LFA-1/ICAM-1 Adhesion Pathway Mediates the Homeostatic Migration of Lymphocytes from Peripheral Tissues into Lymph Nodes through Lymphatic Vessels.

Lymphocyte function-associated antigen-1 (LFA-1) and its endothelial ligand intercellular adhesion molecule-1 (ICAM-1) are important for the migration of lymphocytes from blood vessels into lymph nodes. However, it is largely unknown whether these molecules mediate the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes through lymphatic vessels. In this study, we find that, in naive mice, ICAM-1 is expressed on the sinus endothelia of lymph nodes, but not on the lymphatic vessels of peripheral tissues. In in vivo lymphocyte migration assays, memory CD4+ T cells migrated to lymph nodes from peripheral tissues much more efficiently than from blood vessels, as compared to naive CD4+ T cells. Moreover, ICAM-1 deficiency in host mice significantly inhibited the migration of adoptively transferred wild-type donor lymphocytes from peripheral tissues, but not from blood vessels, into lymph nodes. The migration of LFA-1-deficient donor lymphocytes from peripheral tissues into the lymph nodes of wild-type host mice was also significantly reduced as compared to wild-type donor lymphocytes. Furthermore, the number of memory T cells in lymph nodes was significantly reduced in the absence of ICAM-1 or LFA-1. Thus, our study extends the functions of the LFA-1/ICAM-1 adhesion pathway, indicating its novel role in controlling the homeostatic migration of lymphocytes from peripheral tissues into lymph nodes and maintaining memory T cellularity in lymph nodes.

Inhibition of VEGF (Vascular Endothelial Growth Factor)-A or its Receptor Activity Suppresses Experimental Aneurysm Progression in the Aortic Elastase Infusion Model.

OBJECTIVE: We examined the pathogenic significance of VEGF (vascular endothelial growth factor)-A in experimental abdominal aortic aneurysms (AAAs) and the translational value of pharmacological VEGF-A or its receptor inhibition in aneurysm suppression. Approaches and Results: AAAs were created in male C57BL/6J mice via intra-aortic elastase infusion. Soluble VEGFR (VEGF receptor)-2 extracellular ligand-binding domain (delivered in Ad [adenovirus]-VEGFR-2), anti-VEGF-A mAb (monoclonal antibody), and sunitinib were used to sequester VEGF-A, neutralize VEGF-A, and inhibit receptor tyrosine kinase activity, respectively. Influences on AAAs were assessed using ultrasonography and histopathology. In vitro transwell migration and quantitative reverse transcription polymerase chain reaction assays were used to assess myeloid cell chemotaxis and mRNA expression, respectively. Abundant VEGF-A mRNA and VEGF-A-positive cells were present in aneurysmal aortae. Sequestration of VEGF-A by Ad-VEGFR-2 prevented AAA formation, with attenuation of medial elastolysis and smooth muscle depletion, mural angiogenesis and monocyte/macrophage infiltration. Treatment with anti-VEGF-A mAb prevented AAA formation without affecting further progression of established AAAs. Sunitinib therapy substantially mitigated both AAA formation and further progression of established AAAs, attenuated aneurysmal aortic MMP2 (matrix metalloproteinase) and MMP9 protein expression, inhibited inflammatory monocyte and neutrophil chemotaxis to VEGF-A, and reduced MMP2, MMP9, and VEGF-A mRNA expression in macrophages and smooth muscle cells in vitro. Additionally, sunitinib treatment reduced circulating monocytes in aneurysmal mice. CONCLUSIONS: VEGF-A and its receptors contribute to experimental AAA formation by suppressing mural angiogenesis, MMP and VEGF-A production, myeloid cell chemotaxis, and circulating monocytes. Pharmacological inhibition of receptor tyrosine kinases by sunitinib or related compounds may provide novel opportunities for clinical aneurysm suppression.

Hypoxia-inducible factor 1 in clinical and experimental aortic aneurysm disease.

OBJECTIVE: Mural angiogenesis and macrophage accumulation are two pathologic hallmarks of abdominal aortic aneurysm (AAA) disease. The heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) is an essential regulator of angiogenesis and macrophage function. In this study, we investigated HIF-1 expression and activity in clinical and experimental AAA disease. METHODS: Human aortic samples were obtained from 24 AAA patients and six organ donors during open abdominal surgery. Experimental AAAs were created in 10-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase (PPE). Expression of HIF-1α and its target gene messenger RNA (mRNA) levels were assessed in aneurysmal and control aortae. The HIF-1α inhibitors 2-methoxyestradiol and digoxin, the prolyl hydroxylase domain-containing protein (PHD) inhibitors cobalt chloride and JNJ-42041935, and the vehicle alone as control were administered daily to mice at varying time points beginning before or after PPE infusion. Influences on experimental AAA formation and progression were assessed by serial transabdominal ultrasound measurements of aortic diameter and histopathologic analysis at sacrifice. RESULTS: The mRNA levels for HIF-1α, vascular endothelial growth factor A, glucose transporter 1, and matrix metalloproteinase 2 were significantly increased in both human and experimental aneurysm tissue. Tissue immunostaining detected more HIF-1α protein in both human and experimental aneurysmal aortae compared with respective control aortae. Treatment with either HIF-1α inhibitor, beginning before or after PPE infusion, prevented enlargement of experimental aneurysms. Both HIF-1α inhibition regimens attenuated medial elastin degradation, smooth muscle cell depletion, and mural angiogenesis and the accumulation of macrophages, T cells, and B cells. Whereas mRNA levels for PHD1 and PHD2 were elevated in experimental aneurysmal aortae, pharmacologic inhibition of PHDs had limited effect on experimental aneurysm progression. CONCLUSIONS: Expression of HIF-1α and its target genes is increased in human and experimental AAAs. Treatment with HIF-1α inhibitors limits experimental AAA progression, with histologic evidence of attenuated mural leukocyte infiltration and angiogenesis. These findings underscore the potential significance of HIF-1α in aneurysm pathogenesis and as a target for pharmacologic suppression of AAA disease.

Inhibition or deletion of angiotensin II type 1 receptor suppresses elastase-induced experimental abdominal aortic aneurysms.

OBJECTIVE: Angiotensin (Ang) II type 1 receptor (AT1) activation is essential for the development of exogenous Ang II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic animals. Experimental data derived from this modeling system, however, provide limited insight into the role of endogenous Ang II in aneurysm pathogenesis. Consequently, the potential translational value of AT1 inhibition in clinical AAA disease management remains incompletely understood on the basis of the existing literature. METHODS: AAAs were created in wild-type (WT) and AT1a knockout (KO) mice by intra-aortic infusion of porcine pancreatic elastase (PPE). WT mice were treated with the AT1 receptor antagonist telmisartan, 10 mg/kg/d in chow, or the peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9662, 3 mg/kg/d through oral gavage, beginning 1 week before or 3 days after PPE infusion. Influences on aneurysm progression as well as mechanistic insights into AT1-mediated pathogenic processes were determined using noninvasive ultrasound imaging, histopathology, aortic gene expression profiling, and flow cytometric analysis. RESULTS: After PPE infusion, aortic enlargement was almost completely abrogated in AT1a KO mice compared with WT mice. As defined by a ≥50% increase in aortic diameter, no PPE-infused, AT1a KO mouse actually developed an AAA. On histologic evaluation, medial smooth muscle cellularity and elastic lamellae were preserved in AT1a KO mice compared with WT mice, with marked attenuation of mural angiogenesis and leukocyte infiltration. In WT mice, telmisartan administration effectively suppressed aneurysm pathogenesis after PPE infusion as well, regardless of whether treatment was initiated before or after aneurysm creation or continued for a limited or extended time. Telmisartan treatment was associated with reduced messenger RNA levels for CCL5 and matrix metalloproteinases 2 and 9 in aneurysmal aortae, with no apparent effect on PPARγ-regulated gene expression. Administration of the PPARγ antagonist GW9662 failed to "rescue" the aneurysm phenotype in telmisartan-treated, PPE-infused WT mice. Neither effector T-cell differentiation nor regulatory T-cell cellularity was affected by telmisartan treatment status. CONCLUSIONS: Telmisartan effectively suppresses the progression of elastase-induced AAAs without apparent effect on PPARγ activation or T-cell differentiation. These findings reinforce the critical importance of endogenous AT1 activation in experimental AAA pathogenesis and reinforce the translational potential of AT1 inhibition in medical aneurysm disease management.

A silk-based encapsulation platform for pancreatic islet transplantation improves islet function in vivo.

The success of pancreatic islet (PI) transplantation is challenged by PI functional damage during the peritransplantation period. A silk-based encapsulation platform including mesenchymal stromal cells (MSCs) was evaluated for islet cell delivery in vivo. Islet equivalents (IEQs) were transplanted into the epididymal fat pads of mice with streptozotocin-induced diabetes. Three PI combinations were tested: (A) co-encapsulated in silk with MSCs; (b) encapsulated in silk alone; or (c) pelleted. Blood glucose levels were monitored and intraperitoneal glucose tolerance test (IPGTT) was performed upon return to euglycaemia. Grafts were removed for histology and cytokine content analysis. Mice with PI grafts in silk showed a prompt return to euglycaemia. IPGTT was significantly improved with PI in silk with MSCs, compared to PI in silk alone or pelleted. Both Th1 and Th2 cytokines were increased in PI grafts in silk, but Th1 cytokines were decreased significantly with PI and MSC co-encapsulation. Histological analysis showed osteogenesis and chondrogenesis in the silk grafts containing MSCs. Future studies will evaluate MSC stability and function in vivo and improve silk biocompatibility for applications in islet transplantation. Copyright © 2015 John Wiley & Sons, Ltd.

Diagnostic Dilemma in the Treatment of a Fatal Case of Bloody Diarrhea.

Although diarrhea is the most commonly reported pediatric illness in the United States, mortality is usually a rare and unexpected event. We report the case of a healthy 13-month-old male that succumbed to a diarrheal illness of unclear etiology. Presenting signs included frequent nonbloody stools that progressed to frankly bloody stools over 72 hours. Associated symptoms included fever, tenesmus, relief with stool passage, and significant fatigue. On examination, the patient appeared tired and lay with legs curled toward his chest. The abdominal exam was remarkable for hypoactive bowel sounds, diffuse tenderness to palpation without guarding or rebound pain, and intermittent prolapse of rectal tissue. Abdominal plain films demonstrated a paucity of bowel gas, especially in the rectum; and ultrasound revealed thickening of bowel loops in the left lower quadrant. Abdominal computed tomography scan showed decreased enhancement of the mucosa of the rectosigmoid colon. The patient deteriorated rapidly with cardiorespiratory arrest occurring 48 hours after admission. Despite a protracted effort at cardiopulmonary resuscitation, perfusing heart rate or rhythm could not be reestablished. Autopsy revealed infarction and necrosis of the rectosigmoid colon with invasive gram-negative bacilli. Here we present his perplexing case, diagnostic evaluations, and suggest a unifying diagnosis.

Humanized thymidine kinase-NOG mice can be used to identify drugs that cause animal-specific hepatotoxicity: a case study with furosemide.

Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)-NOG mice with humanized livers could prevent this unfortunate outcome (i.e., "rescue" a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide.

Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity.

Due to the substantial interspecies differences in drug metabolism and disposition, drug-induced liver injury (DILI) in humans is often not predicted by studies performed in animal species. For example, a drug (bosentan) used to treat pulmonary artery hypertension caused unexpected cholestatic liver toxicity in humans, which was not predicted by preclinical toxicology studies in multiple animal species. In this study, we demonstrate that NOG mice expressing a thymidine kinase transgene (TK-NOG) with humanized livers have a humanized profile of biliary excretion of a test (cefmetazole) drug, which was shown by an in situ perfusion study to result from interspecies differences in the rate of biliary transport and in liver retention of this drug. We also found that readily detectable cholestatic liver injury develops in TK-NOG mice with humanized livers after 1 week of treatment with bosentan (160, 32, or 6 mg/kg per day by mouth), whereas liver toxicity did not develop in control mice after 1 month of treatment. The laboratory and histologic features of bosentan-induced liver toxicity in humanized mice mirrored that of human subjects. Because DILI has become a significant public health problem, drug safety could be improved if preclinical toxicology studies were performed using humanized TK-NOG.

VCAM-1/α4ß1 integrin interaction is crucial for prompt recruitment of immune T cells into the brain during the early stage of reactivation of chronic infection with Toxoplasma gondii to prevent toxoplasmic encephalitis.

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4ß1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8ß, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4(+) and CD8(+) T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4ß1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

BACKGROUND: Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. METHODS AND FINDINGS: Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. CONCLUSIONS: FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants. Please see later in the article for the Editors' Summary.

CCR7 directs the recruitment of T cells into inflamed pancreatic islets of nonobese diabetic (NOD) mice.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing ß cells in the pancreatic islets. The migration of T cells from blood vessels into pancreas is critical for the development of islet inflammation and ß cell destruction in T1D. To define the roles of C-C chemokine receptor type 7 (CCR7) in recruitment of T cells into islets, we used laser capture microdissection to isolate tissue from inflamed islets of nonobese diabetic (NOD) mice and uninflamed islets of BALB/c and young NOD mice. RT-PCR analyses detected mRNAs for CCR7 and its chemokine ligands CCL19 (ELC; MIP-3ß) and CCL21 (SLC) in captures from inflamed, but not from uninflamed, islets. Immunohistology studies revealed that high endothelial venules in inflamed islets co-express CCL21 protein and MAdCAM-1 (an adhesion molecule that recruits lymphocytes into islets). Desensitization of lymphocyte CCR7 blocked about 75 % of T cell migration from the bloodstream into inflamed islets, but had no effect on B cell migration into islets. These results indicate that CCR7 and its ligands are important in the recruitment of T cells into inflamed islets and thus in the pathogenesis of T1D.

Protein polymer hydrogels: effects of endotoxin on biocompatibility.

Protein polymer-based hydrogels have shown potential for tissue engineering applications, but require biocompatibility testing for in vivo use. Enzymatically crosslinked protein polymer-based hydrogels were tested in vitro and in vivo to evaluate their biocompatibility. Endotoxins present in the hydrogel were removed by Trition X-114 phase separation. The reduction of endotoxins decreased TNF-α production by a macrophage cell line in vitro; however, significant inflammatory response was still present compared to collagen control gels. A branched PEG molecule and dexamethasone were added to the hydrogel to reduce the response. In vitro testing showed a decrease in the TNF-α levels with the addition of dexamethasone. In vivo implantations into the epididymal fat pad of C57/BL6 mice, however, indicated a decreased inflammatory mediated immune response with a hydrogel treated with both PEGylation and endotoxin reduction. This study demonstrates the importance of endotoxin testing and removal in determining the biocompatibility of biomaterials.

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model.

INTRODUCTION: Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease with complex etiopathogenesis. Despite extensive studies to understand the disease process utilizing human and mouse models, the intersection between these species remains elusive. To address this gap, we utilized a novel systems biology approach to identify disease-related gene modules and signaling pathways that overlap between humans and mice. METHODS: Parotid gland tissues were harvested from 24 pSS and 16 non-pSS sicca patients and 25 controls. For mouse studies, salivary glands were harvested from C57BL/6.NOD-Aec1Aec2 mice at various times during development of pSS-like disease. RNA was analyzed with Affymetrix HG U133+2.0 arrays for human samples and with MOE430+2.0 arrays for mouse samples. The images were processed with Affymetrix software. Weighted-gene co-expression network analysis was used to identify disease-related and functional pathways. RESULTS: Nineteen co-expression modules were identified in human parotid tissue, of which four were significantly upregulated and three were downregulated in pSS patients compared with non-pSS sicca patients and controls. Notably, one of the human disease-related modules was highly preserved in the mouse model, and was enriched with genes involved in immune and inflammatory responses. Further comparison between these two species led to the identification of genes associated with leukocyte recruitment and germinal center formation. CONCLUSION: Our systems biology analysis of genome-wide expression data from salivary gland tissue of pSS patients and from a pSS mouse model identified common dysregulated biological pathways and molecular targets underlying critical molecular alterations in pSS pathogenesis.

Text-mining applied to autoimmune disease research: the Sjögren's syndrome knowledge base.

BACKGROUND: Sjögren's syndrome is a tissue-specific autoimmune disease that affects exocrine tissues, especially salivary glands and lacrimal glands. Despite a large body of evidence gathered over the past 60 years, significant gaps still exist in our understanding of Sjögren's syndrome. The goal of this study was to develop a database that collects and organizes gene and protein expression data from the existing literature for comparative analysis with future gene expression and proteomic studies of Sjögren's syndrome. DESCRIPTION: To catalog the existing knowledge in the field, we used text mining to generate the Sjögren's Syndrome Knowledge Base (SSKB) of published gene/protein data, which were extracted from PubMed using text mining of over 7,700 abstracts and listing approximately 500 potential genes/proteins. The raw data were manually evaluated to remove duplicates and false-positives and assign gene names. The data base was manually curated to 477 entries, including 377 potential functional genes, which were used for enrichment and pathway analysis using gene ontology and KEGG pathway analysis. CONCLUSIONS: The Sjögren's syndrome knowledge base ( http://sskb.umn.edu) can form the foundation for an informed search of existing knowledge in the field as new potential therapeutic targets are identified by conventional or high throughput experimental techniques.

Alpha4beta7 integrin/MAdCAM-1 adhesion pathway is crucial for B cell migration into pancreatic lymph nodes in nonobese diabetic mice.

Although B cells are crucial antigen-presenting cells in the initiation of T cell autoimmunity to islet beta cell autoantigens in type 1 diabetes (T1D), adhesion molecules that control migration of B cells into pancreatic lymph nodes (PanLN) in the nonobese diabetic (NOD) mouse model of human T1D have not been defined. In this study, we found that B cells from PanLN of 3-4-week-old female NOD mice expressed high levels of alpha(4) integrin and LFA-1 and intermediate levels of beta(7) integrin; half of B cells were L-selectin(high). In short-term in vivo lymphocyte migration assays, B cells migrated from the bloodstream into PanLN more efficiently than into peripheral LNs. Moreover, antibodies to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) and alpha(4)beta(7) integrin inhibited >90% of B cell migration into PanLN. In contrast, antibodies to peripheral node addressin, L-selectin or LFA-1 partially inhibited B cell migration into PanLN. Furthermore, one intraperitoneal injection of anti-MAdCAM-1 antibody into 3-week-old NOD mice significantly inhibited entry of B cells into PanLN for at least 2 weeks. Taken together, these results indicate that the alpha(4)beta(7) integrin/MAdCAM-1 adhesion pathway plays a predominant role in migration of B cells into PanLN in NOD mice. Thus, specific blockage of alpha(4)beta(7) integrin/MAdCAM-1 adhesion pathway-mediated B cell migration may be a potential treatment for T1D.

Removal of Toxoplasma gondii cysts from the brain by perforin-mediated activity of CD8+ T cells.

Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts, and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8(+) immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8(+) T cells does not require their production of interferon-gamma, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8(+) T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.

Expression of endothelia and lymphocyte adhesion molecules in bronchus-associated lymphoid tissue (BALT) in adult human lung.

BACKGROUND: Bronchus-associated lymphoid tissue (BALT) is the secondary lymphoid tissue in bronchial mucosa and is involved in the development of bronchopulmonary immune responses. Although migration of lymphocytes from blood vessels into secondary lymphoid tissues is critical for the development of appropriate adaptive immunity, the endothelia and lymphocyte adhesion molecules that recruit specific subsets of lymphocytes into human BALT are not known. The aim of this study was to determine which adhesion molecules are expressed on lymphocytes and high endothelial venules (HEVs) in human BALT. METHODS: We immunostained frozen sections of BALT from lobectomy specimens from 17 patients with lung carcinoma with a panel of monoclonal antibodies to endothelia and lymphocyte adhesion molecules. RESULTS: Sections of BALT showed B cell follicles surrounded by T cells. Most BALT CD4+ T cells had a CD45RO+ memory phenotype. Almost all BALT B cells expressed alpha4 integrin and L-selectin. In contrast, 43% of BALT T cells expressed alpha4 integrin and 20% of BALT T cells expressed L-selectin. Almost all BALT lymphocytes expressed LFA-1. HEVs, which support the migration of lymphocytes from the bloodstream into secondary lymphoid tissues, were prominent in BALT. All HEVs expressed peripheral node addressin, most HEVs expressed vascular cell adhesion molecule-1, and no HEVs expressed mucosal addressin cell adhesion molecule-1. CONCLUSION: Human BALT expresses endothelia and lymphocyte adhesion molecules that may be important in recruiting naive and memory/effector lymphocytes to BALT during protective and pathologic bronchopulmonary immune responses.

PPAR-delta senses and orchestrates clearance of apoptotic cells to promote tolerance.

Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and the silent disposal of apoptotic cells is unknown. Here we show that peroxisome proliferator-activated receptor-delta (PPAR-delta) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-delta decreases expression of opsonins such as complement component-1qb (C1qb), resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific Ppard(-/-) mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-delta has a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.

Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor beta.

Tumor-associated macrophages (TAMs) are frequently found in glioblastomas and a high degree of macrophage infiltration is associated with a poor prognosis for glioblastoma patients. However, it is unclear whether TAMs in glioblastomas promote tumor growth. In this study, we found that folate receptor beta (FR beta) was expressed on macrophages in human glioblastomas and a rat C6 glioma implanted subcutaneously in nude mice. To target FR beta-expressing TAMs, we produced a recombinant immunotoxin consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FR beta monoclonal antibody and Pseudomonas exotoxin A. Injection of the immunotoxin into C6 glioma xenografts in nude mice significantly depleted TAMs and reduced tumor growth. The immunotoxin targeting FR beta-expressing macrophages will provide a therapeutic tool for human glioblastomas.

Systems biology analysis of Sjögren's syndrome and mucosa-associated lymphoid tissue lymphoma in parotid glands.

OBJECTIVE: To identify key target genes and activated signaling pathways associated with the pathogenesis of Sjögren's syndrome (SS) by conducting a systems analysis of parotid glands manifesting primary SS or primary SS/mucosa-associated lymphoid tissue (MALT) lymphoma phenotypes. METHODS: A systems biology approach was used to analyze parotid gland tissue samples obtained from patients with primary SS, patients with primary SS/MALT lymphoma, and subjects without primary SS (non-primary SS controls). The tissue samples were assessed concurrently by gene-expression microarray profiling and proteomics analysis, followed by weighted gene-coexpression network analysis. RESULTS: Gene-coexpression modules related to primary SS and primary SS/MALT lymphoma were significantly enriched with genes known to be involved in the immune/defense response, apoptosis, cell signaling, gene regulation, and oxidative stress. Detailed functional pathway analyses indicated that primary SS-associated modules were enriched with genes involved in proteasome degradation, apoptosis, signal peptides of the class I major histocompatibility complex (MHC), complement activation, cell growth and death, and integrin-mediated cell adhesion, while primary SS/MALT lymphoma-associated modules were enriched with genes involved in translation, ribosome biogenesis and assembly, proteasome degradation, class I MHC signal peptides, the G13 signaling pathway, complement activation, and integrin-mediated cell adhesion. Combined analyses of gene expression and proteomics data implicated 6 highly connected "hub" genes for distinguishing primary SS from non-primary SS, and 8 hub genes for distinguishing primary SS/MALT lymphoma from primary SS. CONCLUSION: Systems biology analyses of the parotid glands from patients with primary SS and those with primary SS/MALT lymphoma revealed pathways and molecular targets associated with disease pathogenesis. The identified gene modules/pathways provide further insights into the molecular mechanisms of primary SS and primary SS/MALT lymphoma. The identified disease-hub genes represent promising targets for therapeutic intervention, diagnosis, and prognosis.