Garret A. FitzGerald, Patrick Loll
Matthew L. Springer, Timothy R. Brazelton, Helen M. Blau
In vitro polarized human Th2 cells preferentially express the chemokine receptors CCR3, CCR4, and CCR8 and migrate to their ligands: eotaxin, monocyte-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC), and I-309. We have studied the expression of chemokines and chemokine receptors in the airway mucosa of atopic asthmatics. Immunofluorescent analysis of endobronchial biopsies from six asthmatics, taken 24 hours after allergen challenge, demonstrates that virtually all T cells express IL-4 and CCR4. CCR8 is coexpressed with CCR4 on 28% of the T cells, while CCR3 is expressed on eosinophils but not on T cells. Expression of the CCR4-specific ligands MDC and TARC is strongly upregulated on airway epithelial cells upon allergen challenge, suggesting an involvement of this receptor/ligand axis in the regulation of lymphocyte recruitment into the asthmatic bronchi. In contrast to asthma, T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis produce IFN-γ and express high levels of CXCR3, while lacking CCR4 and CCR8 expression. These data support the role of CCR4, of its ligands MDC and TARC, and of CCR8 in the pathogenesis of allergen-induced late asthmatic responses and suggest that these molecules could be considered as targets for therapeutic intervention.
Paola Panina-Bordignon, Alberto Papi, Margherita Mariani, Pietro Di Lucia, Gianluca Casoni, Cinzia Bellettato, Cecilia Buonsanti, Deborah Miotto, Cristina Mapp, Antonello Villa, Gianluigi Arrigoni, Leonardo M. Fabbri, Francesco Sinigaglia
Acute graft-versus-host disease (GVHD) is a major complication of bone marrow transplantation (BMT) and is characterized by hematopoietic dysfunction, immunosuppression, and tissue injury in the skin, liver, and intestinal mucosa. Hepatocyte growth factor (HGF), originally identified and cloned as a potent mitogen for hepatocytes, induces mitogenic and antiapoptotic activity in various epithelial cells and promotes hematopoiesis. Working in a murine model of acute GVHD, we performed repeated transfection of the human HGF cDNA into skeletal muscle and showed that this treatment inhibited apoptosis of intestinal epithelial cells and donor T-cell infiltration into the liver, thereby ameliorating the enteropathy and liver injury caused by acute GVHD. HGF also markedly suppressed IFN-γ and TNF-α expression in the intestine and liver and decreased the serum IL-12. Furthermore, extramedullary hematopoiesis by donor cells was increased, and the survival rate was improved. These results suggest that HGF may be useful for controlling acute GVHD after allogeneic BMT.
Takanori Kuroiwa, Eizo Kakishita, Teruaki Hamano, Yasuro Kataoka, Yoshifumi Seto, Nobuo Iwata, Yasufumi Kaneda, Kunio Matsumoto, Toshikazu Nakamura, Takahiro Ueki, Jiro Fujimoto, Tsuyoshi Iwasaki
IL-4, an anti-inflammatory cytokine, inhibits osteoclast differentiation, but the basis of this effect has been unclear. Osteoclastogenesis requires activation of RANK, which exerts its biologic effect via activation of NF-κB. NF-κB activation is manifested by nuclear translocation and binding to DNA, events secondary to phosphorylation and dissociation of IκBα. It is shown here that IL-4 reduces NF-κB nuclear translocation by inhibiting IκB phosphorylation, thus markedly inhibiting NF-κB DNA binding activity and blocking osteoclastogenesis entirely. Residual translocation of NF-κB in the presence of IL-4, however, suggests that nuclear mechanisms must primarily account for inhibition of NF-κB DNA binding and blockade of osteoclastogenesis. To address this issue, this study examined whether IL-4–induced STAT6 transcription factor blocks NF-κB transactivation. The results show that excess unlabeled consensus sequence STAT6, but not its mutated form, inhibits NF-κB binding. Furthermore, exogenously added STAT6 protein inhibits NF-κB/DNA interaction. Further supporting a role for STAT6 in this process are the findings that IL-4 fails to block osteoclastogenesis in STAT6–/– mice but that this blockade can be restored with addition of exogenous STAT6. Thus, IL-4 obliterates osteoclast differentiation by antagonizing NF-κB activation in a STAT6-dependent manner.
It has been proposed that in the liver, chylomicron remnants (lipoproteins carrying dietary lipid) may be sequestered before being internalized by hepatocytes. To study this, chylomicron remnants labeled with a fluorescent dye were perfused into isolated livers of LDL receptor–deficient (LDLR-deficient) mice (Ldlr–/–) and examined by confocal microscopy. In contrast to livers from normal mice, there was clustering of the chylomicron remnants on the cell surface in the space of Disse. These remnant clusters colocalized with clusters of LDLR-related protein (LRP) and could be eliminated by low concentrations of receptor-associated protein, an inhibitor of LRP. When competed with ligands of heparan sulfate proteoglycans (HSPGs), the remnant clusters still appeared but were fewer in number, although syndecans (membrane HSPGs) colocalized with the remnant clusters. This suggests that the clustering of remnants is not dependent on syndecans but that the syndecans may modify the binding of remnants. These results establish that sequestration is a novel process, the clustering of remnants in the space of Disse. The clustering involves remnants binding to the LRP, and this may be stabilized by binding with syndecans, eventually followed by endocytosis.
Kenneth C.-W. Yu, Wei Chen, Allen D. Cooper
Myocyte loss in the ischemically injured mammalian heart often leads to irreversible deficits in cardiac function. To identify a source of stem cells capable of restoring damaged cardiac tissue, we transplanted highly enriched hematopoietic stem cells, the so-called side population (SP) cells, into lethally irradiated mice subsequently rendered ischemic by coronary artery occlusion for 60 minutes followed by reperfusion. The engrafted SP cells (CD34–/low, c-Kit+, Sca-1+) or their progeny migrated into ischemic cardiac muscle and blood vessels, differentiated to cardiomyocytes and endothelial cells, and contributed to the formation of functional tissue. SP cells were purified from Rosa26 transgenic mice, which express lacZ widely. Donor-derived cardiomyocytes were found primarily in the peri-infarct region at a prevalence of around 0.02% and were identified by expression of lacZ and α-actinin, and lack of expression of CD45. Donor-derived endothelial cells were identified by expression of lacZ and Flt-1, an endothelial marker shown to be absent on SP cells. Endothelial engraftment was found at a prevalence of around 3.3%, primarily in small vessels adjacent to the infarct. Our results demonstrate the cardiomyogenic potential of hematopoietic stem cells and suggest a therapeutic strategy that eventually could benefit patients with myocardial infarction.
Kathyjo A. Jackson, Susan M. Majka, Hongyu Wang, Jennifer Pocius, Craig J. Hartley, Mark W. Majesky, Mark L. Entman, Lloyd H. Michael, Karen K. Hirschi, Margaret A. Goodell
Mitochondrial trifunctional protein (MTP) is a hetero-octamer of four α and four β subunits that catalyzes the final three steps of mitochondrial long chain fatty acid β-oxidation. Human MTP deficiency causes Reye-like syndrome, cardiomyopathy, or sudden unexpected death. We used gene targeting to generate an MTP α subunit null allele and to produce mice that lack MTP α and β subunits. The Mtpa–/– fetuses accumulate long chain fatty acid metabolites and have low birth weight compared with the Mtpa+/– and Mtpa+/+ littermates. Mtpa–/– mice suffer neonatal hypoglycemia and sudden death 6–36 hours after birth. Analysis of the histopathological changes in the Mtpa–/– pups revealed rapid development of hepatic steatosis after birth and, later, significant necrosis and acute degeneration of the cardiac and diaphragmatic myocytes. This mouse model documents that intact mitochondrial long chain fatty acid oxidation is essential for fetal development and for survival after birth. Deficiency of MTP causes fetal growth retardation, neonatal hypoglycemia, and sudden death.
Jamal A. Ibdah, Hyacinth Paul, Yiwen Zhao, Scott Binford, Ken Salleng, Mark Cline, Dietrich Matern, Michael J. Bennett, Piero Rinaldo, Arnold W. Strauss
The development of transplant arteriosclerosis (TA) is today’s most important problem in clinical organ transplantation. Histologically, TA is characterized by perivascular inflammation and progressive intimal thickening. Current thought on this process of vascular remodeling assumes that neointimal vascular smooth muscle (VSM) cells and endothelium in TA are graft-derived, holding that medial VSM cells proliferate and migrate into the subendothelial space in response to signals from inflammatory cells and damaged graft endothelium. Using MHC class I haplotype-specific immunohistochemical staining and single-cell PCR analyses, we show that the neointimal α-actin–positive VSM cells in rat aortic or cardiac allografts are of recipient and not of donor origin. In aortic but not in cardiac allografts, recipient-derived endothelial cells (ECs) replaced donor endothelium. Cyclosporine treatment prevents neointima formation and preserves the vascular media in aortic allografts. Recipient-derived ECs do not replace graft endothelium after cyclosporine treatment. We propose that, although it progresses beyond the needs of functional repair, TA reflects the activity of a normal healing process that restores vascular wall function following allograft-induced immunological injury.
Jan-Luuk Hillebrands, Flip A. Klatter, Bart M.H. van den Hurk, Eliane R. Popa, Paul Nieuwenhuis, Jan Rozing
Statins are inhibitors of the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. In addition to reducing LDL cholesterol, statin treatment increases the levels of the antiatherogenic HDL and its major apolipoprotein apoA-I. Here, we investigated the molecular mechanisms of apoA-I regulation by statins. Treatment with statins increased apoA-I mRNA levels in human HepG2 hepatoma cells, and this effect was reversed by the addition of mevalonate, implicating HMG-CoA reductase as the relevant target of these drugs. Pretreatment with Actinomycin D abolished the increase of apoA-I mRNA, indicating that statins act at the transcriptional level. Indeed, statins increased the human apoA-I promoter activity in transfected cells, and we have identified a statin response element that coincides with a PPARα response element known to confer fibrate responsiveness to this gene. The statin effect could be abolished not only by mevalonate, but also by geranylgeranyl pyrophosphate, whereas inhibition of geranylgeranyl transferase activity or treatment with an inhibitor of the Rho GTP-binding protein family increased PPARα activity. Using dominant negative forms of these proteins, we found that Rho A itself mediates this response. Because cotreatment with statins and fibrates activated PPARα in a synergistic manner, these observations provide a molecular basis for combination treatment with statins and fibrates in coronary heart disease.
Geneviève Martin, Hélène Duez, Christophe Blanquart, Vincent Berezowski, Philippe Poulain, Jean-Charles Fruchart, Jamila Najib-Fruchart, Corine Glineur, Bart Staels
Acute generalized exanthematous pustulosis (AGEP) is an uncommon eruption most often provoked by drugs, by acute infections with enteroviruses, or by mercury. It is characterized by acute, extensive formation of nonfollicular sterile pustules on erythematous background, fever, and peripheral blood leukocytosis. We present clinical and immunological data on four patients with this disease, which is caused by different drugs. An involvement of T cells could be implied by positive skin patch tests and lymphocyte transformation tests. Immunohistochemistry revealed a massive cell infiltrate consisting of neutrophils in pustules and T cells in the dermis and epidermis. Expression of the potent neutrophil-attracting chemokine IL-8 was elevated in keratinocytes and infiltrating mononuclear cells. Drug-specific T cells were generated from the blood and skin of three patients, and phenotypic characterization showed a heterogeneous distribution of CD4/CD8 phenotype and of T-cell receptor Vβ-expression. Analysis of cytokine/chemokine profiles revealed that IL-8 is produced significantly more by drug-specific T cells from patients with AGEP compared with drug-specific T cells from patients that had non-AGEP exanthemas. In conclusion, our data demonstrate the involvement of drug-specific T cells in the pathomechanism of this rather rare and peculiar form of drug allergy. In addition, they indicate that even in some neutrophil-rich inflammatory responses specific T cells are engaged and might orchestrate the immune reaction.
Markus Britschgi, Urs C. Steiner, Simone Schmid, Jan P.H. Depta, Gabriela Senti, Andreas Bircher, Christoph Burkhart, Nikhil Yawalkar, Werner J. Pichler
Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2–activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2–mediated secretion from the salivary glands. Intravenous calcitonin gene–related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2–mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2–mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.
Atsufumi Kawabata, Mitsuhiro Kinoshita, Hiroyuki Nishikawa, Ryotaro Kuroda, Minoru Nishida, Hiromasa Araki, Naoki Arizono, Yasuo Oda, Kazuaki Kakehi
Children with chronic inflammatory diseases experience growth failure and wasting. This may be due to growth hormone resistance caused by cytokine-induced suppression of growth hormone receptor (GHR) gene expression. However, the factors governing inflammatory regulation of GHR are not known. We have reported that Sp1 and Sp3 regulate hepatic GHR expression. We hypothesized that TNF-α suppresses GHR expression by inhibiting Sp1/Sp3 transactivators. LPS administration significantly reduced murine hepatic GHR expression, as well as Sp1 and Sp3 binding to GHR promoter cis elements. TNF-α was integral to this response, as LPS did not affect hepatic Sp1/Sp3 binding or GHR expression in TNF receptor 1–deficient mice. TNF-α treatment of BNL CL.2 mouse liver cells reduced Sp1 and Sp3 binding to a GHR promoter cis element and downregulated activity of a GHR promoter-driven luciferase reporter. Combined mutations within adjacent Sp elements eliminated GHR promoter suppression by TNF-α without affecting overall nuclear levels of Sp1 or Sp3 proteins. These studies demonstrate that murine GHR transcription is downregulated by LPS, primarily via TNF-α–dependent signaling. Evidence suggests that inhibition of Sp transactivator binding is involved. Further investigation of these mechanisms may identify novel strategies for preventing inflammatory suppression of growth.
Lee A. Denson, Ram K. Menon, Angel Shaufl, Himmat S. Bajwa, Carol R. Williams, Saul J. Karpen
TR6 (DcR3) is a new member of the TNF receptor (TNFR) family that lacks a transmembrane domain in its sequence, indicating that it is a secreted molecule. TR6 can bind to FasL and prevent FasL-induced apoptosis; it can also associate with LIGHT, another TNF family member. The role of TR6 in immune responses was investigated in this study. According to flow cytometry, recombinant human TR6-Fc binds to human LIGHT expressed on 293 cells or on activated human T cells and competes with the LIGHT receptor TR2 for the binding to LIGHT on these cells. Human TR6 could cross-react with mouse LIGHT in immunoprecipitation. TR6-Fc also downregulates cytotoxic T lymphocyte activity in vitro and graft-versus-host responses in mice. Moreover, TR6-Fc modulates lymphokine production by alloantigen-stimulated mouse T cells. TR6-Fc ameliorated rejection response to mouse heart allograft. These results indicate that TR6 can dampen T-cell responses to alloantigens. Such regulatory effects of TR6 probably occur via interference with interaction between pairs of related TNF and TNFR family members, LIGHT/TR2 being one of the possible candidate pairs.
Jun Zhang, Theodora W. Salcedo, Xiaochun Wan, Stephen Ullrich, Bugen Hu, Theresa Gregorio, Ping Feng, Shijie Qi, Huifang Chen, Yun Hee Cho, Yuling Li, Paul A. Moore, Jiangping Wu
Marked vasodilation in the kidney and other nonreproductive organs is one of the earliest maternal adaptations to occur during pregnancy. Despite the recognition of this extraordinary physiology for over four decades, the gestational hormone responsible has remained elusive. Here we demonstrate a key role for relaxin, a member of the IGF family that is secreted by the corpus luteum in humans and rodents. Using a gravid rodent model, we employ two approaches to eliminate relaxin or its biological activity from the circulation: ovariectomy and administration of neutralizing antibodies. Both abrogate the gestational elevation in renal perfusion and glomerular filtration, as well as preventing the reduction in myogenic reactivity of isolated, small renal arteries. Osmoregulatory changes, another pregnancy adaptation, are also abolished. Our results indicate that relaxin mediates the renal vasodilatory responses to pregnancy and thus may be important for maternal and fetal health. They also raise the likelihood of a role for relaxin in other cardiovascular changes of pregnancy, and they suggest that, like estrogen, relaxin should be considered a regulator of cardiovascular function.
J. Novak, L.A. Danielson, L.J. Kerchner, O.D. Sherwood, R.J. Ramirez, P.A. Moalli, K.P. Conrad