InterLeukin-12 In Multiple Sclerosis

Multiple Sclerosis (MS) is widely accepted as a systemic T-Cell-mediated AutoImmune Disease with a T-Helper Type-1 (Th1) profile of Cytokine production.

We addressed the question whether InterLeukin-12 (IL-12), as a central mediator of Th1-Cell activities, is detectable in Sera of MS patients, and if there is any association with disease activity.

We analyzed 171 Sera of patients with MS and Meningitis, and healthy controls. IL-12 p40 protein was detectable at low levels in MS patients (median 43 pg/ml) and controls (median 49 pg/ml).

Analysing different disease courses and activities, a significant elevation in stable Primary/Progressive MS cases compared with controls (median 66 pg/ml) was found. IL-12 p40 protein was not detectable in CerebroSpinal Fluid probes of 10 patients.

We conclude that the function of IL-12 in MS depends on expression and degradation of the different proteins. These could act ProInflammatory as well as limiting the disease process.

Copyright Lippincott Williams & Wilkins

CoStimulatory molecules B7-1 (CD80) and B7-2 (CD86) are differently involved in T-Cell stimulation. In Chronic Experimental AutoImmune EncephaloMyelitis (EAE) model of Multiple Sclerosis (MS), B7-1 was preferentially involved in PathoPhysiology of relapses.

We used Reverse Transcription Polymerase Chain Reaction (RT-PCR) to amplify the mRNA coding for these molecules in CerebroSpinal Fluid (CSF) Cells and Peripheral Blood MonoNuclear Cells (PBMC) from 18 MS patients and 21 Other Neurological patients.

In CSF cells of MS cases, B7-1 mRNA was only detected in some patients who showed clinical signs of acute relapse at the time of the Spinal Tap, while B7-2 mRNA was widely detectable without difference between active or stable MS and controls.

mRNA coding for Transforming Growth Factor-ß (TGF-ß) was detectable in the majority of cases.

With higher expression in CSF cells of MS and Other Inflammatory Neurological Diseases (OIND) than in NonInflammatory controls, and higher expression in PBMC of MS patients than in all other cases.

Finally, mRNA coding for InterLeukin (IL)-12p40 was only detected in a very few number of MS and Inflammatory cases.

These findings were related to previous detection of other Cytokines in the same cases, showing relationships in CSF cells between high expression of B7-1, IL-12p40 and TNF-.

Treatment with MonoClonal Anti-IL-12 AntiBody injected on day 0, 7 and 10 after immunization with Myelin Oligodendrocyte Glycoprotein (MOG) peptide 35-55 in NOD mice.

Resulted in significant suppression of the development and the severity of the chronic Relapsing/Remitting Experimental AutoImmune EncephaloMyelitis (EAE) both clinically and histologically.

The Spleen Cells from Anti-IL-12 AntiBody treated mice displayed markedly inhibited MOG35-55 specific proliferation and IFN-γ production.

MOG35-55 specific AntiBody production was enhanced by anti-IL-12 AntiBody treatment.

These results suggest that IL-12 is critically involved in the PathoGenesis of MOG-induced EAE and that AntiBody to IL-12 could be an effective therapeutic agent in the clinical treatment of AutoImmune DeMyelinating Diseases such as Multiple Sclerosis (MS).

The objective of this study was to determine whether AutoReactive T-Cells in patients with Multiple Sclerosis (MS) are polarized and committed in their differentiation to a stable Cytokine phenotype or whether the Cytokine secretion can be altered.

We examined the Cytokines secreted by Myelin Basic Protein (MBP) as compared to Tetanus Toxoid-reactive (TT) T-Cells in 12 patients with Relapsing/Remitting MS (RR-MS), 9 patients with Chronic/Progressive MS (CP-MS), and 14 normal individuals.

A total of 5094 short term T-Cell lines to MBP and TT were generated in the presence of growth conditions promoting Th1 (IL-12/alpha-IL-4 mAb) or Th2 (IL-4/alpha-IL-12 mAb) Cytokine secretion.

Antigen-specific Cytokine secretion from normals and MS patients could be shifted to a Th1 or Th2 type phenotype depending upon culture conditions, indicating that the phenotype of MBP reactive T-Cells can be altered even in longstanding Chronic/Progressive MS.

There were no significant differences in the Cytokine patterns secreted by MBP reactive T-Cells in patients with MS as compared to normal individuals.

However, CP-MS patients tended to have fewer MBP reactive T-Cells secreting IL-4 when cultured with IL-12/anti-IL-4 mAb and more IFN-γ (IFN-γ) secreting MBP reactive T-Cells when cultured with IL-4/anti-IL-12 mAb as compared to both normal controls and RR-MS.

Suggesting thaT-Cells from these patients might be more polarized or that fewer undifferentiated MBP-reactive cells are present in these individuals.

The most striking observation was that in contrast to the RR-MS patients and normal controls, almost none of the MBP reactive T-Cells secreting Cytokines in CP-MS incorporated 3[H]Thymidine.

This may be due to chronic in vivo stimulation in the presence of IL-12, or because these T-Cells may have entered a terminally differentiated state.

Nonetheless, the ability to alter the Cytokine secretion of AutoReactive T-Cell lines even in longstanding AutoImmune Disease indicates that Cytokine therapy might have therapeutic benefits by switching the function of Myelin reactive T-Cells such that they are Non-Pathogenic.

Multiple Sclerosis (MS), a disease of the human Central Nervous System, is believed to be a T-Cell mediated AutoImmune Disorder with Genetic and Environmental influences.

InterLeukin-12 (IL-12), a ProInflammatory Cytokine produced primarily by Antigen Presenting Cells, a potent inducer of Interferon-gamma (IFN-γ), and other Th1 Cytokines that may play an important role in MS PathoGenesis.

We have investigated IL-12 production induced by the T-Cell independent pathway in untreated and IFN-ß treated MS patients, healthy individuals, and Other Neurological Disease (OND) patients in response to the human Pathogen Staphylococcus Aureus.

We report that Peripheral Blood MonoNuclear Cells (PBMC), from untreated MS patients, produce normal amounts of the biologically active IL-12 p70 heterodimer.

But significantly less free IL-12 p40 heavy chain than PBMC from both healthy and disease controls, when challenged in vitro with Staphylococcus Aureus.

Both mRNA expression of the inducible IL-12 p40 chain and protein levels were found to be reduced in untreated MS patients.

No decrease in the production of the IL-12 p40 was seen in MS patients on IFN-ß therapy.

The decreased production of IL-12 p40 heavy chain is not attributed to increased IL-10 secretion, a defect in the production of Cytokines by Macrophages or the number of Cytokine producing cells.

The factor(s) responsible for the decrease in p40 remain to be determined.

Since IL-12 p40 antagonizes the biological activity of IL-12 in vitro and in vivo, identification of a defect in the 'natural' antagonist of IL-12, may provide the basis for Immune therapy.

Canine Distemper Virus (CDV) infection in dogs is commonly associated with DeMyelinating LeukoEncephalitis (DL).

Although the mechanism of primary DeMyelination in Distemper remains undetermined recent studies showed a direct Virus-induced CytoLysis in early Non-Inflammatory and Immune-mediated mechanisms in Inflammatory lesions.

To further investigate the PathoGenesis of this MorbilliVirus-induced DeMyelination. The expression of a variety of Cytokine mRNA species:

1. InterLeukin-beta (IL-1ß) IL-2, IL-6, IL-10, IL-12
2. Tumor Necrosis Factor-alpha (TNF-)
3. Transforming Growth Factor-beta (TGF-ß1)
4. Interferon-gamma (IFN-γ)

NeuroPathology revealed the whole spectrum of Distemper DL lesions from acute to chronic alterations, however, most plaques lacked active DeMyelination.

Three control animals were devoid of any Cytokine expression, whereas in Distemper animals IL-10 transcripts were found in nine dogs with Acute and Chronic lesions.

IL-6, TNF, and TGF mRNA was found in six, four, and three animals, respectively.

IL-12 and IFN-γ, suggestive of a T-Helper1 (Th1)-like dominated Immune Response, were detected only in one animal with chronic lesions.

Summarized, TNF and IL-6, associated with disease exacerbation, and IL-10 and TGF, indicative of remission, were often observed simultaneously in Distemper DL and could not be assigned to a specific disease stage.

However IL-10 mRNA remained the most frequently detected Cytokine indicating a stage of inactivity in most animals investigated.

It has been shown that ProInflammatory and AntiInflammatory Cytokines correlate with disease activity in Multiple Sclerosis (MS).

To establish whether such correlations depend on the disease stage, in relation to monthly clinical and Magnetic Resonance Imaging monitoring.

We assessed in a longitudinal fashion, by competitive polymerase chain reaction, in unstimulated Peripheral Blood MonoNuclear Cells of Relapsing/Remitting (RR) and Secondary/Progressive (SP) MS patients:

1. The expression of InterLeukin-12 (IL-12) (p40 and p35)
2. Tumor Necrosis Factor-alpha (Tumor Necrosis Factor-)
3. Interferon-gamma (IFN-gamma (IFN-γ)
4. IL-10 mRNA

MS patients had increased levels of IL-12p40 and decreased levels of IL-10 mRNA compared with controls; this difference was most pronounced in SP patients.

Both RR and SP patients had increased levels of IL-12p40 mRNA compared with controls during the development of active lesions.

Moreover, in RR MS an increase was found before relapse. IL-12p35 mRNA was decreased in both groups, and in relation to disease activity it showed a pattern different from IL-12p40 mRNA.

In RR MS, IL-10 mRNA was low 4 weeks before Magnetic Resonance Imaging activity and 6 weeks before relapse; a significant increase to normal levels was noted when active lesions became apparent.

In contrast, SP patients showed low IL-10 mRNA levels constitutively, suggesting that IL-10 plays an important role in the control of disease progression.

Comment in: Ann Neurol 1999 Jun;45(6):689-92

Interferon-beta (IFN-ß) has been shown to favorably alter the disease course of Relapsing/Remitting Multiple Sclerosis (RRMS) patients.

Although its mode of action is still unclear, there is ample evidence from in vitro studies that IFN-ß directly modulates the function of Immune Cells.

We analyzed here the effects of IFN-ß treatment on Immune functions in vivo in a group of 25 RRMS patients who received IFN-ß (8 MIU) on alternate days.

At baseline and at 1, 3 and 6 months from the start of the treatment, parameters for differentiation and activation states of both Monocytes and T-Lymphocytes were assessed.

A transient increase was seen in Plasma (p) InterLeukin-10 (IFN-ß) level whereas pIL-12 (p40) was not affected. A similar change was found in the ability of Monocytes to secrete these Cytokines in vitro.

Notably, patients who in vitro readily responded to IFN-ß with enhanced IL-10 production had the highest pIL-10 levels.

Concerning T-Cell differentiation, flow cytometric analysis of Cytokine production showed that treatment with IFN-ß moderately decreased the mean percentages of CD8pos T-Cells producing IL-2 and IFN-γ and CD8neg T-Cells producing IL-4 (p<0.05 for all Cytokines).

Whereas a more significant decline was seen in the mean percentage of CD8neg T-Cells producing IFN-γ (p<0.01).

This resulted in a significant lower ratio T-Helper (Th1)) vs. T-Helper (Th2) type cells in the CD8⁺pos T-Cell subset (p<0.05), but not in the CD8⁺neg T-Cell subset.

Finally, IFN-ß treatment resulted in an initial rise in the mean percentage of CD95pos T-Cells and in a gradual increase in the mean level of soluble CD95 (sCD95) in Plasma (p<0.01).

Additional in vitro studies showed that IFN-ß indeed rapidly (within 24 h) upregulates CD95 expression on both primed and unprimed T-Cells and augments the release of sCD95 in culture supernatants.

Thus, we confirm here that IFN-ß treatment leads to similar changes in Cytokine production of T-Cells and Monocytes as previously described in vitro.

Enhanced IL-10 secretion may downmodulate Cytokine secretion by activated T-Cells and in this way dampen newly-induced and/or ongoing Immune Responses.

In addition, we identified a novel effect of IFN-ß treatment, i.e., induction of CD95 expression. The augmentation of CD95 expression may directly interfere with T-Cell selection, notably of AutoAggressive T-Cells.

Future studies are needed to show whether this increased CD95 expression indeed leads to increased Apoptosis of Immune Cells.

Gamma delta T-Lymphocytes are thought to play a role in the PathoGenesis of Multiple Sclerosis (MS) contributing to DeMyelination and Fibrosis in the Central Nervous System.

In this study, we show that, in MS patients with active disease, the percentage of circulating V delta 2⁺ delta T-Cells coexpressing NKRP1A is significantly increased compared with healthy donors.

V delta 2⁺ and V delta 1⁺ T-Cells were sorted from MS patients and healthy volunteers and cloned.

At variance with V delta 1⁺ clones, all V delta 2⁺ clones expressed NKRP1A, which was strongly up-regulated upon culture with IL-12; this effect was neutralized by specific anti-IL-12 Abs.

No up-regulation of NKRP1A by IL-12 was noted on V delta 1⁺ clones.

RNase protection assay showed that IL-12R beta 2 subunit transcript was significantly less represented in V delta 1⁺ than V delta 2⁺ clones.

This finding may explain the different effect exerted by IL-12 on these clones.

In TransEndothelial migration assays, V delta 2⁺ NKRP1A⁺ clones migrated more effectively than V delta 1⁺ clones, and this migratory potential was enhanced following culture with IL-12.

Migration was strongly inhibited by the F(ab')2 of an anti-NKRP1A Ab, suggesting that this lectin is involved in the migration process.

We also show that, in freshly isolated PBMC from MS patients, the migrated population was enriched for V delta 2⁺ NKRP1A⁺ cells.

We conclude that the expression of NKRP1A on V delta 2⁺ cells is associated with increased ability to migrate across the Vascular Endothelium and that this phenomenon may be regulated by IL-12present in the MicroEnvironment.

Experimental Allergic EncephaloMyelitis (EAE) is an Inflammatory, CD4⁺ Th1-mediated AutoImmune Disease, which serves as a model for Multiple Sclerosis.

We examined the effect of a novel Anti-Inflammatory drug, Lisofylline (LSF), on EAE induced either by injection of mouse Spinal Cord homogenate or following transfer of Myelin Basic Protein-reactive T-Cells.

Orally administered LSF significantly inhibited EAE in both cases, decreasing peak clinical scores by >70% and >80%, respectively.

In addition, analysis of representative Spinal Cord sections from LSF-treated mice showed complete lack of DeMyelination and Lymphocyte infiltration.

The reduction in EAE correlated with the inhibition of Th1 differentiation by LSF in vivo, as indicated by a reduction in T-Cell IFN-γ production ex vivo after Ag restimulation.

The inhibition of Th1 differentiation in vivo is consistent with a block in IL-12 receptor signaling, because LSF blocked IL-12-driven Th1 differentiation and T-Cell proliferation in vitro, yet had no effect on IL-12 secretion from APCs ex vivo or in vitro.

Experimental Allergic EncephaloMyelitis (EAE) and Theiler's Murine EncephaloMyelitis Virus (TMEV) disease are two DeMyelinating Diseases of the Central Nervous System (CNS) that serve as animal models for Multiple Sclerosis.