Regulation of T(H)2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells.

Although cognate encounters between antigen-bearing dendritic cells (DCs) that express the chemokine receptor CCR7 and CCR7(+) naive T cells take place in the T cell zone of lymph nodes, it is unknown whether the colocalization of DCs and T cells in the T cell area is required for the generation of effector cells. Here we found that after infection with an intestinal nematode, antigen-bearing DCs and CD4(+) T cells upregulated the chemokine receptor CXCR5 and localized together outside the T cell zone by a mechanism dependent on the chemokine CXCL13, B cells and lymphotoxin. Notably, lymphotoxin-expressing B cells, CXCR5-expressing DCs and T cells, and CXCL13 were also necessary for development of interleukin 4 (IL-4)-producing type 2 helper T cells (T(H)2 cells), which suggests that T(H)2 differentiation can initiate outside the T cell zone.

Lymphotoxin-α promotes tumor angiogenesis in HNSCC by modulating glycolysis in a PFKFB3-dependent manner.

Tumor angiogenesis is critical for tumor progression as the new blood vessels supply nutrients and facilitate metastasis. Previous studies indicate tumor associated lymphocytes, including B cells and T cells, contribute to tumor angiogenesis and tumor progression. The present study aims to identify the function of Lymphotoxin-α (LT-α), which is secreted by the activated lymphocytes, in the tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). The coculture system between HNSCC cell line Cal27 and primary lymphocytes revealed that tumor cells promoted the LT-α secretion in the cocultured lymphocytes. In vitro data further demonstrated that LT-α promoted the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) by enhancing the PFKFB3-mediated glycolytic flux. Genetic and pharmacological inhibition of PFKFB3 suppressed the enhanced proliferation and migration of HUVECs. We further identified that LT-α induced PFKFB3 expression was dependent on the TNFR/NF-κB signaling pathway. In addition, we proved that PFKFB3 blockade decreased the density of CD31 positive blood vessels in HNSCC xenografts. Finally, the results from the human HNSCC tissue array revealed that the expression of LT-α in HNSCC samples positively correlated with microvessel density, lymphocytes infiltration and endothelial PFKFB3 expression. In conclusion, infiltrated lymphocyte secreted LT-α enhances the glycolysis of ECs in a PFKFB3-dependent manner through the classical NF-κB pathway and promotes the proliferation and migration of ECs, which may contribute to the aberrant angiogenesis in HNSCCs. Our study suggests that PFKFB3 blockade is a promising therapeutic approach for HNSCCs by targeting tumor angiogenesis.

Alcohol-Induced Interleukin-17 Expression Causes Murine Lung Fibroblast-to-Myofibroblast Transdifferentiation via Thy-1 Down-Regulation.

BACKGROUND: Alcohol exposure induces TGFß1 and renders the lung susceptible to injury and disrepair. We determined that TGFß1 regulates myofibroblast differentiation through the loss of Thy-1 expression and consequent induction of α-SMA. TGFß1 is important for T helper 17 (Th17) differentiation and IL-17 secretion, which in turn participates in tissue repair. We hypothesized that alcohol induces Th17 differentiation via TGFß1 and that IL-17 produced by these cells contributes to the development of profibrotic lung myofibroblasts. METHODS: Primary lung fibroblasts (PLFs) were treated with alcohol, TGFß1, and IL-17 and then analyzed for Thy-1 expression and cell morphology. Naïve and Th17-polarized CD4+ T cells were exposed to alcohol and assessed for IL-17 expression. CD4+ T cells from alcohol-fed mice were analyzed for Th17 and IL-17 expression. Lungs of control-fed, bleomycin-treated and alcohol-fed, bleomycin-treated mice were analyzed for IL-17 protein expression. RESULTS: Alcohol-treated PLFs expressed lower levels of Thy-1 than untreated cells. TGFß1 or IL-17 exposure suppressed PLF Thy-1 expression. When administered together, TGFß1 and IL-17 additively down-regulated Thy-1 expression. Exposure of naïve and Th17-polarized CD4+ T cells to alcohol induced the Th17 phenotype and augmented their production of IL-17. CD4+ Th17+ levels are elevated in the peripheral compartment but not in the lungs of alcohol-fed animals. Treatment of the PLFs with IL-17 and alcohol induced α-SMA expression. Induction of α-SMA and myofibroblast morphology by IL-17 occurred selectively in a Thy-1- fibroblast subpopulation. Chronic alcohol ingestion augmented lung-specific IL-17 expression following bleomycin-induced lung injury. CONCLUSIONS: Alcohol exposure skews T cells toward a Th17 immune response that in turn primes the lung for fibroproliferative disrepair through loss of Thy-1 expression and induction of myofibroblast differentiation. These effects suggest that IL-17 and TGFß1 contribute to fibroproliferative disrepair in the lung and targeting these proteins could limit morbidity and mortality following lung injury in alcoholic individuals.

mTORC1 activation in B cells confers impairment of marginal zone microarchitecture by exaggerating cathepsin activity.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTß) and lymphotoxin receptor (LTßR) expression indicated that LTßR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTßR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTßR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.

Wound healing potential of naringin ointment formulation via regulating the expression of inflammatory, apoptotic and growth mediators in experimental rats.

CONTEXT: Wound healing is a consequence of a complex process involving inflammatory, proliferative, and remodeling phases. Naringin, a flavanone glycoside, is associated with modulation of various oxido-inflammatory and growth factors. AIM: The aim of this study is to evaluate the wound-healing activity of naringin ointment formulation (NOF) on experimental wound models. MATERIALS AND METHODS: A soft paraffin-based cream containing 1, 2, and 4% (w/w) naringin was formulated and evaluated for physicochemical characters. Excision wounds and incisions wounds were used to study the topical effect of NOF for 20 d (once a day) on various biochemical, molecular, and histological parameters. RESULTS: NOF (2 and 4%, w/w) treatment showed a significant decrease (p < 0.05) in wound area and epithelization period whereas the rate of wound contraction increased significantly (p < 0.05). The altered levels of oxido-nitrosative stress (SOD, GSH, MDA, MPO, and NO) were significantly (p < 0.05) restored by NOF. Treatment produced a significant increase (p < 0.05) in tensile strength, hydroxyproline content, and protein content. TNF-α, IL-1ß, IL-6, IL-8, NF-κB, smad-7, and Bax mRNA expression were significantly down-regulated (p < 0.05) by NOF, whereas polymerase gamma (pol-γ), smad-3, VEGF and TGF-ß, and collagen-1 mRNA expressions were significantly up-regulated (p < 0.05) by NOF. Histological alterations in wound skin were also restored by NOF. CONCLUSION: NOF exerts wound healing potential via down-regulated expression of inflammatory (NF-κB, TNF-α, and ILs), apoptotic (pol-γ and Bax), and up-regulated growth factor (VEGF and TGF-ß) expression, thus modulating collagen-1 expression to induce angiogenesis leading to wound healing.

IL-13 immunotoxin accelerates resolution of lung pathological changes triggered by silica particles in mice.

Instillation of silica into the lungs of rodents results in pathological changes that strongly mimic human silicosis, an occupational lung disease marked by restrictive airway obstruction, inflammation, and fibrosis. Because IL-13 is a pivotal proinflammatory and fibrogenic cytokine, we examined whether a recombinant immunotoxin comprised of human IL-13 and a mutated form of Pseudomonas exotoxin (IL-13-PE) might affect pathological features of experimental silicosis. Mice received a single intranasal instillation of silica particles and were treated with intranasal IL-13-PE every other day from days 21 to 27 postsilica. The sensitivity of putative cell targets to IL-13-PE was also assessed in in vitro settings. Upregulation of IL-13, its receptor subunits IL-13Rα1 and IL-13Rα2, and shared receptor IL-4Rα were associated with development of granulomatous lung inflammation triggered by silica. IL-13-PE inhibited silica-induced granuloma and fibrotic responses noted at 24 h and 15 d after the last treatment. Upregulation of TNF-α, TGF-ß, and chemokines, as well as increased collagen deposition and airway hyperreactivity to methacholine were all clearly sensitive to IL-13-PE. In addition, IL-13-PE inhibited both IL-13-induced proliferation of cultured lung fibroblasts from silicotic mice and silica-induced IL-8 generation from A549 cells. In conclusion, our findings show that therapeutic treatment with IL-13-PE can reverse important pathological features caused by inhalation of silica particles, suggesting that this recombinant immunotoxin is a promising molecular template in drug discovery for the treatment of silicosis.

A stimulation-dependent alternate core promoter links lymphotoxin α expression with TGF-ß1 and fibroblast growth factor-7 signaling in primary human T cells.

Lymphotoxin (LT)-α regulates many biologic activities, yet little is known of the regulation of its gene. In this study, the contribution to LTA transcriptional regulation of the region between the transcription and translation start sites (downstream segment) was investigated. The LTA downstream segment was found to be required for, and alone to be sufficient for, maximal transcriptional activity in both T and B lymphocytes. The latter observation suggested that an alternate core promoter might be present in the downstream segment. Characterization of LTA mRNAs isolated from primary and from transformed human T cells under different stimulation conditions identified eight unique transcript variants (TVs), including one (LTA TV8) that initiated within a polypyrimidine tract near the 3' end of the downstream segment. Further investigation determined that the LTA downstream segment alternate core promoter that produces the LTA TV8 transcript most likely consists of a stimulating protein 1 binding site and an initiator element and that factors involved in transcription initiation (stimulating protein 1, TFII-I, and RNA polymerase II) bind to this LTA region in vivo. Interestingly, the LTA downstream segment alternate core promoter was active only after specific cellular stimulation and was the major promoter used when human T cells were stimulated with TGF-ß1 and fibroblast growth factor-7. Most importantly, this study provides evidence of a direct link for crosstalk between T cells and epithelial/stromal cells that has implications for LT signaling by T cells in the cooperative regulation of various processes typically associated with TGF-ßR and fibroblast growth factor-R2 signaling.

IL-12Rß2 is essential for the development of experimental cerebral malaria.

A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12Rß2 in ECM development. C57BL/6 mice deficient for IL-12Rß2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12Rß2, no neurologic sign of ECM developed upon PbA infection. Although wild-type mice developed distinct brain microvascular pathology, ECM-resistant, IL-12Rß2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12Rß2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-α, TNF-α, and IFN-γ in the brain after PbA infection. Therefore, IL-12Rß2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12Rß2 and lymphotoxin-α, TNF-α, and IFN-γ expression, key cytokines for ECM pathogenesis.

Complement C5a regulates IL-17 by affecting the crosstalk between DC and gammadelta T cells in CLP-induced sepsis.

Complement 5a (C5a) and Interleukin-17 (IL-17) are two important inflammatory mediators in sepsis. Here we studied the mechanisms underlying regulation of IL-17 by anaphylatoxin C5a. We found that C5a blockade increased the survival rate of mice following cecal ligation and puncture (CLP)-induced sepsis and decreased IL-17 expression in vivo. IL-17 was secreted mainly by gammadelta T cells in this model. Importantly, our data suggest that C5a participates in the regulation of IL-17 secretion by gammadelta T cells. Dendritic cells (DC) were found to act as a "bridge" between C5a and gammadelta T cells in a mechanism involving IL-6 and transforming growth factor beta (TGF-beta). These results imply that C5a affects the crosstalk between DC and gammadelta T cells during sepsis development, and this may result in a large production of inflammatory mediators such as IL-17.

Cytokine mRNA profile of alveolar T lymphocytes and macrophages in patients with systemic sclerosis suggests a local Tr1 response.

The development of an autoimmune disease like systemic sclerosis (SSc) is suspected to be driven by an activated T lymphocyte subset, expressing a cytokine profile specific to the disease. To further characterize the type of immune reaction in SSc, we searched for a broad panel of cytokine messenger ribonucleic acids (mRNAs) in T lymphocytes and monocytes/macrophages from paired samples of bronchoalveolar lavage fluid and peripheral blood in 18 patients and 16 age- and sex-matched controls. RNA from CD3(+) T lymphocytes and CD14(+) monocytes/macrophages was examined by means of the reverse transcriptase polymerase chain reaction. SSc alveolar T lymphocytes expressed a cytokine profile suggestive of a mixed Th1/Th2 reaction, showing an increased frequency of mRNA for interleukin (IL)-10, IL-6 and interferon (IFN)γ, while IL-1ß, IFNγ and tumour necrosis factor ß were expressed in blood T lymphocytes in a higher percentage of patients with SSc than controls. SSc alveolar T cells expressed IL-10 mRNA more often than peripheral T cells, a phenomenon not found in controls and which may point at local IL-10 activation/response in SSc lung. Transforming growth factor ß mRNA was present in all alveolar as well as peripheral blood T cell samples in patients and controls. The cytokine mRNA profile in SSc with interstitial lung disease (ILD) was similar to the profile found in SSc without ILD. Our findings point at a mixed Th1/Th2 reaction in SSc and may indicate regulatory T 1 cell activation/response in the lungs of patients with SSc.

[Transcriptional regulation of TNF/LT locus in immune cells].

Tumor necrosis factor (TNF) is one of the most important proinflammatory cytokines. It demonstrates a complex pattern of tissue-specific expression and behaves as a product of immediate early transcriptional response in macrophages. These properties have made the regulation of TNF gene, as well as regulation of tightly linked related lymphotoxin alpha (LTalpha) and beta (LTbeta) genes the object of thorough investigation for more than two decades. Some aspects of TNF/LT locus regulation, such as the role of distal TNF-promoter and of NF-kappaB factors in TNF gene transcription, still remain the object of discussion. Moreover, several recent studies uncovering the molecular mechanisms of immediate early gene activation and directly related to TNF gene regulation have not been reflected in published reviews yet. Here we briefly overview the modern concepts of transcriptional regulation of the TNF/LT locus, with an accent on new data and unanswered questions.

Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes.

We present evidence that human peripheral blood lymphocytes, free of contaminating monocytes, rapidly produce high levels of tumor necrosis factor (TNF) when stimulated with phorbol diester and calcium ionophore, and lower but significant levels of TNF when stimulated with mitogens. These two types of inducers act preferentially on T cells, both CD4+ and CD8+. NK cells produce TNF only when stimulated with phorbol diester and calcium ionophore, and they do so at a much lower level than T cells. The procedures used in the purification of lymphocytes and the differential ability to respond to various inducers allow us to exclude that monocytes or basophils contaminating the lymphocyte preparation participate in the production of TNF. In particular, LPS, a potent inducer of TNF production from monocytes, is unable to induce significant levels of TNF in the lymphocyte preparations. The TNF produced by lymphocytes has antigenic, physicochemical, and biochemical characteristics identical to those of the TNF produced by myeloid cell lines or monocytes upon stimulation with LPS. LT is also produced by lymphocyte preparations. Production of TNF and LT proteins in response to the different inducers is paralleled by accumulation of cytoplasmic TNF and LT mRNA. Both at mRNA and at protein levels, stimulation of T lymphocytes with phorbol diester and calcium ionophore preferentially induces TNF, whereas mitogen stimulation preferentially induces LT. Our data suggest that the TNF and LT genes, two closely linked genes encoding two partially homologous proteins with almost identical biological functions, are independently regulated in lymphocytes.

Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis.

Recent studies have shown that mucocutaneous leishmaniasis (MCL), a severe and debilitating form of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis infection, is accompanied by high circulating levels of tumor necrosis factor (TNF)-alpha. Analysis of TNF polymorphisms in Venezuelan ACL patients and endemic unaffected controls demonstrates a high relative risk (RR) of 7.5 (P < 0.001) of MCL disease in homozygotes for allele 2 of a polymorphism in intron 2 of the TNF-beta gene, especially in females (RR = 9.5; P < 0.001) compared with males (RR = 4; P < 0.05). A significantly higher frequency (P < 0.05) of allele 2 at the -308-basepair TNF-alpha gene polymorphism was also observed in MCL patients (0.18) compared with endemic control subjects (0.069), again associated with a high relative risk of disease (RR = 3.5; P < 0.05) even in the heterozygous condition. Because both the TNF-alpha and TNF-beta polymorphisms have previously been linked with functional differences in TNF-alpha levels, these data suggest that susceptibility to the mucocutaneous form of disease may be directly associated with regulatory polymorphisms affecting TNF-alpha production.

Production of tumor necrosis factor/cachectin by human T cell lines and peripheral blood T lymphocytes stimulated by phorbol myristate acetate and anti-CD3 antibody.

The induction of mRNA synthesis and accumulation of TNF/cachectin and lymphotoxin (LT) mRNAs in T leukemic cell lines and freshly isolated T cells were studied by Northern blot analyses. Without stimulation, TNF mRNA was barely detected in four T cell lines (CEM, KE4, MT-1, and SKW-3) and not detectable in Molt-4 and Jurkat cells, while a considerable amount of TNF mRNA was observed in HSB-2 cells. When stimulated by PMA, these T cell lines accumulated varying levels of TNF mRNA. All seven T cell lines expressed LT mRNA when unstimulated and responded well to PMA by increased accumulation of LT mRNA. The calcium ionophore A23187 by itself had no effect on TNF and LT mRNA accumulations in these cell lines. The CD3+ T cell lines did not respond to anti-CD3 mAb T3-II alone. However, A23187 and mAb T3-II further elevated TNF and LT mRNA accumulations in PMA-treated T cell lines. Synergism between PMA and mAb T3-II was modest in the CD3+ cell lines. A slight difference in kinetics of TNF and LT mRNA accumulations was noted. In addition, heterogeneities in TNF and LT expressions by these cell lines in responses to PMA and other stimuli were observed. In monocyte-depleted peripheral blood T cell populations. PMA was able to induce both TNF and LT mRNA syntheses. This effect was potentiated markedly by the addition of anti-CD3 mAb T3-II. This synergistic response to anti-CD3 mAb and PMA provided further evidence that T cells were the source of TNF synthesis in these cultures. There was a difference in the kinetics of TNF mRNA accumulation and that of LT mRNA. Maximal accumulation of TNF mRNA occurred at 4 h while 8-18 h was required for maximal LT mRNA accumulation. IL-2 mRNA accumulated at an intermediate peak time of 4-8 h. Western blot analyses and cytotoxicity assays with L cells as targets indicated that these T cell lines and peripheral blood T cells secreted TNF. These results provide further evidence that human T cells are capable of making TNF as well as LT under appropriate stimulations. Their productions are an integral part of T cell response to activation signals. In addition, it appears that the production of these two closely related molecules is independently regulated.

Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies.

Lymphokine synthesis patterns of a panel of 19 T cell clones have been evaluated, using mRNA hybridization methods to examine 11 different mRNAs induced by Con A. The two types of CD4+ Th cell clone described previously were clearly distinguished by this procedure, and the differences between the two types have now been extended to six induced products. With minor exceptions, only Th1 clones synthesized mRNA for IL-2, IFN-gamma, and lymphotoxin, and only Th2 clones synthesized mRNA for IL-4, IL-5, and another induced gene, P600. Four more induced products were expressed preferentially but not uniquely by one or another type of clone: mRNAs for GM-CSF, TNF, and another induced, secreted product (TY5) were produced in larger amounts by Th1 clones, whereas preproenkephalin was preferentially expressed by Th2 clones. IL-3 was produced in similar amounts by both types of clone. mAbs were used to establish three bioassays that were functionally monospecific for IL-2, IL-3, and IL-4, and a new anti-IFN gamma mAb, XMG1.2, was used to establish an ELISA for IFN-gamma. These four assays were used to show that secreted protein and mRNA levels correlated well for all cell lines. The implications of these findings for normal T cells are discussed.

Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production.

Since a dysregulated synthesis of tumor necrosis factor alpha (TNF-alpha) may be involved in the pathogenesis of autoimmune diseases, it was of interest to precisely locate the recently reported NcoI restriction fragment length polymorphism (RFLP) of the TNF-alpha region. However, by mapping of 56.8 kb of overlapping cosmid clones and direct sequencing, we could localize the polymorphic NcoI restriction site within the first intron of the TNF-beta gene and not in the TNF-alpha gene. To study whether regulatory mechanisms are affected by this polymorphism, we analyzed the TNF-alpha/TNF-beta production of phytohemagglutinin-stimulated peripheral blood mononuclear cells of individuals homozygous for the TNF-beta NcoI RFLP by ELISA and concomitant Northern blot analysis. On days 2-4 after stimulation with mitogen, the TNFB*1 allele corresponding to a 5.3-kb NcoI fragment presented with a significantly higher TNF-beta response. A mRNA analysis demonstrated that higher protein levels of TNF-beta correlate also with increased amounts of TNF-beta transcripts. No allelic association was found in respect to TNF-alpha production. To further investigate a possible allelic influence on transcription, we determined the DNA sequence of 2 kb of the 5' portion of our cloned TNFB*2 allele and compared it with the available TNF-beta sequences. By computer-aided recognition motif search of DNA binding factors, we report putative binding sites conserved between mouse and man in the 5' flanking region as well as in intron 1 of the TNF-beta gene, found also in other cytokine promoter sequences. In addition, by polymerase chain reaction amplification and sequencing of 740 bp of the 5' part of TNF-beta of individuals typed homozygously for the NcoI RFLP, we could show that amino acid position 26 is conserved as asparagine in the TNFB*1 and as threonine in the TNFB*2 sequence. A previously reported, EcoRI RFLP in the 3' untranslated region of TNF-beta does not segregate with either of the two alleles. Thus, four TNFB alleles can be defined at the DNA level.

Evaluation of Tear Protein Markers in Dry Eye Disease with Different Lymphotoxin-Alpha Expression Levels.

PURPOSE: To compare tear protein markers between normal subjects and patients with dry eye (DE) and high and low lymphotoxin-alpha (LT-α) levels. DESIGN: Prospective cross-sectional study. METHODS: Patients with DE were divided into low (≤700 pg/mL) and high (>700 pg/mL) LT-α groups. Twelve protein markers were measured by microsphere-based immunoassay and ocular surface parameters were determined in right eyes (33 high LT-α DE, 27 low LT-α DE, and 20 control eyes) and left eyes (21 high LT-α DE, 39 low LT-α DE, and 20 control eyes). RESULTS: In both eyes, tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-1ß, IL-1 receptor antagonist (IL-1Ra), IL-17A, and IL-12/23 p40 levels in high LT-α DE were significantly higher (P < .01) than in low LT-α DE. Significant correlations identified in high LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = 0.43, P = .013), IL-1ß (R = 0.48, P = .005), and IL-12/23 p40 (R = 0.50, P = .003), IL-12/23 p40 with ocular surface disease index (R = 0.35, P = .049), and epidermal growth factor with corneal fluorescein staining score (R = -0.36, P = .038). Significant correlations in low LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = -0.39, P = .046), TNF-α (R = -0.39, P = .047), and IL-17A (R = -0.48, P = .013), ocular surface disease index with TNF-α (R = -0.47, P = .017) and IL-17A (R = -0.46, P = .018), and IL-6 with tear breakup time (R = -0.40, P = .044). Lastly, IL-1Ra levels significantly increased in DE patients, positively correlated with temporal conjunctival hyperemia index, and negatively correlated with Schirmer I test (P < .05). CONCLUSIONS: Our study identified tear IL-1Ra level as a potential biomarker to replace the Schirmer I test. Multiple tear protein marker levels increased in high LT-α DE, indicating that high LT-α DE might have a different pathogenesis.

Induction of lymphotoxin-alpha by interleukin-12 p40 homodimer, the so-called biologically inactive molecule, but not IL-12 p70.

Interleukin-12 (IL-12) p70 (p40:p35) is a bioactive cytokine and its biological functions are becoming clear. On the other hand, the IL-12 p40 homodimer (p40(2)) was considered an inactive or inhibitory molecule and its functions are poorly understood. It has been reported that increased expression of lymphotoxin-alpha (Lt-alpha) in the central nervous system as well as in peripheral immune cells is associated with multiple sclerosis and experimental allergic encephalomyelitis. Here we describe that p40(2) induces the expression of Lt-alpha in primary mouse and human microglia, BV-2 microglial cells, splenic macrophages, RAW 264.7 cells and splenic T cells. Interestingly, IL-12 p70 was either unable to induce Lt-alpha or was a very weak inducer of Lt-alpha in these cell types. Consistently, p40(2), but not p70, induced Lt-alpha promoter-driven luciferase activity in microglial cells. Among various stimuli tested, p40(2) emerged as the most potent followed by IL-16, lipopolyaccharide and double-stranded RNA in inducing the activation of Lt-alpha promoter in microglial cells. Furthermore, an increase in Lt-alpha messenger RNA expression by overexpression of p40, but not p35, complementary DNA and induction of Lt-alpha expression by p40(2) in microglia isolated from IL-12p35(-/-) mice confirm that p40, but not p35, is responsible for the induction of Lt-alpha. Finally, by using primary microglia from IUL-12 receptor beta1 deficient (IL-12Rbeta1(-/-)) and IL-12Rbeta2(-/-) mice, we demonstrate that p40(2) induced the expression of Lt-alpha in microglia and macrophages via IL-12Rbeta1, but not IL-12Rbeta2. These studies delineate a novel biological function of p40(2) that is absent in IL-12.

Overexpression of lymphotoxin in T cells induces fulminant thymic involution.

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Variability in the response of human dendritic cells stimulated with Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Dendritic cells are able to prime and polarize naïve T cells towards either a T helper 1 or a T helper 2 response, depending on the antigen type and concentration, the costimulatory signals and the local cytokine millieu. In this investigation, we analyzed the response of human dendritic cells to stimulation with different concentrations of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans. MATERIAL AND METHODS: Using different concentrations of P. gingivalis ATCC33277 and A. actinomycetemcomitans ATCC 33384, we determined the expression of the maturation markers CD80 and CD86 from purified human dendritic cells by flow cytometry. We also evaluated the mRNA expression levels for the cytokines interleukin-1beta, interleukin-2, interleukin-5, interleukin-6, interleukin-10, interleukin-12, interleukin-13, interferon-gamma, tumor necrosis factor-alpha and tumor necrosis factor-beta by quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Both P. gingivalis and A. actinomycetemcomitans led to dendritic cell maturation, but the expression of CD80 was higher when the dendritic cells were stimulated with A. actinomycetemcomitans. Although both pathogens induced a T helper 1 pattern of cytokine expression, A. actinomycetemcomitans-stimulated dendritic cells expressed interleukin-1beta, interleukin-12, interferon-gamma, tumor necrosis factor-alpha and tumor necrosis factor-beta at lower bacterial concentrations than P. gingivalis. While 10(6) bacteria/mL of P. gingivalis or 10(4) bacteria/mL of A. actinomycetemcomitans induced expression of interleukin-12p40, the expression of other cytokines required 10 to 100-fold higher concentrations of bacteria. CONCLUSION: These results demonstrate that A. actinomycetemcomitans is a more potent immunogen than P. gingivalis because, at least in vitro, it induces stronger differentiation and activation of dendritic cells. In addition, our data also show that for a given strain, the bacterial load determines the pattern of cytokines that are expressed.