensor digitorum longus (EDL) muscle at birth, activity in the diaphragm than in the extensor digitorum longus (EDL) muscle at birth.

Aging↗
PubMed1990

Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression.

Although chronic severe asthma is characterized by increased smooth muscle mass in the airways, the physiological stimuli that promote airway smooth muscle (ASM) proliferation (hyperplasia) or increase ASM protein expression (hypertrophy) are unknown. We examined the effects of histamine, an autocoid associated with airway hyperresponsiveness, on protein synthesis, myosin heavy chain expression, and cell proliferation in cultured canine ASM cells. In confluent ASM cells, histamine significantly increased incorporation of [35S]-methionine in protein. Maintenance of the proportion of smooth muscle-specific myosin heavy chain to total myosin heavy chain suggested a nonspecific increase in contractile protein expression. DNA synthesis, as measured by [3H]thymidine incorporation, was significantly increased by histamine in a concentration-dependent manner. Cell proliferation paralleled [3H]thymidine incorporation; histamine significantly increased cell numbers at 24 and 48 h of stimulation. Because growth of mesenchymal-derived cells is associated with transcription of c-fos mRNA, we examined whether histamine altered expression of this proto-oncogene. Histamine-treated cells showed marked increases in expressions of steady-state c-fos mRNA, with a time course of mRNA induction similar to cells exposed to platelet-derived growth factor or serum, known smooth muscle and fibroblast cell mitogens. Therefore, histamine is an ASM mitogen with an action similar to other mesenchymal cell growth factors and may play a role in the hyperplasia of ASM in asthma.

Animals↗
PubMed1989

A human airway smooth muscle cell line that retains physiological responsiveness.

We report the development of a nontransformed line of human airway smooth muscle cells retaining smooth muscle-specific contractile protein expression and physiological responsiveness to agonists implicated in inflammatory airway diseases. Specific responses to histamine, leukotrienes, bradykinin, platelet-activating factor, substance P, and thromboxane analogues are demonstrated as well as functional coupling to beta-adrenergic receptors. The cell line was characterized using indirect immunofluorescence, as well as electrophoretic separation and immunoblot analysis of smooth muscle-specific actin. Functional responses were assessed by measurements of cytosolic calcium and stimulation of adenosine 3',5'-cyclic monophosphate production. The cells retain their responsiveness over many population doublings and should be a useful model to examine specific receptor-effector mechanisms, as well as the effects of neurohumoral agents on the regulation of airway smooth muscle growth and differentiation.

Actins↗