Annalisa Chiocchetti

Defective T cell Fas function in patients with multiple sclerosis

Background: Fas (CD95) triggers programmed cell death and is involved in shutting off the immune response. Inherited deleterious mutations hitting Fas or its signaling pathway cause autoimmune/lymphoproliferative syndrome (ALPS). Objective: To assess the possibility that decreased Fas function plays a role in development of MS. Methods: The authors evaluated Fas function in long-term T cell lines (21 days of culture) from 32 patients with relapsing-remitting MS (RRMS), 15 with secondary progressive MS (SPMS), and 15 with primary progressive MS (PPMS) by assessing cell survival upon Fas triggering by monoclonal antibodies (Mab). Results: Fas-induced cell death was significantly lower in all patient groups than in controls, and lower in SPMS than in RRMS. Moreover, 8/15 patients with PPMS, 10/15 with SPMS, and 8/32 with RRMS were frankly resistant to Fas. Frequency of resistance to Fas-induced cell death was significantly higher in all patient groups than in controls (2/75), and higher in SPMS than in RRMS. The findings that the parents of two Fas-resistant patients were Fas-resistant and that fusion of T cells from two Fas-resistant patients with Fas-sensitive HUT78 cells gave rise to Fas-resistant hybrid lines suggest that Fas-resistance is due to inherited alterations of the Fas signaling pathway, with production of molecules exerting a dominant negative effect on a normal Fas system. Conclusions: Defects of the immune response shutting-off system may be involved in the pathogenesis of MS, particularly in its progressive evolution.

Osteopontin gene haplotypes correlate with multiple sclerosis development and progression

Osteopontin (OPN) is an inflammatory cytokine highly expressed in multiple sclerosis (MS) plaques. In a previous work, we showed that four OPN polymorphisms form three haplotypes (A, B, and C) and that homozygotes for haplotype-A display lower OPN levels than non-AA subjects. In this work, we evaluated the distribution of these OPN haplotypes in 425 MS patients and 688 controls. Haplotype-A homozygotes had about 1.5 lower risk of developing MS than non-AA subjects. Clinical analysis of 288 patients showed that AA patients displayed slower switching from a relapsing remitting to a secondary progressive form and milder disease with slower evolution of disability. MS patients displayed increased OPN serum levels, which were partly due to the increased frequency of non-AA subjects. Moreover in AA patients, OPN levels were higher than in AA controls and similar to those found in both non-AA patients and controls, which suggests a role of the activated immune response. These data suggest that OPN genotypes may influence MS development and progression due to their influence on OPN levels.

Osteopontin is Increased in the Cerebrospinal Fluid of Patients with Alzheimer's Disease and Its Levels Correlate with Cognitive Decline

Inflammation is believed to play a role in Alzheimers disease (AD). Osteopontin (OPN) is a molecule involved in macrophage recruitment and activation and implicated in neurodegeneration. In order to elucidate the role of OPN in AD, we evaluated its levels in serum and cerebrospinal fluid (CSF) of 67 AD patients, 46 frontotemporal dementia (FTD) patients, and 69 controls. We found that OPN levels: i) are significantly increased in the CSF of AD patients; ii) correlate with MMSE score; and iii) are higher in the early disease phases ( 2 years). These findings support a role of OPN in AD pathogenesis.

GREEN FLUORESCENT PROTEIN AS A REPORTER OF GENE EXPRESSION IN TRANSGENIC MICE

We used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a reporter of gene expression in transgenic mice. The GFP coding sequence was placed under the control of the human hemopexin and the mouse beta1 integrin promoter that were previously studied in transgenic mice using the lacZ reporter gene. We showed that GFP has a higher degree of sensitivity compared to the lacZ reporter gene allowing to identify cells with low and otherwise undetectable beta-galactosidase activity. Thus we showed the potentiality of GFP in replacing lacZ as a reporter gene to investigate promoter mapping and gene regulation in transgenic mice.

Variations of the perforin gene in patients with multiple sclerosis

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13–3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10–1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways.

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4+ Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti‐H4/ICOS antibodies strongly enhanced CD3‐mediated tyrosine phosphorylation of ZAP‐70, ζ, or Vav, as well as extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3‐kinase (PI‐3K) to H4/ICOS was enhanced by H4/ICOS cross‐linking, and PI‐3K inhibitors inhibited ERK and JNK activation andIL‐4/IL‐10 secretion, but not p38 MAP kinase or ZAP‐70 activation. H4/ICOS‐mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP‐70, MAP kinase activation, or IL‐4/IL‐10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP‐70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL‐4, but not IL‐10 secretion. ERK or JNKinhibitors partially inhibited IL‐4 and IL‐10 secretion, while PKC or p38 inhibitors had no significant effects on IL‐4 or IL‐10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.

Serum levels of osteopontin are increased in SIRS and sepsis

ObjectiveIn sepsis, dysregulation of the immune response leads to rapid multiorgan failure and death. Accurate and timely diagnosis is lifesaving and should discriminate sepsis from the systemic inflammatory response syndrome (SIRS) caused by non-infectious agents. Osteopontin acts as an extracellular matrix component or a soluble cytokine in inflamed tissues. Its exact role in immune response and sepsis remains to be elucidated. Therefore, we investigated the role of osteopontin in SIRS and sepsis.DesignProspective, observational study.SettingIntensive care unit of a university hospital.Patients and participantsFifty-six patients with SIRS or sepsis and 56 healthy subjects were enrolled.InterventionsWe analyzed the serum levels of osteopontin and TH1–TH2 cytokines and investigated the role of osteopontin on interleukin 6 secretion by monocytes.Measurements and main resultsSerum osteopontin levels were strikingly higher in patients than in controls and in sepsis than in SIRS, and decreased during the resolution of both the disorders. Receiver operating characteristic curves showed that osteopontin levels have discriminative power between SIRS and sepsis with an area under the curve of 0.796. Osteopontin levels directly correlated with those of interleukin 6 and in vitro, recombinant osteopontin increased interleukin 6 secretion by monocytes in both the absence and presence of high doses of lipopolysaccharide.ConclusionThese data suggest that osteopontin might be a mediator involved in the pathogenesis of SIRS and sepsis, possibly by supporting interleukin 6 secretion.Descriptor45. SIRS/Sepsis: clinical studies.

Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes.

1 The effects of L‐glutamate on activation‐induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin‐2; 8 days) T lymphocytes were studied by measuring anti‐CD3 monoclonal antibody (10 μg ml−1; 18 h)‐induced cell apoptosis (Annexin V and propidium iodide staining). 2 L‐Glutamate (1 × 10−8–1 × 10−4 M) significantly (P0.01) inhibited AICD in a concentration‐dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). 3 The L‐glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)‐ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)‐3,5‐DHPG; 2.0 × 10−5 M for CHPG. 4 Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. 5 L‐Glutamate (1 × 10−6 M; 18 h) significantly (P0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6 Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T‐cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L‐glutamate‐mediated inhibition of AICD was exerted on T cells. 7 These data depict a novel role for L‐glutamate in the regulation of the immune response through group I mGlu receptor‐mediated mechanisms.

Variations of the Perforin Gene in Patients With Type 1 Diabetes

OBJECTIVE—Perforin plays a key role in cell-mediated cytotoxicity. Mutations of its gene, PRF1, cause familial hemophagocytic lymphohistiocytosis but have also been associated with lymphomas and the autoimmune/lymphoproliferative syndrome. The aim of this work was to investigate the role of PRF1 variations in type 1 diabetes. RESEARCH DESIGN AND METHODS—We typed for the N252S and A91V variations in an initial population of 352 type 1 diabetic patients and 816 control subjects and a second population of 365 patients and 964 control subjects. Moreover, we sequenced the coding sequence and intron-exons boundaries in 200 patients and 300 control subjects. RESULTS—In both cohorts, allelic frequency of N252S was significantly higher in patients than in control subjects (combined cohorts: 1.5 vs. 0.4%; odds ratio 6.68 [95% CI 1.83–7.48]). Sequencing of the entire coding region detected one novel mutation in one patient, causing a P477A amino acid change not detected in 199 patients and 300 control subjects. Typing for HLA-DQA1 and DQB1 alleles showed that type 1 diabetes–predisposing DQα/DQβ heterodimers were less frequent in patients carrying N252S or P477A than in those carrying wild-type PRF1. We previously found that natural killer (NK) activity is not decreased in most N252S heterozygotes, but we detected one whose NK activity was normal at the age of 12 but strikingly low in early childhood. Here, we discovered that NK function was low in three heterozygotes in early childhood, one homozygous adult, and in the subject carrying P477A. CONCLUSIONS—These data suggest that N252S and possibly other PRF1 variations are susceptibility factors for type 1 diabetes development.

ICOS cooperates with CD28, IL-2 and IFN-γ and modulates activation of human naïve CD4+ T cells

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4+ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti‐CD3 mAb. ICOS strikingly potentiated secretion of IL‐2, IFN‐γ, IL‐10, and TNF‐α, but not IL‐4, promoted by optimal stimulation of CD3+CD28, and it was the key switching‐factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL‐2. In these conditions, blockade of IL‐2 and IFN‐γ showed that ICOS builds up a positive feedback loop with IFN‐γ, which required IL‐2 and was inhibited by IL‐4. By contrast, in the absence of CD28 triggering or exogenous IL‐2, ICOS‐induced costimulation mainly supported expression of TGF‐β1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4+ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL‐2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.