Production of IL-16 correlates with CD4+ Th1 inflammation and phosphorylation of axonal cytoskeleton in multiple sclerosis lesions

BACKGROUND: Multiple sclerosis (MS) is a central nervous system-specific autoimmune, demyelinating and neurodegenerative disease. Infiltration of lesions by autoaggressive, myelin-specific CD4+Th1 cells correlates with clinical manifestations of disease. The cytokine IL-16 is a CD4+ T cell-specific chemoattractant that is biased towards CD4+ Th1 cells. IL-16 precursor is constitutively expressed in lymphocytes and during CD4+ T cell activation; active caspase-3 cleaves and releases C-terminal bioactive IL-16. Previously, we used an animal model of MS to demonstrate an important role for IL-16 in regulation of autoimmune inflammation and subsequent axonal damage. This role of IL-16 in MS is largely unexplored. Here we examine the regulation of IL-16 in relation to CD4+ Th1 infiltration and inflammation-related changes of axonal cytoskeleton in MS lesions. METHODS: We measured relative levels of IL-16, active caspase-3, T-bet, Stat-1 (Tyr (701)), and phosphorylated NF(M+H), in brain and spinal cord lesions from MS autopsies, using western blot analysis. We examined samples from 39 MS cases, which included acute, subacute and chronic lesions, as well as adjacent, normal-appearing white and grey matter. All samples were taken from patients with relapsing remitting clinical disease. We employed two-color immunostaining and confocal microscopy to identify phenotypes of IL-16-containing cells in frozen tissue sections from MS lesions. RESULTS: We found markedly increased levels of pro- and secreted IL-16 (80 kD and 22 kD, respectively) in MS lesions compared to controls. Levels of IL-16 peaked in acute, diminished in subacute, and were elevated again in chronic active lesions. Compared to lesions, lower but still appreciable IL-6 levels were measured in normal-appearing white matter adjacent to active lesions. Levels of IL-16 corresponded to increases in active-caspase-3, T-bet and phosphorylated Stat-1. In MS lesions, we readily observed IL-16 immunoreactivity confined to infiltrating CD3+, T-bet+ and active caspase-3+ mononuclear cells. CONCLUSION: We present evidence suggesting that IL-16 production occurs in MS lesions. We show correlations between increased levels of secreted IL-16, CD4+ Th1 cell inflammation, and phosphorylation of axonal cytoskeleton in MS lesions. Overall, the data suggest a possible role for IL-16 in regulation of inflammation and of subsequent changes in the axonal cytoskeleton in MS

Role of IL-16 in CD4(+) T cell-mediated regulation of relapsing multiple sclerosis

In an important article published in Nature Medicine, Liu and colleagues described a novel CD4+ FoxA1+ regulatory T (Treg) cell population as distinct regulators of relapsing-remitting multiple sclerosis (RRMS) and experimental autoimmune encephalomyelitis (EAE). CD4+ FoxA1+ Treg cells appear as key regulators of responsiveness to therapy with interferon beta (IFN-β) in RRMS patients. Data indicate that CD4+ FoxA1+ FOXP3− Treg cells develop within the central nervous system (CNS), and a potential of cerebellar granule neurons (CGN) in generation of CD4+ FoxA1+ PD-L1hiFOXP3− Treg cells from encephalitogenic CD4+ T cells. A CD4 co-receptor specific ligand, IL-16, governs trafficking and biological properties of CD4+ T cells irrespective of their activation state. Functions of IL-16, relevant to Treg cells, include expansion of CD4+CD25+ T cells in long-term cultures with IL-2, de novo induction of FOXP-3 and migration of FOXP-3+ T cells. IL-16 is highly conserved across species including human and mouse. CGN and neurons in hippocampus contain neuronal-IL-16 (NIL-16), splice variant of immune IL-16, and express CD4 molecule. In a CD4-dependent manner, IL-16 supports cultured CGN survival. Concomitant studies of RRMS lesions and corresponding MOG35–55-induced relapsing EAE in (B6 × SJL)F1 (H-2b/s) mice discovered similar roles of IL-16 in regulation of relapsing disease. In RRMS and EAE relapse, peak levels of IL-16 and active caspase-3 correlated with CD4+ T cell infiltration and levels of T-bet, Stat-1(Tyr701), and phosphorylated neurofilaments of axonal cytoskeleton [NF (M + H) P], suggesting a role of locally produced IL-16 in regulation of CD4+ Th1 inflammation and axonal damage, respectively. IL-16 was abundantly present in CD4+ T cells, followed by CD20+ B, CD8+ T, CD83+ dendritic cells, and Mac-1+ microglia. Apart from lesions, bioactive IL-16 was located in normal-appearing white matter (NAWM) and normal-appearing grey matter (NAGM) in RRMS brain and spinal cord. A cytokine IL-16 emerges as an important regulator of relapsing MS and EAE. Better understanding of immune cell-neuron interactions mediated by IL-16 will foster development of more specific CD4+ T cell subset-targeted therapies to prevent or ameliorate progression of neuroinflammation and axonal and neuronal damage. Translational studies necessitate corresponding EAE models

Immunopathology of CD4+ T Cell-Mediated Autoimmune Responses to Central Nervous System Antigens: Role of IL-16

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating and degenerative disease of the central nervous system (CNS). While etiology of the disease remains unknown, genetic susceptibility and autoimmune mechanisms in the initiation and progression of the disease have been strongly suggested. Experimental autoimmune encephalomyelitis (EAE) is commonly used to study immune regulation of MS. Infiltration by CD4+ T cells, through blood-brain barrier (BBB), precedes the onset and relapses of MS. CNS migration and homing patterns of T cells are tightly synchronized by astrocyte and microglia derived cytokines and chemokines. Autoimmune, CNS antigenreactive, infiltrating T cells produce and locally release cytokines including but not limited to IFNγ, IL-2, IL-6, IL-16, IL-17, TNFα, and chemokines including CCL2, CCL5 and CXCL10. Chemokine mediated chemotaxis is exclusive for activated cell state and most chemokines do not discriminate between distinct cell types. Conversely, a cytokine IL-16 is a CD4-specific cytokine-ligand and exclusively induces chemotaxis of CD4+T cells, by binding and signaling through CD4, regardless of T cell activation state. In this article we focus on CD4+ T cell-mediated autoimmune responses to CNS antigens because of their importance for immunopathology of MS and EAE. We focus on autoimmune responses to myelin oligodendrocyte glycoprotein (MOG) because of its relevance for immunopathology of MS. We emphasize a role of IL-16 in regulation of CD4+T cell mediated autoimmune responses to MOG in EAE and MS. While a role of IL-16 in regulation of other CD4+T cell mediated autoimmune diseases has been established, its role in regulation of MS remains to be determined. Emerging data from our laboratories have indicated that IL-16-mediated CD4+ T cell chemoattraction has a significant role in regulation of CD4+ T cell-mediated autoimmune responses to CNS antigens. We propose an important function of this cytokine in regulation of relapsing-remitting EAE

The Discovery of Cometary Activity in Near-Earth Asteroid (3552) Don Quixote

The near-Earth object (NEO) population, which mainly consists of fragments from collisions between asteroids in the main asteroid belt, is thought to include contributions from short-period comets as well. One of the most promising NEO candidates for a cometary origin is near-Earth asteroid (3552) Don Quixote, which has never been reported to show activity. Here we present the discovery of cometary activity in Don Quixote based on thermal-infrared observations made with the Spitzer Space Telescope in its 3.6 and 4.5 {\mu}m bands. Our observations clearly show the presence of a coma and a tail in the 4.5 {\mu}m but not in the 3.6 {\mu}m band, which is consistent with molecular band emission from CO2. Thermal modeling of the combined photometric data on Don Quixote reveals a diameter of 18.4 (-0.4/+0.3) km and an albedo of 0.03 (-0.01/+0.02), which confirms Don Quixote to be the third-largest known NEO. We derive an upper limit on the dust production rate of 1.9 kg s^-1 and derive a CO2 gas production rate of (1.1+-0.1)10^26 molecules s^-1. Spitzer IRS spectroscopic observations indicate the presence of fine-grained silicates, perhaps pyroxene rich, on the surface of Don Quixote. Our discovery suggests that CO2 can be present in near-Earth space over a long time. The presence of CO2 might also explain that Don Quixote's cometary nature remained hidden for nearly three decades.Comment: 40 pages, 8 figures, accepted by Ap

Increased Frequency and Compromised Function of T Regulatory Cells in Systemic Sclerosis (SSc) Is Related to a Diminished CD69 and TGFβ Expression

Contains fulltext : 80239.pdf (publisher's version ) (Open Access)BACKGROUND: Regulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc). METHODS/PRINCIPAL FINDINGS: Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 20) or diffuse cutaneous SSc (dcSSc, n = 48). Further subdivision was made between early dcSSc (n = 24) and late dcSSc (n = 24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25(high)CD127(-) and CD4CD25(low)CD127(high) and CD3(+) cells. Suppressive function was correlated with CD69 surface expression and TGFbeta secretion/expression. The frequency of CD4(+)CD25(+) and CD25(high)FoxP3(high)CD127(neg) T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma. CONCLUSIONS/SIGNIFICANCE: These results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFbeta expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis

Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study

BACKGROUND: Cryopreservation of peripheral blood mononuclear cells has been used to preserve and standardize immunologic measurements for multicenter studies, however, effects of cryopreservation on cytokine responses are incompletely understood. In designing immunologic studies for a new multicenter birth cohort study of childhood asthma, we performed a series of experiments to determine the effects of two different methods of cryopreservation on the cytokine responses of cord and peripheral blood mononuclear cells. RESULTS: Paired samples of PBMC were processed freshly, or after cryopreservation in a Nalgene container (NC) or a controlled-rate freezer (CRF). Although there were some differences between the methods, cryopreservation inhibited PHA-induced IL-10 secretion and Der f 1-induced IL-2 secretion, and augmented PHA-induced IL-2 secretion and spontaneous secretion of TNF-α. In separate experiments, NC cryopreservation inhibited secretion of several cytokines (IL-13, IL-10, IFN-γ, TNF-α) by PHA-stimulated cord blood mononuclear cells. With the exception of PHA-induced IL-13, results from fresh and cryopreserved cord blood samples were not significantly correlated. Finally, in reproducibility studies involving processing of identical cell samples in up to 4 separate laboratories, variances in cytokine responses of fresh cells stimulated at separate sites did not exceed those in cryopreserved cells stimulated at a central site. CONCLUSION: Collectively, these studies indicate that cryopreservation can affect mononuclear cell cytokine response profiles, and that IL-10 secretion and antigen-induced responses may be especially vulnerable. These studies also demonstrate that mononuclear cell responses can be standardized for performance in a small number of laboratories for multicenter studies, and underscore the importance of measuring reproducibility and of testing whether cryopreservation techniques alter specific immunologic outcomes