Mast Cells In Multiple Sclerosis

Nitric Oxide (NO) plays diverse roles in physiological and pathological processes. During Immune and inflammatory responses, for example in Asthma, NO is generated at relatively high and sustained levels by the inducible form of Nitric Oxide Synthase (NOS-2).

NOS-2 derived NO regulates the function, growth, death and survival of many Immune and inflammatory cell types. In the case of Mast Cells, NO suppresses Antigen-induced degranulation, mediator release, and Cytokine expression.

The action of NO on Mast Cells is time dependent, requiring several hours, and noncGMP mediated, most probably involving chemical modification of proteins.

NO inhibits a number of Mast Cell-dependent inflammatory processes in vivo, including Histamine mediated VasoDilatation, VasoPermeation and Leucocyte-Endothelial Cell attachment.

In human Asthma and animal models of Lung inflammation the role of NO is harder to define. However, although there are conflicting data, the balance of evidence favors a predominantly protective role for NO.

Mimicking or targeting NO dependent pathways may prove to be a valuable therapeutic approach to Mast Cell mediated diseases.

Background
In Asthma and other Allergic Disorders, the activation of Mast Cells by IgE and Antigen induces the cells to release Histamine and other mediators of inflammation, as well as to produce certain Cytokines and Chemokines.

To search for new Mast Cell products, we used complementary DNA microarrays to analyze gene expression in human umbilical cord blood-derived Mast Cells stimulated via the high-affinity IgE receptor (Fc Epsilon;RI).

Results
One to two hours after Fc Epsilon;RI-dependent stimulation, more than 2,400 genes (about half of which are of unknown function) exhibited 2-200 fold changes in expression.

The transcriptional program included changes in the expression of IL-11 and at least 30 other Cytokines and Chemokines. Human Mast Cells secreted 130-529 pg of IL-11/106 cells by 6 h after stimulation with anti-IgE.

Conclusion
Our initial analysis of the transcriptional program induced in in vitro-derived human Mast Cells stimulated via the Fc Epsilon;RI has identified many products that heretofore have not been associated with this cell type.

But, which may significantly influence Mast Cell function in IgE-associated host responses. We also have demonstrated that Mast Cells stimulated via the Fc Epsilon;RI can secrete IL-11.

Based on the previously reported biological effects of IL-11, our results suggest that production of IL-11 may represent one link between IgE-dependent Mast Cell activation in subjects with allergic Asthma.

And the development of a spectrum of structural changes, in the airways of these individuals; such changes, collectively termed "airway remodeling," can constitute an important long term consequence of Asthma.

InterLeukin-6 (IL-6) is a PleioTropic Cytokine (26 kDa) that originally was named Interferon-beta 2 or B-Cell-Stimulating Factor or differentiating B-Cell Factor inducing ImmunoGlobulin production. IL-6 is produced in many diseases.

After secretion, IL-6 binds to its Receptor IL-6R alpha (gp 80), the IL-6R alpha complex then recruits the signal-transducing beta-subunit (gp 130), which is the functional complex for signal transduction.

In addition, activation of Th2 cells or Mast Cells also produce IL-6, which mediates Immune Responses, Inflammation, Acute Phase Responses, Hematopoiesis, Cancer, Inflammatory Bowel Disease, etc. IL-6 also is a crucial Cytokine for Mast Cell maturation.

Human cord blood CD34⁺ cells differentiate and grow into Mast Cells in the presence of Stem Cell Factor (SCF) and IL-6, causing increases in cell size, frequency of Chymase positive cells, and IntraCellular Histamine levels when compared with cells treated with SCF alone.

Activated Mast Cells increase IL-6 mRNA associated with protein kinase C (PKC) activity. IL-6 also up-regulates Histamine production rather than increases its storage and is an important inducing factor for the expression of ImmunoGlobulin E (IgE) Fc epsilon RI.

Mast Cells are multifunctional, tissue-dwelling cells capable of secreting a wide variety of mediators.

They develop from bone marrow-derived progenitor cells, primed with Stem Cell Factor (SCF), which mediates its actions by interacting with the SCF Receptor or c-kit on the cell surface.

Mast Cells continue their maturation and differentiation in peripheral tissue, developing into two well described subsets of cells, MCT and MCTC Cells, varying in content of Tryptase and Chymase as well as in ImmunoBiology.

Mast Cells are activated by numerous stimuli, including Antigen (acting via the high affinity IgE Receptor, Fc?RI), Superoxides, Complement Proteins, Neuropeptides and Lipoproteins resulting in activation and degranulation.

Following activation, these cells express mediators such as Histamine, Leukotrienes and Prostanoids, as well as Proteases, and many Cytokines and Chemokines, pivotal to the genesis of an inflammatory response.

Recent data suggests that mast cells may play an active role in such diverse diseases as AtheroSclerosis, Malignancy, Asthma, Pulmonary Fibrosis and Arthritis.

Mast cells directly interact with bacteria and appear to play a vital role in host defense against pathogens.

Drugs, such as GlucoCorticoids, Cyclosporine and Cromolyn have been demonstrated to have inhibitory effects on Mast Cell degranulation or mediator release.

Homeostatic mechanisms regulating Mast Cell numbers and function in peripheral tissues have largely focused on Cytokines, such as Stem Cell Factor, InterLeukin (IL-3), IL-4, and IL-10, which regulate Mast Cell maintenance and proliferation.

Despite these advances, little attention has been paid to the mechanisms that mediate mature Mast Cell turnover, and control of Mast Cell hyperplasia generated during Th2-mediated responses.

These are important issues, as Mast Cells are now known to be multi-functional Effector Cells, that have the capacity to mediate both Innate and Th2-induced Immune Responses.

Numerous secretagogues may elicit Mast Cells to release a large number of important mediators that can cause chronic inflammation.

Therefore, how Mast Cell Homeostasis is regulated may have significant effects on normal physiology, and contribute to the genesis of inflammatory disease.

Our laboratory has characterized an in vitro model of Mast Cell Homeostasis, by which long-term exposure of murine bone-marrow-derived Mast Cells to the Th2-derived Cytokines IL-3, IL-4, and IL-10, will induce downregulation of critical Mast Cell Effector Proteins such as Kit and Fc Epsilon-RI, followed by Mast Cell Apoptosis.

These data offer a novel role for Th2 Cytokines, acting to both initiate and resolve Mast Cell activation and proliferation. Loss of these signals may contribute to a multitude of diseases, such as MastoCytosis and Allergy.

In addition to their well characterized role in Allergic Inflammation, recent data confirm that Mast Cells play a more extensive role in a variety of Immune Responses.

However, their contribution to AutoImmune and Neurologic Disease processes has not been investigated.

Experimental Allergic EncephaloMyelitis (EAE) and its human disease counterpart, Multiple Sclerosis, are considered to be CD4⁺ T-Cell-mediated AutoImmune Diseases affecting the Central Nervous System.

Several lines of indirect evidence suggest that Mast Cells could also play a role in the PathoGenesis of both the human and murine disease.

Using a Myelin Oligodendrocyte Glycoprotein (MOG)-induced model of acute EAE, we show that when compared with their wild-type ConGenic littermates, Mast Cell-deficient W/W(v) mice exhibit significantly:

• Reduced Disease Incidence, Delayed Disease Onset, and Decreased Mean Clinical Scores.

No differences were observed in MOG-specific T-Cell and B-Cell Responses between the two groups, indicating that a global T or B-Cell defect is not present in W/W(v) animals.

Reconstitution of the Mast Cell population in W/W(v) mice restores induction of early and severe disease to wild-type levels, suggesting that Mast Cells are critical for the full manifestation of disease.

These data provide a new mechanism for Immune destruction in EAE and indicate that Mast Cells play a broader role in Neurologic Inflammation.

In order to characterize the phenotype of human Mast Cell Precursors in the peripheral blood Mononuclear fraction and its alterations.

During in vivo Mast Cell differentiation, cells were studied before and during culture with Stem Cell factor or Stem Cell factor-containing cell supernatants.

Prior to culture, 86% of cells were ImmunoReactive for the Monocytic marker CD14, slightly fewer for CD11b and CD64, < 10% expressed Fc EpsilonRI, rare cells were CD34⁺ ( < 0,1%), and none stained for CD1, CD33, c-Kit, and Tryptase.

After 2 wk of culture, there was de novo expression of c-Kit (14% - 43% positive cells), Tryptase (26% - 79%), CD33 (57%), and CD64 (64%), an upregulation of Fc EpsilonRI (23% - 52%), CD11b (93%), and CD68 (95%).

But, no expression of CD34. Levels of mRNA for Fc EpsilonRI and c-Kit were detectable prior to culture and increased during culture, together with de novo expression of Tryptase.

Double staining after 2 wk of culture showed that Fc EpsilonRI-positive cells were mostly CD14⁺ (90%), CD64⁺ (82%), and CD68⁺ (52%) on flow cytometry.

IntraCellular Tryptase activity was first detectable after 1 wk of culture, increased Fc EpsilonRI expression was only detectable by week 2.

Cultured cells acquired the ability to release Histamine during IgE-dependent stimulation, and culture with the c-Kit antibody YB5.B8 resulted in a downregulation of Tryptase and Fc EpsilonRI, but not of c-Kit.

These data show that human Mast Cells develop from c-Kit- and Tryptase-negative precursors in the MyeloMonocytic fraction of peripheral blood.

And that they upregulate, maintain, and share many phenotypic characteristics of cells from the Monocyte/Macrophage lineage during early phases of in vitro differentiation.

Mast Cells play a central role in both Immediate Allergic Reactions and Inflammation. A functional Nerve-Mast Cell interaction has been proposed, given the morphological association between Mast Cells and NeuroPeptide-containing Peripheral Nerves.

We now show that purified rat peritoneal Mast Cells contain large quantities of N-AcetylAspartate (NAA; 747.50 nmol/mg of protein).

Mast Cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and Mastoparan, respectively.

These Secretagogues strongly decreased Mast Cell Histamine content over the same time period, suggesting also that NAA is stored in Secretory Granules. The data are the first to show that NAA is present in an Immune Effector Cell type.

Because NAA may be involved in Myelin synthesis and Glutamyl Peptide metabolism, NAA released from Mast Cells following Nervous or other stimuli could participate in NeuroImmune interactions.

Mast Cells in Multiple Sclerosis plaques may contribute to the reported elevations in Brain NAA in this disease.

In summary, Mast Cell interactions in the Nervous System are relevant to both physiological processes (i.e. Reproduction) and pathologic states (i.e. Inflammatory DeMyelination, Painful Disorders, Toxic and Metabolic Disease, and Tumor Angiogenesis).

Their physiologic roles may contribute to gender-related vulnerability to Inflammatory Disease and may modulate sensitivity to Pain. Mast Cells are universally involved in tissue repair and they release and respond to Trophic Factors such as NGF.

These cells also produce and react to Cytokines, and thus appear to play a role in tissue degeneration as well as repair.

In certain Neurological Diseases, i.e. Multiple Sclerosis and Guillain-Barre Syndrome, the ability of Mast Cell Proteases to degrade specific Myelin proteins suggests that these cells are agents, rather than bystanders, in the DeMyelinative process.

Even more intriguing is their recently identified capacity to process Bacterial Antigen as efficiently as activated Macrophages, suggesting that a more critical role than previously suspected might be considered for Mast Cells in CNS and PNS DeMyelination.

In experimental metabolic disorders such as Galactose Intoxication and Thiamine Deficiency, Mast Cells appear to play a Pathogenic role.

Thus, in Galactose Intoxication, altered BNB Vascular Permeability occurs in conjunction with Mast Cell Proliferation and DeGranulation.

While in Thiamine Deficiency, increased Histamine levels have been reported in the rat Thalamus (79) and are associated with cell death and proliferation as well as Mast Cell DeGranulation (Powell and Langlais, unpublished observations).

Structural interactions between Mast Cells and a variety of other cells have been observed, as well as close approximation of Mast Cells to Nerve endings in tissues in which Mast Cells are especially active.

Due to their Paracrine nature, Mast Cells can modulate events in their MicroEnvironment through explosive DeGranulation, piecemeal DeGranulation, or "TransGranulation" as they insert granules into neighboring cells.

Lastly, these cells play specific roles in reparative processes, e.g. AngioGenesis, and are active in NeoPlastic states, including von Recklinghausen's Disease (NeuroFibroMatosis).

Their involvement may have been underestimated in NeuroPathological studies, to date, by a reliance on staining techniques that are inadequate for identifying DeGranulated and therefore activated Mast Cells (4).

More exacting HistoChemical and ImmunoStaining procedures will help to fully realize the extent of their participation in physiological and pathological processes.

Mast Cells and their potent chemical mediators are known to initiate and modulate a number of important Inflammatory cascades.

With respect to the Central Nervous System, the role of Mast Cells as participants in the promotion and resolution of Inflammation has been widely underestimated.

Mast Cell-derived Histamine, Serotonin, Kallikreins, and Tumor Necrosis Factor-alpha (TNF-) can enhance MicroVascular Permeability, Leukocyte Rolling, Adhesion, and ExtraVasation of Inflammatory Cells into the Brain and Spinal Cord.

Mast Cell mediators may play an important role in AutoImmune EncephaloMyelitis and Multiple Sclerosis by promoting the entry of AutoReactive T-Cells and the recruitment of nonspecific Monocytes across the Blood-Brain Barrier.

Mast Cells may invade the Brain as a consequence of a childhood infection or predisposition, and it is proposed that Multiple Sclerosis arises due to the effect of various mediators (Histamine and Protease) released from the PeriVascular Mast Cells after stimulation by some diet factor.

Multiple Sclerosis (MS) lesions are associated with infiltration of T-Lymphocytes and Macrophages that appear to mediate Myelin destruction and Gliosis (Scarring).

Mast Cells are located PeriVascularly in the Brain, are juxtaposed to Neurons, and have been shown to secrete VasoActive and Inflammatory Mediators in response to NeuroPeptides and direct Nerve stimulation.

Mast Cells have been previously identified in MS lesions, are activated by Myelin Basic Protein, and can participate in the regulation of Blood-Brain Barrier permeability, as well as in Myelin destruction.

Here, CerebroSpinal Fluid from MS patients and controls with other Neurologic Diseases was assayed for Histamine, its major metabolite MethylHistamine, and the specific Mast Cell marker, Tryptase. Histamine and MethylHistamine were not elevated in MS.

However, the Mast Cell specific proteolytic enzyme Tryptase was significantly elevated in MS, suggesting that Mast Cell activation may be involved in the PathoPhysiology of this Disease.