Macrophages of Multiple Sclerosis Patients Display Deficient SHP-1 Expression and Enhanced Inflammatory Phenotype

George P Christophi; Michael Panos; Chad A Hudson; Rebecca L Christophi; Ross C Gruber; Akos T Mersich; Scott D Blystone; Burk Jubelt; Paul T Massa

Disclosures

Lab Invest. 2009;89(7):742-759. 

In This Article

Abstract and Introduction

Abstract

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-κB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-κB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-κB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-κB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

Introduction

Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system (CNS) that remains a major cause of disability.[1] Several studies demonstrated that MS lesions contain multiple leukocyte cell types including lymphocytes, macrophages, and dendritic cells all of which are believed to contribute to lesion formation by various distinct and interacting mechanisms.[2,3] Among these leukocyte subsets, infiltrating macrophages have been identified as major effectors of demyelination in both MS and animal models for MS.[4,5,6,7,8,9,10,11] In accord with these studies, it was recently described that a predominant mechanism of demyelination in MS is macrophage mediated.[12]

These findings have stimulated intense interest on the various effector functions of macrophages in lesion formation including signaling events that draw these cells into the CNS white matter and trigger the effector mechanisms by which these cells destroy myelin. For instance, macrophages have been identified as the major responders to CNS chemokines, producers of a number of proinflammatory cytokines, chemokines, and toxic molecules known to promote demyelination,[7,13,14,15,16,17,18] and the major cells involved in phagocytosis/degradation of myelin sheathes. Interestingly, both leukocytes and CNS plaques of MS patients contain activated transcription factors such as NF-κB,[19,20] STAT1,[20,21,22] and STAT6,[23,24,25] which can lead to enhanced expression of these inflammatory effector functions in macrophages. Based on our previous work, we propose that modulation of inflammatory signaling via these multiple transcriptional pathways may be deficient in leukocytes, including inflammatory macrophages of MS patients, and that this deficiency is responsible for susceptibility to inflammatory demyelinating processes within the CNS.

SHP-1 is a protein tyrosine phosphatase with two SH2 domains which acts as a negative regulator of both innate and acquired immune cytokine signaling via NF-κB,[26,27,28] STAT1,[29,30] and STAT6.[31,32,33,34] Mice genetically lacking SHP-1 (motheaten mice) display myelin deficiency, which may be mediated by increased innate inflammatory mediators in the CNS.[35,36] Furthermore, motheaten mice are highly susceptible to experimentally induced demyelinating disease.[37,38,39] Taken together, these studies indicate that SHP-1 is a key regulator of inflammation in the CNS that may be relevant to the pathogenesis of MS. Indeed, others and we reported that SHP-1 expression and function is deficient in leukocytes of MS patients compared to normal human subjects.[22,25]

We have shown that SHP-1-deficient mice uniquely display an unusually rapid CNS demyelination associated with extensive white matter cellular infiltration and clinical paralysis within the first week of Theiler's murine encephalomyelitis virus (TMEV) infection compared to their wild-type littermates.[37] Recently, we have shown that SHP-1-deficient mice show a profound and predominant infiltration of blood-derived macrophages into the CNS following TMEV infection and that these macrophages are concentrated to areas of demyelination.[9] Importantly, these macrophages possessed an inflammatory profile in vivo with an unusual coactivation of STAT6, STAT1, and NF-κB and corresponding responsive proinflammatory genes. Because of the rapidity of demyelination in this model, we proposed that SHP-1-deficient macrophages possess a particularly pronounced demyelinating phenotype. Accordingly, macrophage/monocyte depletion with clodronate liposomes resulted in a significant delay in the onset of clinical symptoms and decreased inflammation and demyelination in the spinal cords of SHP-1-deficient mice. Those studies suggested that SHP-1 is an important regulator of CNS inflammatory demyelination acting predominantly to control recruitment of macrophages into the CNS and a broad modulation of macrophage inflammatory effector functions.

In the light of recent studies demonstrating the importance of macrophages in mediating demyelination in MS[12] and our recent studies in SHP-1-deficient mice cited above,[9] it became imperative to characterize the inflammatory profile of macrophages of MS patients compared to normal subjects. This study demonstrates that the expression of SHP-1 is deficient in macrophages of MS patients compared to normal subjects. Corresponding with this deficiency, we show that phosphorylation of STAT6 and STAT1 and activation of NF-κB are higher in macrophages of MS patients compared to those of normal subjects. In accordance with this activation, several STAT6-, STAT1-, and NF-κB-responsive genes that are important for inflammatory demyelination were increased in macrophages of MS patients following cytokine stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in macrophages of normal human subjects using siRNA resulted in an increased activation of the above transcription factors and increased inflammatory gene expression to levels seen in macrophages of MS patients. Taken together, we propose the potential involvement of SHP-1 deficiency in mediating the augmented inflammatory profile in macrophages seen in MS patients and contributing to the pathogenesis of MS.

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