Matrix MetalloProteinases

Expression of 4 Integrin by AutoReactive T-Cells is critical for their ability to induce EAE, an AutoImmune Disease of the Central Nervous System in mice, used as a model to study human Multiple Sclerosis.

Having previously identified one role for 4 Integrin in Adhesion-mediated induction of Matrix MetalloProteinase-2 (MMP-2), an enzyme that degrades the SubEndothelial Basement Membrane Matrix (Parenchyma).

We investigated independent roles for MMP-2 and 4 Integrin during EAE.

The data suggest that expression of 4 Integrin by auto-reactive T-Cells is important not only in mediating MMP-2 induction to facilitate entry into the CNS, but also plays a role in maintaining residency within the CNS.

Introduction
At what stage in the PathoGenesis of Multiple Sclerosis (MS) does the damage to Axons occur, and why should there be any Axon loss at all in what is thought to be principally an Axon sparing DeMyelinating Disease?

A recently described new technique for investigating Axon damage depends for its ability on the ImmunoReactivity of Amiloid Precursor Protein (APP), which has been shown to be more sensitive than silver stains for detecting damaged Axons.

Development
We used APP ImmunoReactivity as a method to investigate whether Axon damage occurs in acute MS Lesions.

The results of our APP staining showed that the expression of APP in MS lesions is associated with acute MS lesions and the active border of less acute lesions.

There was little, if any, APP expression in the chronic Lesions. If we accept that the APP staining represents irreversible damage to some Axons, the next question is what factors are responsible for mediating damage to Axons in MS?

Matrix MetalloProteinases (MMP) are expressed by Macrophages in acute MS lesions and in the active borders of active chronic Lesions.

The injection of highly-purified MMP into the Brain results in DeMyelination, Blood-Brain Barrier breakdown, and Axonal Loss.

Moreover, the inhibition of the MMP activity reduces the severity of MS-like lesions in experimental models. Thus the properties and distribution of these Enzymes make them rational targets for therapeutic intervention.

Conclusion
Whatever mechanism proves to be responsible for Axonal damage in MS, it is clear that this disease should, perhaps, be more appropriately recognized as a primary DeMyelinating entity with associated primary Axonal Loss.

The Immunological effects of high-dose MethylPrednisolone in attacks of Multiple Sclerosis and acute Optic Neuritis have only been examined in a few randomized, controlled trials.

We studied Immunological changes in 50 patients with Optic Neuritis or Multiple Sclerosis who underwent Lumbar Puncture before and 1 week after completing a 15-day course of oral high-dose MethylPrednisolone treatment.

Treatment resulted in:

1. Decrease in the concentration of MBP
2. Decrease in the Serum concentration of
   • ImmunoGlobulin G (IgG)
   • Intrathecal IgG synthesis
3. Increase in the CerebroSpinal Fluid concentration of Transforming Growth Factor-beta1
4. Changes in the expression of CD25, CD26, and HLA-DR on CD4⁺ T-Cells

No effect was seen on the CerebroSpinal Fluid Leucocyte count or the CerebroSpinal Fluid activity of Matrix MetalloProteinase-9 (MMP-9).

The lack of a persistent effect on CerebroSpinal Fluid Leukocyte recruitment and MMP-9 activity, despite changes in IgG synthesis, T-Cell activation, and Cytokine production, suggests that modulation of the function of inflammatory cells may contribute to the clinical efficacy of oral high-dose MethylPrednisolone treatment in Optic Neuritis and Multiple Sclerosis.

Interferon-beta (IFN-ß) has a beneficial influence on the course of Multiple Sclerosis (MS) and has become standard treatment of this disease, though its mechanisms of action are incompletely understood.

This study examines the effect of IFN-ß treatment on the Cytokines IL-6, TNF-, IFN- and IL-10; the MetalloProteinases MMP-3, -7 and -9 and the Tissue Inhibitor of MetalloProteinase-1 (TIMP-1).

IFN-ß treatment resulted in decreased numbers of MonoNuclear Cells (MNC) secreting IL-6 and TNF- and expressing mRNA of MMP-3 and MMP-9 compared to pretreatment levels.

On the contrary, numbers of IL-10 secreting MNC and TIMP-1 mRNA expression were augmented during IFN-ß; therapy.

Whether the down-regulatory effects on pro-inflammatory and upregulatory effects on anti-inflammatory molecules are a direct result of IFN-ß on the Immune System or secondary to clinical stabilization of MS pathology induced by IFN-ß remains to be evaluated.

Chemokines and Matrix MetalloProteinases (MMPs) appear to be crucial in Leukocyte recruitment to the Central Nervous System in Multiple Sclerosis (MS).

CCR5 delta32, a truncated allele of the CC Chemokine Receptor CCR5 Gene encoding a non-functional receptor, did not confer protection from MS.

CCR5 delta32 was, however, associated with a lower risk of recurrent clinical disease activity. High CSF levels of MMP-9 activity were also associated with recurrent disease activity.

These results directly link Intrathecal inflammation to disease activity in patients with MS, suggesting that treatments targeting CCR5 or treatment with MMP Inhibitors may attenuate disease activity in MS.

Objective

1. Compare monthly Serum levels of Matrix MetalloProteinase-9 (MMP-9) and Tissue Inhibitor of MMP-type 1 (TIMP-1) in Relapsing/Remitting MS (RRMS) versus healthy controls.
   
   
2. Determine the relationship among monthly Serum levels of MMP-9 and TIMP-1 and MRI activity.

Background
Activated T-Cells and Macrophages secrete MMPs that may facilitate their migration across Vascular SubEndothelial basement membranes into the CNS.

The Serum concentration of MMP-9 is reported to be higher in patients with RRMS than healthy controls.

Methods
Monthly evaluations including Gadolinium-enhanced (Gd⁺) Brain MRI and measures of Serum MMP-9 and TIMP-1 were performed for up to 15 months in 24 patients with RR/MS and for up to 4 months in 10 controls.

Results
Serum MMP-9 but not TIMP-1 levels are elevated in RR/MS patients compared to healthy controls (p = 0.025, p = 0.61).

In a univariate analysis, high MMP-9 and low TIMP-1 levels precede appearance of new Gd⁺ lesions (respectively; odds ratio = 3.3, p = 0.008; odds ratio = 2.2, p = 0.086).

In a multivariate analysis, in comparison to months when MMP-9 is low and TIMP-1 high, MRI scans obtained the month following high MMP-9 and low TIMP-1 Serum concentrations are more likely to report new Gd⁺ lesions (p = 0.0006, odds ratio = 21.5).

Conclusion
An increase in the activity of Matrix MetalloProteinase-9 (MMP-9) relative to Tissue Inhibitor of MMP-type 1 (TIMP-1) may be related to formation of new MS lesions.

Suggesting that Serum levels of MMP-9 and TIMP-1 may be surrogate markers of disease activity in Relapsing/Remitting MS.

Comments:

• Comment in: Neurology 1999 Oct 22;53(7):1380-1

Multiple Sclerosis (MS) and Stroke pathology are characterized Blood-Brain Barrier breakdown, Leukocyte emigration, and tissue destruction.

Each process is thought to involve the Matrix MetalloProteinases (MMP), but little is known of their expression.

We undertook to investigate whether MMP expression is dependent on the nature of the CNS lesion and whether expression would coincide with the HistoPathology.

MS or Cerebral-Infarct tissue was examined for the presence of Gelatinase-A, Gelatinase-B, Matrilysin and Stromelysin-1.

Gelatinases A and B and Matrilysin expression was found to be up-regulated in Microglia/Macrophages within acute MS lesions.

In active-chronic MS lesions, Matrilysin and Gelatinase-A expression was pronounced in the active borders.

In chronic MS lesions, the expression of Matrilysin was confined to Macrophages within PeriVascular cuffs. The pattern of MMP expression in Infarct lesions differed considerably.

Gelatinase-B was strongly expressed by Neutrophils in tissue from patients up to 1 week after an infarct, whereas Gelatinase-A and Matrilysin staining was much less marked.

From 1 week to 5 years, Neutrophils were absent and the large number of Macrophages present were expressing Matrilysin and Gelatinase A.

Only a low level of Gelatinase-A and Matrilysin expression was observed in normal Brain controls.

Thus, MMPs are expressed in inflammatory lesions in the CNS, but their individual expression is dependent on the nature and chronicity of the Lesion.

However, the general pattern of expression, in PeriVascular cuffs and in active lesions, supports a role for these Enzymes as mediators of Blood-Brain Barrier breakdown and tissue destruction, both in MS and in Cerebral Ischemia.

The Matrix MetalloProteinases (MMPs) are a family of at least 14 Zinc-dependent Enzymes which are known to degrade the protein components of ExtraCellular Matrix.

In addition, MMPs and related Enzymes can also process a number of cell surface Cytokines, receptors, and other soluble proteins.

In particular we have shown that the release of the pro-inflammatory Cytokine, Tumor Necrosis Factor-, from its membrane-bound precursor is an MMP-dependent process.

MMPs are expressed by the inflammatory cells which are associated with CNS Lesions in animal models of Multiple Sclerosis (MS) and in tissue from patients with the disease.

MMP expression will contribute to the tissue destruction and inflammation in MS. Drugs which inhibit MMP activity are effective in animal models of MS and may prove to be useful therapies in the clinic.

Contrast-enhanced MRI in patients with MS shows that increased permeability of the Blood-Brain Barrier (BBB) commonly occurs. The changes in Capillary permeability often precede T₂-weighted MRI evidence of tissue damage.

In animal studies, IntraCerebral injection of the Matrix MetalloProteinase (MMP) 72-kDa type IV Collagenase (Gelatinase A) opens the BBB by disrupting the Basal Lamina around Capillaries.

Steroids affect production of endogenous MMPs and Tissue Inhibitors to MetalloProteinases (TIMPs).

To determine the role of MMP activity in BBB damage during acute exacerbations of MS, we measured MMPs in the CSF of patients with MS.

Patients (n = 7) given Steroids to treat an acute episode of MS had CSF sampled before and after 3 days of MethylPrednisolone (1 g/day).

Patients had a graded Neurologic Examination and Gadolinium-enhanced MRI before treatment. CSF studies included total protein, cell count, and a DeMyelinating profile.

We measured levels of MMPs, Urokinase-type PlasMinogen Activator (uPA), and TIMPs by Zymography, Reverse Zymography, and Western blots.

The MMP, 92-kDa type IV Collagenase (Gelatinase B), fell from 216 +/- 70 before Steroids to 54 +/- 26 relative lysis zone units (p < 0.046) after treatment. Similarly, uPA dropped from 3880 +/- 800 to 2655 +/- 353 (p < 0.03).

Four patients with Gadolinium enhancement on MRI had the most pronounced drop in Gelatinase B and uPA.

Western Immunoblots showed an increase in a complex of Gelatinase B and TIMPs after treatment, suggesting an increase in a TIMP (p < 0.05).

Reverse Zymography of CSF samples showed that Steroids increased a TIMP with a molecular weight similar to that of mouse TIMP-3 (p = 0.053).

Our results suggest that increased Gelatinase B is associated with an open BBB on MRI.

Steroids may improve Capillary function by reducing activity of Gelatinase B and uPA and increasing levels of TIMPs.

Matrix MetalloProteinases (MMPs) comprise a family of ProteoLytic enzymes that are involved in remodeling of the ExtraCellular Matrix (ECM) of many tissues.

They have been implicated in degradation of Vascular basement membranes thereby facilitating Leukocyte migration into inflammatory sites.

To determine the cellular localization and levels of MMPs in the normal human Central Nervous System (CNS), Multiple Sclerosis (MS) lesions, and other conditions, cryostat sections of CNS samples were Immunostained with AntiSera to MMP-1, -2, -3 and -9.

In control White Matter the principal cells that express the MMPs were PeriVascular and Parenchymal Microglia.

Cellular MMP expression was also found in sporadic Microglial nodules in MS White Matter. Most CNS MicroVessel Endothelial Cells expressed MMP-3 and -9 but not MMP-1 or -2.

The majority of Macrophages in active MS and Necrotic lesions were MMP-l-, -2-, -3-, and -9-positive whereas chronic MS lesions had fewer MMP-positive Macrophages.

Small numbers of Astrocytes were MMP-2-, -3- and -9-positive in acute and chronic MS lesions.

These data suggest that Microglia-derived MMPs may mediate turnover of the CNS ECM under normal conditions and in Microglial nodules.

In sites of CNS tissue injury there is complex and dynamic regulation of MMP expression by different cell populations.

In MS lesions MMP-mediated ProteoLysis may contribute to breakdown of the Blood-Brain Barrier and Leukocyte migration into the CNS.

In situ Immune activation, DeMyelination, metabolism of bioactive Peptides, and the formation of an ECM that does not promote ReMyelination or Axonal repair.

The ProteoLytic activities of Matrix MetalloProteinases and PlasMinogen Activators as well as their inhibitors are important in maintaining the integrity of the ExtraCellular Matrix (ECM).

Cell-ECM interactions influence cell proliferation, differentiation, adhesion and migration.

In the Nervous System, ProteoLysis of the ECM is involved in Neuronal Cell migration in the developing Cerebellum and in Neurite outgrowth.

Likewise, in pathological conditions such as Brain Tumor growth and invasion, Leukocyte infiltration into Brain Tumors, Leukocyte trafficking in the Central Nervous System in inflammatory diseases such as Multiple Sclerosis and Viral Encephalitis.

And in Nerve DeMyelination, Matrix-degrading Proteinases and their inhibitors have been implicated.

An understanding of Cell-ECM interactions and ECM degradation in diseases of the Nervous System would provide new insight for drug design and other forms of therapy.

In this study, we employed a sensitive activity assay system to measure CerebroSpinal Fluid (CSF) and Serum levels of active Matrix MetalloProteinase-9 (MMP-9) in 37 Relapsing/Remitting (RR), 15 Secondary/Progressive (SP) and nine Primary/Progressive (PP) Multiple Sclerosis (MS) patients

Grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity.

We also studied, as Neurological controls, 48 patients with Other Inflammatory Neurological Disorders (OIND) and 48 with Non-Inflammatory Neurological Disorders (NIND).

To assess active MMP-9/TIMP-1 circuit, CSF and Serum levels of MMP-9 Tissue Inhibitor TIMP-1 were quantified by ELISA in the same patient population.

CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and Intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P < 0.05, < 0.02 and < 0.02, respectively).

Serum active MMP-9/TIMP-1 ratio was higher in MS (P < 0.01) and OIND (P < 0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P < 0.05).

More importantly, Serum active MMP-9 mean levels, serum active MMP-9/TIMP-1 ratio and intrathecal production of active MMP-9 were increased in MS patients with clinical (P < 0.001, < 0.001 and < 0.05, respectively) and MRI (P < 0.001, < 0.001 and < 0.02, respectively) disease activity.

Whereas CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P < 0.02 and < 0.01, respectively).

Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS Immune dysregulation.

In addition, our results indicate that CSF and Serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity.

In particular, Serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.