Elevated plasma levels and monocyte-associated expression of CD137 ligand in patients with acute atherothrombotic stroke.

BACKGROUND: CD137 ligand (CD137L) is expressed by various immune cells and exists in membrane-bound and soluble forms. Recently, CD137L was found to be localized to macrophages in human atherosclerotic lesions and CD137L levels were much higher in atherosclerotic lesions than in normal arteries. However, the role of CD137L with different forms in atherothrombotic stroke remains unclear. PATIENTS AND METHODS: The soluble CD137L (sCD137L) protein and CD137L expression on monocytes were analyzed by an enzyme-linked immunosorbent assay and flow cytometry in peripheral blood of patients with acute ischemic atherosclerotic stroke, atherosclerosis controls and normal controls. RESULTS: During the initial 24h after onset, the stroke patients had elevated plasma sCD137L levels (133.2 pg/ml) and CD137L expression on monocytes [7.9 ± 4.1%, 7.0 ± 4.0 mean fluorescence intensity (MFI)] as compared with normal controls (75 pg/ml, p < 0.05; 4.6 ± 2.4%, 4.1 ± 2.7 MFI, p < 0.05). CONCLUSIONS: The dysregulation of CD137L expression may reflect a persistent chronic inflammatory response that may have been induced during early stages of the disease. Our results strongly suggest that the abnormal expression of CD137L on monocytes may lead to dyregulated CD137L/CD137 signaling and consequently form part of a positive-feedback, inflammation-promoting circuit in stroke, while the elevated sCD137L protein levels may function as a self-regulatory mechanism of CD137/CD137L interaction and costimulation.

Decreased 4-1BB expression on CD4+CD25 high regulatory T cells in peripheral blood of patients with multiple sclerosis.

As a tumour necrosis factor receptor superfamily member, 4-1BB (CD137) is preferentially expressed in CD4+CD25+ regulatory T cells (Tregs) and has been suggested to play an important role in regulating the generation or function of Tregs. Recent studies of human Tregs have shown that blood CD4+CD25(high) T cells were much closer to Tregs in terms of their functionality. Furthermore, CD4+CD25(high) Tregs have been found to have a decreased effector function in patients with multiple sclerosis (MS). In this study, we examined the expression of 4-1BB and soluble 4-1BB (s4-1BB) protein levels in the peripheral blood of MS patients. Compared with healthy controls, MS patients had decreased 4-1BB expression in their CD4+C25(high) Tregs and increased plasma s4-1BB protein levels. Moreover, the plasma s4-1BB levels of MS patients were shown to be inversely correlated with the 4-1BB surface expression of CD4+CD25(high) Tregs. The down-regulated 4-1BB expression on CD4+CD25(high) Tregs of MS patients may be involved in the impaired immunoactivity of these Tregs. The elevated s4-1BB levels may, at least in part, function as a self-regulatory attempt to inhibit antigen-driven proliferation of Tregs or their immunosuppressive activity.

Enhanced plasma levels of LIGHT in patients with acute atherothrombotic stroke.

OBJECTIVES: As a member of the tumor necrosis factor superfamily (TNFSF), LIGHT (TNFSF14) was recently found to be associated with platelets and released upon activation. Increased plasma levels of LIGHT have been reported in patients with myocardial infarction and unstable angina. The aim of the study was to analyze plasma levels of LIGHT in acute ischemic atherosclerotic stroke. MATERIALS AND METHODS: The soluble LIGHT protein was analyzed by an enzyme-linked immunosorbent assay in peripheral blood of patients with acute ischemic atherosclerotic stroke (n = 20), asymptomatic carotid stenosis (n = 19) and normal controls (n = 23). RESULTS: During the initial 24 h after onset, the stroke patients had an increased plasma LIGHT levels as compared with normal controls. Moreover, the plasma LIGHT levels of the stroke patients were correlated with blood platelet count (r = 0.6341, P = 0.0027). CONCLUSION: The elevated LIGHT levels may reflect a persistent chronic inflammatory response that may have been induced during early stages of the disease. We speculate that this derangement of LIGHT may be important for atherogenetic process of ischemic stroke.

Increased soluble 4-1BB ligand (4-1BBL) levels in peripheral blood of patients with multiple sclerosis.

4-1BB ligand (4-1BBL; CD137L) is a member of the tumour necrosis factor superfamily expressed primarily on antigen presenting cells such as B cells, macrophages and dendritic cells. Its engagement with the receptor 4-1BB (CD137) has been shown to promote T-cell activation and regulate proliferation and survival of T cells. The role of the costimulatory molecule in multiple sclerosis (MS) remains unclear. In this study, the expression of 4-1BBL and soluble 4-1BBL (s4-1BBL) protein levels were analysed in peripheral blood of MS patients. Compared with healthy controls, MS patients had an increase in both plasma s4-1BBL protein levels and expression of 4-1BBL in CD14(+) monocytes. In contrast, myelin basic protein-reactive T-cell proliferation was not found to be inhibited by the use of an anti-4-1BBL antibody. The elevated s4-1BBL protein levels in the MS patients may function as a self-regulatory mechanism of 4-1BB/4-1BBL interaction and costimulation.

Lack of association of human chemokine receptor gene polymorphisms CCR2-64I and CCR5-Delta32 with autoimmune Addison's disease.

The attraction of leukocytes to tissues is essential for inflammation and the initiation of the autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytokines. We investigated whether human chemokine receptor gene polymorphisms, namely CCR5-Delta32 and CCR2-64I, are associated with susceptibility to autoimmune Addison's disease. Genotyping was performed in 56 patients and 127 healthy controls by a new method using pyrosequencing for CCR2-64I and by polymerase chain reaction and detecting gel for CCR5-Delta32. None of the CCR2 or CCR5 alleles was found to be associated, either positively or negatively, with disease risk. Our results indicate that the CCR2-64I and CCR5-Delta32 gene polymorphisms do not play a major role in conferring genetic risk for, and/or protection against, autoimmune Addison's disease.

Expression of the B cell-attracting chemokine CXCL13 in the target organ and autoantibody production in ectopic lymphoid tissue in the chronic inflammatory disease Sjögren's syndrome.

Sjögren's syndrome is an autoimmune disease characterized by lymphocytic infiltrates resembling secondary lymphoid organs in salivary glands. In this study, we demonstrate the expression of the lymphoid tissue homing chemokine CXCL13 (BCA-1/BLC), which has attracting properties for B cells and subsets of activated T cells, in salivary glands of patients with Sjögren's syndrome using immunohistochemistry and in situ hybridization. CXCL13 expression was primarily observed in epithelial cells in acini and ducts of inflamed glands while its receptor, CXCR5 (BLR-1), was expressed on the infiltrating mononuclear cells. In addition, cells producing antibodies against one of the major autoantigens in Sjögren's syndrome, Ro 52, were identified at the periphery of the follicular infiltrates indicating that the ectopic lymphoid tissue is directly involved in the disease process. Identification of CXCL13 and CXCR5 in salivary glands suggests that the target organ plays an essential role in the inflammatory process by recruiting B and T cells. These results also provide a molecular mechanism by which lymphoid neogenesis and ectopic germinal centre formation might occur in the glands of these patients, which may be the key step in the development of the chronic inflammatory process in Sjögren's syndrome.

Lymphoid neogenesis: de novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines.

Chronic inflammation is a complex pathophysiological process with accumulation of mononuclear cells seen in response to invading pathogens, neoplastic transformation, or autoimmune recognition of self-antigens. The inflammatory process has evolved to facilitate effective elimination of pathogens and tumors and it is normally transient and turned off when the causative stimulus has been eliminated. Occasionally, however, the process is sustained for a long time and can lead to severe tissue damage. This is seen in organ-specific autoimmune diseases such as rheumatoid arthritis, Sjögren's syndrome, and Hashimoto's thyroiditis, but also in infectious diseases such as Helicobacter pylori-induced gastritis. Disturbingly, many of these chronic inflammatory diseases are associated with an increased risk for neoplastic transformation and development of lymphomas. This review summarizes experimental evidence suggesting that chronic inflammation involves ectopic de novo formation of organized lymphoid tissue and that this lymphoid neogenesis is regulated by expression of homing chemokines.

Lymphotoxin-alpha deficiency completely protects C57BL/6 mice from developing clinical experimental autoimmune myasthenia gravis.

A complete prevention of clinical experimental autoimmune myasthenia gravis (EAMG) was observed in lymphotoxin (LT)-alpha deficient (LT-alpha(-/-)) mice compared to LT-alpha(+/+) mice when immunized with acetylcholine receptor. However, only a partial prevention of clinical EAMG incidence was observed in LT-beta(-/-) mice compared to LT-beta(+/+) mice. LT-alpha(-/-)and LT-beta(-/-) mice had lower mean titers of total IgG, IgG(1), IgG(2a) and IgG(2b) and higher or equal mean titers of IgM anti-AChR antibodies compared to controls. Therefore, LT-alpha(-/-)and LT-beta(-/-) AChR immunized mice are capable of mounting a primary (IgM) humoral immune response to AChR, but are less capable of switching to the pathogenic anti-AChR IgG isotypes. LT could play a significant role in the pathogenesis of myasthenia gravis.

Tumor necrosis factor a-11 and DR15-DQ6 (B*0602) haplotype increase the risk for cervical intraepithelial neoplasia in human papillomavirus 16 seropositive women in Northern Sweden.

HLA genes have been shown to be associated with cervical intraepithelial neoplasia (CIN), a precursor of cervical cancer. The human papillomaviruses (HPV) types 16 and 18 are the major environmental cause of this disease. Because the immune system plays an important role in the control of HPV infection, the association of polymorphic HLA could lead to a different immune response to control the development of cervical cancer. The aim of this study was to analyze the association between CIN and a microsatellite polymorphism of tumor necrosis factor (TNFa) taking HPV exposure and CIN-associated HLA haplotypes into account. In a nested case-control study in northern Sweden, 64 patients and 147 controls matched for age and sex and derived from the same population-based cohort were typed for TNFA, HLA-DR, and DQ and assayed for antibodies to HPV types 16 and 18. TNFa polymorphism was not associated with CIN per se. However, there was a significant increase in the frequency of TNFa-11 among HPV16-positive and HLA DR15-DQ6 (B*0602) patients compared with HPV16- and HLA-DQ6-negative patients (odds ratios, 5.4 and 9.3, respectively). The relative risk for CIN conferred by the combination of TNFa-11, HLA-DQ6, and HPV 16 positivity was 15. Our study suggests that the TNFa-11 allele is associated with HPV16 infection and associated with CIN in combination with HLA-DQ6 but not by itself.

Lymphoid tissue homing chemokines are expressed in chronic inflammation.

Secondary lymphoid tissue chemokine (SLC) and B lymphocyte chemoattractant (BLC) are homing chemokines that have been implicated in the trafficking of lymphocytes and dendritic cells in lymphoid organs. Lymphotoxin-alpha (LTalpha), a cytokine crucial for development of lymphoid organs, is important for expression of SLC and BLC in secondary lymphoid organs during development. Here we report that transgenic expression of LTalpha induces inflammation and ectopic expression of SLC and BLC in the adult animal. LTbeta was not necessary for induction of BLC and SLC in inflamed tissues, whereas, in contrast, tumor necrosis factor receptor-1 was found to be important for the LTalpha-mediated induction of these chemokines. The ectopic expression of LTalpha is associated with a chronic inflammation that closely resembles organized lymphoid tissue and this lymphoid neogenesis can also be seen in several chronic inflammatory diseases, including in the pancreas of the prediabetic nonobese diabetic (NOD) mouse. Expression of SLC was also observed in the pancreas of prediabetic NOD mice. This study implicates BLC and SLC in chronic inflammation and presents further evidence that LTalpha orchestrates lymphoid organogenesis both during development and in inflammatory processes.

Localization of the tetrodotoxin-resistant sodium channel NaN in nociceptors.

Tetrodotoxin-resistant sodium currents contribute to the somal and axonal sodium currents of small diameter primary sensory neurons, many of which are nociceptive. NaN is a recently described tetrodotoxin-resistant sodium channel expressed preferentially in IB4-labeled dorsal root ganglion (DRG) neurons. We employed an antibody raised to a NaN specific peptide to show that NaN is preferentially localized along axons of IB4-positive unmyelinated fibers in the sciatic nerve and in axon terminals in the cornea. NaN immunoreactivity was also found at some nodes of Ranvier of thinly myelinated axons of the sciatic nerve, where it was juxtaposed to Kv1.2 potassium channel immunoreactivity. This distribution of NaN is consistent with a role for NaN sodium channels in nociceptive transmission.

Kinetics and cellular origin of cytokines in the central nervous system: insight into mechanisms of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 (H-2b) mice is characterized by early (day 12) acute paralysis, followed by a sustained chronic clinical course that gradually stabilizes. Extensive inflammation and demyelination coincide with clinical signs of disease. To identify the mechanisms of these processes, individual proinflammatory and anti-inflammatory cytokines and chemokines were studied. Sensitive single-cell assays were utilized to determine the cellular origin and kinetics of cytokine production in the CNS. Immunization with MOG35-55 peptide resulted in priming of both Th1 (lymphotoxin, IFN-gamma, and TNF-alpha) and Th2 (IL-4) cells in the spleen. However, only Th1 cells were apparent in the CNS. CD4 T cells that produced IFN-gamma or TNF-alpha were present in the CNS by day 7 after immunization with MOG35-55, peaked at day 20, and then waned. TNF-alpha was also produced in the CNS by Mac-1+ cells. On days 7 and 10 after immunization, the TNF-alpha-producing Mac1+ cells were predominantly microglia. By day 14, a switch occurred in that the Mac1+ TNF-alpha-producing cells had the phenotype of infiltrating macrophages. RANTES, IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1 chemokine mRNA were detected in the CNS by day 8 after immunization. The early presence of monocyte chemotactic protein 1 (MCP-1) in the CNS provides a mechanism for the recruitment of macrophages. These data implicate TNF-alpha production by a continuum of T cells, microglia, and macrophages at various times during the course of disease. The importance of Th1 cytokines is highlighted, with little evidence for a role of Th2 cytokines.

MICA gene polymorphism and the risk to develop cervical intraepithelial neoplasia.

Cervical intraepithelial neoplasia (CIN) is associated with human papillomaviruses (HPV) and the HLA genes. The MICA (MHC class I chain-related gene A) is expressed by keratinocytes and epithelial cells and interacts with gamma delta T cells. It is therefore possible that MICA might influence the pathogenesis of CIN and cervical cancer through presentation of viral or tumor antigens. To investigate this, we determined the MICA transmembrane allele frequencies in a prospective population-based cohort study from the Västerbotten County in northern Sweden. 74 women developed CIN. 153 control women who remained healthy during follow up were matched for age. Five polymorphic microsatellite alleles of MICA were identified by a polymerase chain reaction-based (PCR) technique using fluorescent-labeled primers. MICA A5 and A5.1 were the most common alleles in this population. None of the alleles of MICA were associated with disease. The frequency of MICA allele A5 was higher among HPV 18 seropositive than HPV 18 seronegative patients but this difference was not significant after the correction of p value. In conclusion, microsatellite allele polymorphism of MICA transmembrane part is not associated with cervical intraepithelial neoplasia.

B-cell-deficient mice develop experimental allergic encephalomyelitis with demyelination after myelin oligodendrocyte glycoprotein sensitization.

Myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) is an animal model for the central nervous system disease multiple sclerosis (MS). The roles of individual components of the immune system have not been completely defined in the mouse model, and to determine the role of B cells and Abs in the induction of EAE and demyelination, B cell-deficient muMT (H-2b) mice were immunized with MOG peptide 35-55. The muMT mice were susceptible to MOG-induced EAE and developed a chronic sustained disease, with inflammatory lesions and primary demyelination in the spinal cord, brain, and optic nerves, similar to that seen in wild-type C57BL/6 mice. The inflammatory cells in the central nervous system of muMT mice included both activated and memory T cells and macrophages. The data suggest that B cells and Abs are not necessary for primary demyelination in MOG-induced EAE in mice.

A molecular model of myelin oligodendrocyte glycoprotein.

Myelin oligodendrocyte glycoprotein (MOG) is a protein on the surface of myelin sheaths. It is a putative target of the autoimmune attack in the inflammatory and demyelinating CNS disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. MOG belongs to the immunoglobulin superfamily (IgSF), and its extracellular N-terminal domain contains many conserved IgSF consensus residues seen in immunoglobulin variable region folds. The aim of the present study was to create a molecular model of the extracellular N-terminal domain of mouse MOG. No crystal structure is yet available of MOG, and thus a molecular model would be useful in providing insight into its structure and binding characteristics. Molecular graphics techniques and molecular dynamics with secondary structure-based restraints were used in the construction and refinement of the MOG model. Regions of high prediction confidence were identified, and possible glycosylation, dimerization, complement binding, and antibody-binding regions in MOG were mapped and analyzed.

Polymorphism in tumor necrosis factor genes associated with myasthenia gravis.

The aim of this study was to analyze associations between myasthenia gravis (MG) and polymorphisms in the tumor necrosis factor (TNF) region in 79 Swedish patients and 155 unrelated controls. The frequency of the TNFa2 allele of a microsatellite located 3.5 kb upstream of the lymphotoxin alpha (LT-alpha) gene in the TNF region was found to be increased in overall MG patients compared to controls. The frequency of the short 5.5 kb fragment (TNFB * 1) of a bi-allelic NcoI RFLP polymorphism located at the first intron of the LT-alpha gene was increased in patients with an early onset of disease compared to patients with a later onset.

Structural differences between HLA-DQ molecules associated with myasthenia gravis characterized by molecular modeling.

Myasthenia gravis (MG) is characterized by muscle weakness due to autoimmunity against the nicotinic acetylcholine receptor (nAChR). MG is associated with polymorphisms in HLA-DQ genes and the aim of the present study was to characterize structural differences in the peptide binding groove of HLA-DQ molecules positively and negatively associated with MG. Three dimensional models of the positively associated DQ2 (DQB1*02) and negatively associated DQ6 (DQB1*0603) molecules were constructed by homology modeling techniques. The differences in peptide binding properties were primarily localized to peptide-anchor pockets P7 and P9, which might be of importance for the binding of disease-associated peptides from the nAChR.

Polymorphisms in IL-1beta and IL-1 receptor antagonist genes are associated with myasthenia gravis.

Interleukin 1 (IL-1)beta, TaqI restriction fragment length polymorphism (RFLP) in exon 5 and IL-1 receptor antagonist (IL-1Ra) polymorphism, variable numbers of an 86-bp tandem repeat (VNTR), were analysed in 107 patients with myasthenia gravis (MG) and 82 ethnically matched healthy control (HC) individuals. Positive association was found with IL-1beta TaqI RFLP allele 2 carriage in MG (OR = 2.007), while allele 1 was negatively associated with MG (OR = 0.498). When homozygous individuals for allele 2 were considered, the association was stronger (OR = 4.630), indicating a dose effect of allele 2. Analysis of IL-1beta TaqI RFLP in relation to HLA-B8 demonstrated that the allelic association was more pronounced in patients without HLA-B8 (OR = 2.813). There was no difference in IL-1Ra VNTR allelic distribution in MG patients compared with HC. However, MG patients who were noncarriers of IL-Ra allele 2 had a significantly higher percentage of IL-1beta TaqI RFLP allele 2 carriage (OR = 3.085), while there was no such difference in IL-1Ra allele 2 carriers. Our results demonstrate a new genetic marker in MG, which exerts its maximum effect in patients with the lowest MHC-associated susceptibility. We propose a possible pathogenetic role of IL-1beta and a possible intrinsic dyregulation of IL-1 in MG.