Stress Signal ULBP4, an NKG2D Ligand, Is Upregulated in Multiple Sclerosis and Shapes CD8+ T-Cell Behaviors.

BACKGROUND AND OBJECTIVES: We posit the involvement of the natural killer group 2D (NKG2D) pathway in multiple sclerosis (MS) pathology via the presence of specific NKG2D ligands (NKG2DLs). We aim to evaluate the expression of NKG2DLs in the CNS and CSF of patients with MS and to identify cellular stressors inducing the expression of UL16-binding protein 4 (ULBP4), the only detectable NKG2DL. Finally, we evaluate the impact of ULBP4 on functions such as cytokine production and motility by CD8+ T lymphocytes, a subset largely expressing NKG2D, the cognate receptor. METHODS: Human postmortem brain samples and CSF from patients with MS and controls were used to evaluate NKG2DL expression. In vitro assays using primary cultures of human astrocytes and neurons were performed to identify stressors inducing ULBP4 expression. Human CD8+ T lymphocytes from MS donors and age/sex-matched healthy controls were isolated to evaluate the functional impact of soluble ULBP4. RESULTS: We detected mRNA coding for the 8 identified human NKG2DLs in brain samples from patients with MS and controls, but only ULBP4 protein expression was detectable by Western blot. ULBP4 levels were greater in patients with MS, particularly in active and chronic active lesions and normal-appearing white matter, compared with normal-appearing gray matter from MS donors and white and gray matter from controls. Soluble ULBP4 was also detected in CSF of patients with MS and controls, but a smaller shed/soluble form of 25 kDa was significantly elevated in CSF from female patients with MS compared with controls and male patients with MS. Our data indicate that soluble ULBP4 affects various functions of CD8+ T lymphocytes. First, it enhanced the production of the proinflammatory cytokines GM-CSF and interferon-γ (IFNγ). Second, it increased CD8+ T lymphocyte motility and favored a kinapse-like behavior when cultured in the presence of human astrocytes. CD8+ T lymphocytes from patients with MS were especially altered by the presence of soluble ULBP4 compared with healthy controls. DISCUSSION: Our study provides new evidence for the involvement of NKG2D and its ligand ULBP4 in MS pathology. Our results point to ULBP4 as a viable target to specifically block 1 component of the NKG2D pathway without altering immune surveillance involving other NKG2DL.

Soluble HLA-G molecules are released as HLA-G5 and not as soluble HLA-G1 isoforms in CSF of patients with relapsing-remitting multiple sclerosis.

Cerebrospinal fluid (CSF) levels of sHLA-G (sHLA-G1/HLA-G5) molecules and their soluble isoforms HLA-G5 and sHLA-G1 were measured by ELISA procedures in 68 relapsing-remitting Multiple Sclerosis (RR MS) patients, in 67 patients with other inflammatory neurological disorders (OIND) and in 70 subjects with non-inflammatory neurological disorders (NIND). CSF concentrations of sHLA-G1/HLA-G5 and HLA-G5 were higher in RR MS than in OIND and NIND, and in Magnetic Resonance Imaging (MRI) inactive as compared to MRI active RR MS. Our results indicate that the potential implication of sHLA-G proteins in the resolution of MS intrathecal inflammatory response is probably due to HLA-G5 isoform.

Expression of the immune-tolerogenic major histocompatibility molecule HLA-G in multiple sclerosis: implications for CNS immunity.

HLA-G is a non-classical major histocompatibility complex (MHC) class I antigen with highly limited tissue distribution under non-pathological conditions. Although capable of acting as a peptide-presenting molecule, its strong immune-inhibitory properties identify HLA-G as a mediator of immune tolerance with specific relevance at immune-privileged sites such as trophoblast or thymus. To assess the role of HLA-G in CNS immunity, we investigated its expression in brain specimens from patients with multiple sclerosis (n = 11), meningitis (n = 2) and Alzheimer's disease (n = 2) and non-pathological CNS controls (n = 6). Furthermore, cultured human microglial cells and CSF of patients with multiple sclerosis and controls were assessed. Furthermore, CSF from MS patients and controls, as well as cultured human microglial cells were assessed. Using several HLA-G specific mAb and immunohistochemistry, HLA-G protein was found strongly expressed in brain specimens from patients with multiple sclerosis while it was rarely detectable in the non-pathological control specimens. In multiple sclerosis brain specimens, HLA-G immunoreactivity was observed in acute plaques, in chronic active plaques, in perilesional areas as well as in normal appearing white matter. In all areas microglial cells, macrophages, and in part endothelial cells were identified as the primary cellular source of expression. HLA-G was also found in other disease entities (meningitis, Alzheimer's specimens) where expression correlated to activation and MHC class II expression on microglial cells. Importantly, ILT2, a receptor for HLA-G, was also found in multiple sclerosis brain specimens thus emphasizing the relevance of this inhibitory pathway in vivo. HLA-G mRNA and protein expression and regulation could also be corroborated on cultured human microglial cells in vitro. Further, expression of HLA-G in the CSF of multiple sclerosis patients and controls was analysed by flow cytometry and ELISA. Monocytes represented the main source of cellular HLA-G expression in the CSF. Corresponding to the observations with the tissue specimens, CSF mean levels of soluble HLA-G were significantly higher in multiple sclerosis than in non-inflammatory controls (171 +/- 31 versus 39 +/- 10 U/ml; P = 0.0001). The demonstration of HLA-G and its receptor ILT2 on CNS cells and in areas of microglia activation implicate HLA-G as a contributor to the fundamental mechanisms regulating immune reactivity in the CNS. This pathway may act as an inhibitory feedback aimed to downregulate the deleterious effects of T-cell infiltration in neuroinflammation.

Intrathecal synthesis of soluble class I antigens in multiple sclerosis.

We have studied the intrathecal synthesis of soluble class I antigens (sHLA), reflected by the index IH = (CSF sHLA/serum sHLA)/(CSF albumin/serum albumin), in multiple sclerosis (MS). IH was increased in patients in relapse, but normal in patients in remission; these findings show that there is a high lymphocyte activation within the central nervous system in patients with clinically active MS.

Clinical and MRI disease activity in multiple sclerosis are associated with reciprocal fluctuations in serum and cerebrospinal fluid levels of soluble HLA class I molecules.

The goal of our study was to clarify the contribution of soluble human leukocyte antigens class I (sHLA-I) in multiple sclerosis (MS) immune dysregulation. We retrospectively evaluated by ELISA cerebrospinal fluid (CSF) and serum sHLA-I levels in 79 relapsing-remitting (RR), 26 secondary progressive (SP) and 15 primary progressive (PP) MS patients stratified according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity. One hundred and nine patients with other inflammatory neurological disorders (OIND), 88 with noninflammatory neurological disorders (NIND) and 82 healthy donors were used as controls. An intrathecal synthesis of sHLA-I detected by a specific index was significantly more consistent in MS than in controls, with more pronounced values in MS patients with relapses and MRI enhancing brain lesions. A decrease in serum sHLA-I concentrations was observed in MS patients with demyelinating attacks, while an increase in CSF levels of sHLA-I was found in MS patients with lesional activity on MRI scans. This association between intrathecal synthesis and reciprocal fluctuations of CSF and serum levels of sHLA-I in clinically and MRI active MS seems to suggest a potential role for CSF and serum levels of sHLA-I as a sensitive marker of immune activation taking place both intrathecally and systemically in MS.

Presence of detectable levels of soluble HLA-G molecules in CSF of relapsing-remitting multiple sclerosis: relationship with CSF soluble HLA-I and IL-10 concentrations and MRI findings.

We have investigated the presence of non-classical soluble HLA-G molecules (sHLA-G) in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and the possible relationships between CSF levels of sHLA-G, classical soluble HLA-I (sHLA-I) molecules, IL-10 amounts and Magnetic Resonance Imaging (MRI) findings were evaluated. We studied by ELISA technique the sHLA-I, sHLA-G and IL-10 levels in CSF of 50 relapsing-remitting (RR) MS patients stratified according to clinical and MRI evidence of disease activity. Thirty-six patients with other inflammatory neurological disorders (OIND) and 41 with non-inflammatory neurological disorders (NIND) were used as controls. CSF mean levels were significantly higher in MS and OIND than in NIND for sHLA-I (p<0.001) and in MS than in controls for sHLA-G (p<0.001), with no differences among the various groups for IL-10 mean concentrations. An increase in CSF sHLA-I was found in MS patients with Gd-enhancing lesions (p<0.01), while sHLA-G and IL-10 were more represented in MS patients without lesional activity on MRI scans (p<0.02). In MRI-inactive MS, CSF IL-10 mean concentrations were significantly greater in patients with CSF-detectable levels of sHLA-G than in those without any evidence of CSF sHLA-G expression (p<0.05). Our findings suggest that CSF classical sHLA-I and non-classical sHLA-G levels may modulate MS activity as assessed by MRI acting in opposite directions. The association observed between sHLA-G and IL-10 when Gd-enhancing lesion resolved indicates a potential immunoregulatory role for IL-10 in the control of MS disease activity by shifting the sHLA-I/sHLA-G balance towards sHLA-G response.

Soluble HLA class I and class II molecule levels in serum and cerebrospinal fluid of multiple sclerosis patients.

Increased concentrations of soluble HLA class I and class II molecules (sHLA-I and sHLA-II) have been observed in infectious, inflammatory, and autoimmune diseases. Because autoimmune mechanisms are considered to play a role in the pathogenesis of multiple sclerosis (MS), we decided to dose sHLA-I and sHLA-II in serum and cerebrospinal fluid (CSF) of MS patients comparing their concentrations with those observed in serum and CSF of patients with other neurologic diseases (OND) without evidence of neuroradiologic involvement of central nervous system (CNS) and in serum of healthy donors. The serum concentrations of sHLA-I were higher in both MS and OND patients than in healthy donors (P < 0.05) whereas sHLA-II serum concentrations were lower in MS patients than in both OND patients and healthy donors (P < 0.01). Detectable amounts of sHLA-II were observed in the CSF of 45% of MS patients and in CSF of only 6% of OND patients (P < 0.001). In MS patients a significant correlation between sHLA-I serum and CSF concentrations was observed (P < 0.01), whereas sHLA-II serum and CSF levels did not correlate. In conclusion, alterations of sHLA-I and sHLA-II serum and CSF concentrations are present in MS patients and could be involved in the induction of enhanced susceptibility to develop MS or in MS pathogenesis.

Intrathecal soluble HLA-E correlates with disease activity in patients with multiple sclerosis and may cooperate with soluble HLA-G in the resolution of neuroinflammation.

Expression and function of the immunoregulatory molecule HLA-E was investigated in patients with relapsing-remitting (RR) multiple sclerosis (MS). Serum and cerebrospinal fluid (CSF) soluble (s)HLA-E and -G levels were measured by ELISA in 80 RRMS patients. Controls were patients with other inflammatory neurological disorders (OIND, n = 81) and noninflammatory neurological disorders (NIND, n = 86). Serum sHLA-E concentrations were higher in RRMS than in NIND patients only. CSF sHLA-E concentrations were higher in RRMS than controls. Increased CSF sHLA-E levels were detected in MRI inactive and clinically stable RRMS patients. sHLA-E intrathecal synthesis (ITS) was higher in RRMS than controls, and the number of patients with sHLA-E ITS above cut-off was higher i) in MS than controls, and ii) in clinically stable than clinically active MS patients. sHLA-E CSF levels and ITS correlated with i) the same sHLA-G parameters, and ii) disease duration. HLA-E expression and co-expression with CD markers were investigated in MS plaques from three different cases by immunohistochemistry and confocal microscopy, respectively. Infiltrating T lymphocytes and macrophages, as well as resident microglial cells and astrocytes expressed HLA-E. CSF samples from MS patients were finally tested for inhibitory activity of in vitro CTL and NK cell mediated cytotoxicity. sHLA-E⁺ were more effective than sHLA-E⁻ CSF samples in such inhibition. Maximum inhibition was achieved with sHLA-E⁺/sHLA-G⁺ CSF samples In conclusion, increased sHLA-E CSF levels may play an immunomodulatory role in MS, contributing to the inhibition of intrathecal inflammatory response. The potential of sHLA-E as biomarker of MS activity warrants further investigation.

Potential role of soluble human leukocyte antigen-G molecules in multiple sclerosis.

Nonclassical human leukocyte antigen (HLA)-G antigens in soluble form (sHLA-G) have recently been suggested to have a potential role as immunomodulatory factors in multiple sclerosis (MS), a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system of unknown etiology and supposed autoimmune origin. In MS patients, sHLA-G levels were elevated in cerebrospinal fluid (CSF), intrathecally synthesized, predominantly represented by the HLA-G5 isoform and even more elevated in cases of inactive disease, as determined by magnetic resonance imaging. In MS, CSF sHLA-G concentrations were also related to the formation of an intrathecal anti-inflammatory microenvironment based on a positive correlation to CSF interleukin-10 titers and an inverse association to the levels of antiapoptotic sFas molecules in the CSF. Expression of HLA-G antigens was detected in microglia, macrophages, and endothelial cells within and around MS lesions and was enhanced in microglial cells by T-helper-1 proinflammatory cytokines. A novel subpopulation of naturally occurring CD4(+) and CD8(+) regulatory T cells expressing HLA-G1 and secreting HLA-G5 was identified in the CSF of MS patients. Taken together, these findings seem to indicate that sHLA-G antigens may be implicated in the termination of MS autoimmunity and associated with remission of the disease through their function as anti-inflammatory molecules. However, the mechanisms operating in the immunomodulatory circuit mediated by sHLA-G proteins remain to be clarified.

A CSF biomarker panel for identification of patients with amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with complicated pathogenesis that poses challenges with respect to diagnosis and monitoring of disease progression. OBJECTIVES: To identify a biomarker panel that elucidates ALS disease pathogenesis, distinguishes patients with ALS from neurologic disease controls, and correlates with ALS disease characteristics, and to determine the effect of HFE gene variants, a potential risk factor for sporadic ALS, on the biomarker profile. METHODS: We obtained CSF samples by lumbar puncture from 41 patients with ALS and 33 neurologic disease controls. All patients were genotyped for HFE polymorphisms. We performed a multiplex cytokine and growth factor analysis and immunoassays for iron-related analytes. Classification statistics were generated using a support vector machine algorithm. RESULTS: The groups of patients with ALS and neurologic disease controls were each associated with distinct profiles of biomarkers. Fourteen biomarkers differed between patients with ALS and the control group. The five proteins with the lowest p values differentiated patients with ALS from controls with 89.2% accuracy, 87.5% sensitivity, and 91.2% specificity. Expression of IL-8 was higher in those patients with lower levels of physical function. Expression of beta2-microglobulin was higher in subjects carrying an H63D HFE allele, while expression of several markers was higher in subjects carrying a C282Y HFE allele. CONCLUSIONS: A CSF inflammatory profile associated with amyotrophic lateral sclerosis (ALS) pathogenesis may distinguish patients with ALS from neurologic disease controls, and may serve as a biomarker panel to aid in the diagnosis of ALS pending further validation. Some of these biomarkers differ by HFE genotype.

CSF levels of soluble HLA-G and Fas molecules are inversely associated to MRI evidence of disease activity in patients with relapsing-remitting multiple sclerosis.

Cerebrospinal fluid (CSF) concentrations of soluble human leukocyte antigen class I (HLA-I) (sHLA-I), HLA-G (sHLA-G) and anti-apoptotic Fas (sFas) molecules were measured by enzyme linked immunosorbent assay technique in 65 relapsing-remitting (RR) MS patients classified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. Sixty-four patients with other inflammatory neurological disorders (OIND) and 64 subjects with noninflammatory neurological disorders (NIND) served as controls. CSF concentrations were higher in RRMS and in OIND than in NIND patients for sHLA-I (P < 0.02), greater in RRMS than in OIND and in NIND for sHLA-G (P < 0.001 and P < 0.01, respectively) and lower in RRMS than in OIND and in NIND for sFas (P < 0.001 and P < 0.02, respectively). An increase in CSF levels was identified in MRI active RRMS for sHLA-I (P < 0.01) and in MRI stable RRMS for sHLA-G (P < 0.01), whereas CSF values of sFas were decreased in RRMS without Gd-enhancing lesions (P < 0.02). In MS patients with no evidence of MRI disease activity, a trend towards an inverse correlation was found between CSF concentrations of sHLA-G and sHLA-I and between CSF levels of sHLA-G and sFas. Our results indicate that enhanced CSF levels of sHLA-I antigens most likely represent an indirect manifestation of intrathecal immune activation taking place in neuroinflammation. Conversely, reciprocal fluctuations in CSF sHLA-G and sFas levels observed when MRI disease activity resolved suggest that sHLA-G could play an immunomodulatory role in MS through Fas/FasL-mediated mechanisms.

Intrathecal synthesis of soluble HLA-G and HLA-I molecules are reciprocally associated to clinical and MRI activity in patients with multiple sclerosis.

The aim of this study was to provide further insight into the effective contribution of classical soluble HLA-A, B and C class Ia (sHLA-I) and non-classical soluble HLA-G class Ib (sHLA-G) molecules in immune dysregulation occurring in multiple sclerosis (MS). We evaluated by enzyme-linked immunosorbent assay (ELISA) technique intrathecal synthesis and cerebrospinal fluid (CSF) and serum levels of sHLA-I and sHLA-G in 69 relapsing-remitting (RR), 21 secondary progressive (SP) and 13 primary progressive (PP) MS patients stratified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also tested, as neurological controls, 91 patients with other inflammatory neurological disorders (OIND) and 92 with non-inflammatory neurological disorders (NIND). Eighty-two healthy volunteers served as further controls for sHLA-I and sHLA-G determinations. An intrathecal production of sHLA-I and sHLA-G detected by specific indexes was significantly more frequent in MS patients than in controls (P<0.01). An intrathecal synthesis of sHLA-I was prevalent in clinically (P<0.02) and MRI active (P<0.001) MS, whereas a CSF-restricted release of sHLA-G predominated in clinically (P<0.01) and MRI stable (P<0.001) MS. sHLA-I levels were low in the serum of clinically active (P<0.001) and high in the CSF of MRI active (P<0.01) MS. Conversely, sHLA-G concentrations were decreased in the serum of clinically stable MS (P<0.01) and increased in the CSF of MRI inactive MS (P<0.001). The trends towards a negative correlation observed between CSF and serum concentrations and intrathecal synthesis of sHLA-I and sHLA-G in patients without evidence of clinical and MRI activity confirmed that intrathecal production and fluctuations in CSF and serum concentrations of sHLA-I and sHLA-G were reciprocal in MS. Our results suggest that, in MS, a balance between classical sHLA-I and non-classical sHLA-G products modulating both MRI and clinical disease activity in opposite directions may exist.

Soluble class I antigens in serum and CSF of patients with varicella-zoster virus meningitis.

Soluble class I antigens (sHLA) are secreted by lymphocytes upon activation in vitro. The intrathecal synthesis (ITS) of these molecules has been studied in patients with the varicella-zoster virus (VZV) meningitis. In this paper we describe a sHLA index IH = (CSF sHLA/serum sHLA): (CSF albumin/serum albumin) which is expected to increase only when sHLA is synthesised within the central nervous system (CNS). The IH is elevated in the first week of meningitis, when antibody synthesis is still low, and decreases thereafter. We think IH is an index of early lymphocyte activation within the CNS. The relation of these findings with previous in vitro studies is also discussed.

Soluble HLA class I and class II antigens in patients with multiple sclerosis.

Soluble HLA class I (sHLA-I) and soluble HLA class II (sHLA-II) antigen levels during different stages of disease were investigated in paired serum and cerebrospinal fluid (CSF) samples from 37 patients with multiple sclerosis (MS) using ELISA and Western blot analysis. Soluble HLA-II antigens in the serum of untreated patients with the relapsing-remitting type of MS (RRMS) were found to be significantly elevated in acute relapse as compared to values obtained from patients under steroid treatment, in remission or healthy controls. No significant differences in circulating sHLA-I levels could be detected. In contrast, a trend towards increased intrathecal production of sHLA-I molecules in the CSF was observed in untreated RRMS patients in acute relapse, whereas the levels of soluble HLA-II antigens in the CSF were below the detection limit of the ELISA method. Our observations underline the presence of systemic immune activation in MS patients, as reflected in elevated serum sHLA-II antigen levels, and reveal a dichotomy between sHLA class I and II antigen production in the peripheral blood versus CSF in acute MS. Serial measurements of sHLA-II antigen levels might represent a non-invasive method to assess disease activity in MS patients.

Soluble class 1 antigens (sHLA) in CSF and serum of patients with HIV infection.

sHLA are secreted by B and T lymphocytes upon activation. These antigens are present in serum and cerebrospinal fluid (CSF). The establishment of a sHLA index, IH = (CSF sHLA/serum sHLA)/(CSF albumin/serum albumin) helped us to define that values above 9.7 reflect an intrathecal synthesis of these proteins. IH was significantly increased in a group of patients with intrathecal production of IgG against HIV-1 (HIV) and directly correlated with the synthesis of such antibodies. Therefore, IH seems to be an index of lymphocyte activation in CNS.