Rapidly progressive amyotrophic lateral sclerosis is associated with microglial reactivity and small heat shock protein expression in reactive astrocytes.

AIMS: Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive loss of motor neurons, muscle weakness, spasticity, paralysis and death usually within 2-5 years of onset. Neuroinflammation is a hallmark of ALS pathology characterized by activation of glial cells, which respond by upregulating small heat shock proteins (HSPBs), but the exact underlying pathological mechanisms are still largely unknown. Here, we investigated the association between ALS disease duration, lower motor neuron loss, TARDNA-binding protein 43 (TDP-43) pathology, neuroinflammation and HSPB expression. METHODS: With immunohistochemistry, we examined HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 expression in cervical, thoracic and sacral spinal cord regions in 12 ALS cases, seven with short disease duration (SDD), five with moderate disease duration (MDD), and ten age-matched controls. Expression was quantified using ImageJ to examine HSP expression, motor neuron numbers, microglial and astrocyte density and phosphorylated TDP-43 (pTDP-43+) inclusions. RESULTS: SDD was associated with elevated HSPB5 and 8 expression in lateral tract astrocytes, while HSP16.2 expression was increased in astrocytes in MDD cases. SDD cases had higher numbers of motor neurons and microglial activation than MDD cases, but similar levels of motor neurons with pTDP-43+ inclusions. CONCLUSIONS: Increased expression of several HSPBs in lateral column astrocytes suggests that astrocytes play a role in the pathogenesis of ALS. SDD is associated with increased microgliosis, HSPB5 and 8 expression in astrocytes, and only minor changes in motor neuron loss. This suggests that the interaction between motor neurons, microglia and astrocytes determines neuronal fate and functional decline in ALS.

Heat shock proteins are differentially expressed in brain and spinal cord: implications for multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by demyelination, inflammation and neurodegeneration throughout the central nervous system. Although spinal cord pathology is an important factor contributing to disease progression, few studies have examined MS lesions in the spinal cord and how they differ from brain lesions. In this study we have compared brain and spinal cord white (WM) and grey (GM) matter from MS and control tissues, focusing on small heat shock proteins (HSPB) and HSP16.2. Western blotting was used to examine protein levels of HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 in brain and spinal cord from MS and age-matched non-neurological controls. Immunohistochemistry was used to examine expression of the HSPs in MS spinal cord lesions and controls. Expression levels were quantified using ImageJ. Western blotting revealed significantly higher levels of HSPB1, HSPB6 and HSPB8 in MS and control spinal cord compared to brain tissues. No differences in HSPB5 and HSP16.2 protein levels were observed, although HSPB5 protein levels were higher in brain WM versus GM. In MS spinal cord lesions, increased HSPB1 and HSPB5 expression was observed in astrocytes, and increased neuronal expression of HSP16.2 was observed in normal-appearing GM and type 1 GM lesions. The high constitutive expression of several HSPBs in spinal cord and increased expression of HSPBs and HSP16.2 in MS illustrate differences between brain and spinal cord in health and upon demyelination. Regional differences in HSP expression may reflect differences in astrocyte cytoskeleton composition and influence inflammation, possibly affecting the effectiveness of pharmacological agents.

Heat shock protein expression in cerebral X-linked adrenoleukodystrophy reveals astrocyte stress prior to myelin loss.

AIMS: X-linked adrenoleukodystrophy (X-ALD) is a genetic white matter disorder in which demyelination occurs due to accumulation of very long-chain fatty acids. Inflammation in the brain white matter is a hallmark of the pathology of cerebral X-ALD, but the underlying pathogenic mechanisms are still largely unknown. In other inflammatory demyelinating disorders, such as multiple sclerosis, the expression of heat shock proteins (HSPs) in combination with interferon-γ (IFN-γ) has been suggested to play a prominent role in the initiation of demyelination and inflammation. We therefore investigated these pathways in X-ALD lesions. METHODS: By immunohistochemistry, we examined the expression of small HSPs (HSPB1, HSPB5, HSPB6, HSPB8) and higher molecular weight HSPs (HSPA, HSPD1), and the expression of elements of the IFN-γ pathway on autopsy material of five patients with X-ALD. RESULTS: The expression of the larger HSPs, HSPA and HSPD1, as well as small HSPs is increased in X-ALD lesions compared with normal-appearing white matter. Such upregulation can already be detected before demyelination and inflammation occur, and it is predominant in astrocytes. The IFN-γ pathway does not seem to play a leading role in the observed inflammation. CONCLUSIONS: The finding that astrocytes show signs of cellular stress before demyelination suggests that they play a major role early in the pathogenesis of cerebral X-ALD, and may therefore be involved in the initiation of inflammation and demyelination.

Targeting the tetraspanin CD81 blocks monocyte transmigration and ameliorates EAE.

Leukocyte infiltration is a key step in the development of demyelinating lesions in multiple sclerosis (MS), and molecules mediating leukocyte-endothelial interactions represent prime candidates for the development of therapeutic strategies. Here we studied the effects of blocking the integrin-associated tetraspanin CD81 in in vitro and in vivo models for MS. In an in vitro setting mAb against CD81 significantly reduced monocyte transmigration across brain endothelial cell monolayers, both in rodent and human models. Interestingly, leukocyte as well as endothelial CD81 was involved in this inhibitory effect. To assess their therapeutic potential, CD81 mAb were administered to mice suffering from experimental autoimmune encephalomyelitis (EAE). We found that Eat2, but not 2F7 mAb directed against mouse CD81 significantly reduced the development of neurological symptoms of EAE when using a preventive approach. Concomitantly, Eat2 treated animals showed reduced inflammation in the spinal cord. We conclude that CD81 represents a potential therapeutic target to interfere with leukocyte infiltration and ameliorate inflammatory neurological damage in MS.

Cell biology of autoimmune diseases.

Autoimmune diseases such as insulin-dependent diabetes mellitus, rheumatoid arthritis, and multiple sclerosis are common in the western world and are often devastating diseases which pose serious health problems. The key feature of such diseases is the development and persistence of inflammatory processes in the apparent absence of pathogens, leading to chronic breakdown of selected tissues. To date, no comprehensive explanation can be given for the onset or persistence of autoimmunity. As a rule, the chronic activation of helper T lymphocytes reactive against self proteins appears to be crucial for fueling the destructive autoimmune process, but why this occurs remains to be established. In this review, we present an overview on the rules that govern activation of T lymphocytes and on the factors that control it. The contribution of both genetic and environmental factors are discussed, clarifying that most autoimmune disease are of multifactorial origin. Special emphasis is given to the contribution of infectious events and the role of stress proteins in the process. In attempts to dissect the mechanisms involved in autoimmunity and to develop ways of blocking disease, experimental animal models are widely employed. We describe the various experimental models that exist for the study of multiple sclerosis, diabetes, and other autoimmune diseases and on the experience that has been gained in such models with experimental therapies to block the activation of self-reactive T lymphocytes. The lessons that can be drawn from these studies provide hope that continued efforts will lead to the successful development of antigen-specific strategies which block the development of autoimmunity also in humans.

Multiple sclerosis: an altered immune response or an altered stress response?

The pathogenesis of multiple sclerosis (MS), the major neurological disease of young adults in the Western world, is still poorly understood, and no effective therapy to block MS is available as yet. The clinical symptoms of MS result from inflammatory damage to the insulating myelin sheath of axons in the CNS and-at later stages-to axons themselves. A local autoimmune process involving activation of helper T cells against CNS protein components is likely to be crucial in this development. Especially at the first stages of MS, therapies aimed at the selective downregulation of MS-specific autoimmune responses may contribute to controlling the disease. Key to the success of such approaches is the identification of CNS proteins that are the target of local T cell responses. We recently identified the small heat-shock protein alpha B-crystallin as the single immunodominant myelin antigen in MS-affected myelin. This review discusses the functional and therapeutic implications of this finding along with other data on MS, and hypothesizes that an inappropriate stress response within the CNS itself is crucial as an initiating event in disease development.

Purification of the stress protein alpha B-crystallin and its differentially phosphorylated forms.

The stress protein alpha B-crystallin was recently identified as a component of central nervous system myelin that is strongly immunogenic to human T cells. Stress-induced alpha B-crystallin that accumulates in the central nervous system is phosphorylated and recent evidence indicates that both rodent and human T cells can discriminate between differentially phosphorylated forms of alpha B-crystallin. For immunological studies, therefore, the availability of purified preparations of alpha B-crystallin and its various differentially phosphorylated forms would be especially useful. Here we describe a rapid and simple method for the purification of alpha B-crystallin from adult bovine eye lenses by a combination of size-exclusion chromatography and reversed-phase high-performance liquid chromatography. This yields a preparation of purified alpha B-crystallin that contains all the various differentially phosphorylated forms of the protein. Subsequent anion-exchange chromatography under denaturing conditions permits the separation of these phosphorylated forms of alpha B-crystallin into purified fractions with a defined number of phosphorylated serines.

Expression of alphaB-crystallin in glia cells during lesional development in multiple sclerosis.

The small heat shock protein alphaB-crystallin was recently identified as a dominant human T-cell antigen in myelin derived from multiple sclerosis (MS) patients. Using immunohistochemical techniques, oligodendrocytes as well as astrocytes in MS lesions were shown to express alphaB-crystallin. In the present study we examined the expression of alphaB-crystallin, human natural killer cell marker (HNK-1; as a marker for immature oligodendrocytes) and heat shock protein 60 (hsp60) in glia cells at different stages of MS lesion development i.e. in early active lesions, late active lesions and inactive lesions. The results demonstrate that already at the earliest stages of lesional development a subpopulation of oligodendrocytes express detectable levels of alphaB-crystallin. In active lesions about 5-10% of all oligodendrocytes were found to express alphaB-crystallin, whereas in inactive lesions the relative number of alphaB-crystallin-expressing oligodendrocytes was approximately tenfold less. For astrocytes the relative number of alphaB-crystallin-expressing cells was 40-50% for all three types of lesions. Also, alphaB-crystallin-expressing oligodendrocytes and astrocytes displayed different patterns of distribution in lesional areas. These data suggest different regulatory pathways for alphaB-crystallin expression in either type of glia cell. No correlation was found between expression patterns of HNK-1 and alphaB-crystallin indicating that the subpopulation of alphaB-crystallin-expressing oligodendrocytes consisted of both mature and immature oligodendrocytes. In addition, no correlation was found between expression of hsp60 and alphaB-crystallin in MS lesions suggesting different regulatory pathways for either hsp.

Mistaken self, a novel model that links microbial infections with myelin-directed autoimmunity in multiple sclerosis.

Several findings indicate that infectious events play a role in the pathogenesis of multiple sclerosis (MS). At the same time, T-cell autoimmunity to myelin antigens is widely believed to be crucial to the development of MS lesions. Several mechanisms have been put forward to explain the presumed link between microbial infections and myelin-directed autoimmunity. These include molecular mimicry, bystander activation including epitope spreading and superantigenic activation of T cells. Evidence that either one of these mechanisms actually occurs in MS patients, however, is still weak. Also, none of the above mechanisms explain why MS is unique to humans. We propose an alternative link between microbial infection and myelin autoimmunity, which we refer to as 'mistaken self'. In this mechanism, peripheral microbial infections of lymphoid cells prime the human T-cell repertoire not only to microbial antigens but also to the stress protein alpha B-crystallin that is expressed de novo in infected lymphoid cells. Subsequently, stress-induced accumulation of this self antigen in oligodendocytes/myelin can provoke pro-inflammatory responses as the recruited memory T-cell repertoire then mistakes the self protein for a microbial antigen. In this paper we review the currently available evidence that 'mistaken self' centering on alpha B-crystallin represents a powerful source of anti-myelin autoimmunity in a way that is unique to humans.

Tolerance controls encephalitogenicity of alphaB-crystallin in the Lewis rat.

The myelin-associated protein, alphaB-crystallin, is considered a candidate autoantigen in multiple sclerosis (MS). In the present study, we examined the potential of alphaB-crystallin to induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Attempts to induce EAE with either bovine, rat or murine alphaB-crystallin or alphaB-crystallin peptides consistently failed. Immunization with either autologous rat or murine alphaB-crystallin did not trigger any antigen-specific T cell response. Immunization with bovine alphaB-crystallin or a synthetic peptide representing the cryptic epitope 49-64 did trigger T cell responses but these failed to crossreact with autologous rat alphaB-crystallin. Examination of lymphoid tissues of the Lewis rat revealed constitutive expression of alphaB-crystallin in thymus, spleen, and peripheral lymphocytes. Our data show that in Lewis rats, constitutive lymphoid expression of alphaB-crystallin is associated with a state of nonresponsiveness to autologous alphaB-crystallin that effectively controls the development of EAE in response to this myelin antigen.

Differential expression of stress proteins in human adult astrocytes in response to cytokines.

Various lines of evidence suggest a close relationship between heat shock proteins (hsp) and several autoimmune diseases such as arthritis, diabetes and multiple sclerosis. While enhanced expression of hsp in autoimmune diseases is often regarded as a non-specific bystander effect of the inflammatory process, surprisingly little is known on hsp regulation by inflammatory mediators such as cytokines. In this study cytokine-induced expression of hsp60, hsp27 and alphaB-crystallin was studied in cultures of primary human adult astrocytes at the mRNA as well as at the protein level. We show differential hsp expression patterns in response to pro-inflammatory and immunoregulatory cytokines. Hsp60 expression was found to be enhanced in response to cytokines as diverse as IL-1beta, TNF-alpha, IL-4, IL-6 and IL-10. Upregulation of hsp27, however, was primarily induced by immunoregulatory cytokines like IL-4, IL-6 and TGF-beta whereas alphaB-crystallin expression was found to be enhanced by the pro-inflammatory cytokine TNF-alpha only. None of the cytokines studied was able to enhance expression of all three hsp simultaneously. These results show that in human astrocytes induced expression of hsp27 and alphaB-crystallin is dependent on the presence of a defined set of stimuli, while induced expression of hsp60 is a much less selective event. This highly differential pattern of hsp expression in response to inflammatory mediators known to play an important role in the pathogenesis of autoimmune diseases indicates that hsp responses are specific rather than non-specific bystander responses.

Minor myelin proteins can be major targets for peripheral blood T cells from both multiple sclerosis patients and healthy subjects.

T cell recognition of myelin is likely to play a role in the pathogenesis of multiple sclerosis. Predominant protein components of myelin, myelin basic protein (MBP) and proteolipid protein (PLP), have been considered as possibly relevant autoantigens, especially since both proteins are encephalitogenic in various laboratory animals. It has remained unclear, however, to what extent the numerous minor proteins contained in myelin may serve as targets for human T cell responses to myelin. In this study, the abilities of several minor myelin proteins to trigger proliferative responses of human peripheral blood T cells were compared to that of MBP. By using a water soluble collection of myelin proteins as an antigen, including MBP as the major component, short-term T cell lines were generated. Proliferative responses were determined against the various proteins after their fractionation by HPLC. Short-term T cell lines from both multiple sclerosis patients and healthy control subjects displayed significant responses to several minor myelin proteins but failed to respond to MBP. Only the use of purified MBP as trigger antigen allowed the selective expansion of MBP-specific T cell lines. These findings indicate that minor myelin proteins may act as relevant targets for autoreactive human T cells.

Encephalitogenic and immunogenic potential of the stress protein alphaB-crystallin in Biozzi ABH (H-2A(g7)) mice.

The stress protein alphaB-crystallin is an immunodominant antigen in multiple sclerosis (MS)-affected myelin for human T cells and is expressed at elevated levels in MS lesions. Using bovine alphaB-crystallin and synthetic peptides based on mouse alphaB-crystallin the ability of this stress protein to induce experimental allergic encephalomyelitis (EAE) was screened in Biozzi ABH (H-2A(g7)) mice. While whole alphaB-crystallin and the immunodominant T cell epitopes (49-64, 73-88, 153-168) failed to induce disease the subdominant or cryptic epitope (1-16) was weakly encephalitogenic. The lack of encephalitogenicity of whole protein and dominant epitopes may be due to the low constitutive expression of alphaB-crystallin in the CNS combined with a state of peripheral tolerance suggested by the constitutive expression of alphaB-crystallin in secondary lymphoid tissues in ABH mice. Further evidence for a role of alphaB-crystallin in the progression of chronic relapsing neurological disease is suggested by the development of T cell responses to alphaB-crystallin during MOG-induced relapsing EAE as myelin damage accumulates. Together our data indicate that normal tolerising mechanisms in ABH mice prevent the induction of EAE by alphaB-crystallin while the subdominant or cryptic epitope is able to circumvent these mechanisms and contribute to pathogenic myelin-directed autoimmunity following T cell activation.

The stress kit: a new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human alphaB-crystallin, Hsp27, and Hsp60.

We describe a reverse transcriptase-polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins alphaB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of beta-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of alphaB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.

Anti-alpha B-crystallin immunoreactivity in inflammatory nervous system diseases.

alpha B-Crystallin, a small heat shock protein, is an immunodominant antigen with increased tissue expression in demyelination. To investigate the humoral response against alpha B-crystallin, the sera and CSF samples of patients with multiple sclerosis (MS), Guillain-Barré syndrome (GBS), neuro-Behçet's disease (NBD) and other non-inflammatory neurological diseases (NIND) were screened by enzyme-linked immunosorbent assay for anti-alpha B-crystallin IgG and IgM antibodies. Serum and CSF IgG antibody responses to alpha B-crystallin were significantly elevated only in NBD patients (serum IgG, NBD 1.29 +/- 0.49 vs. NIND 0.95 +/- 0.39, P = 0.01; CSF IgG, NBD 1.22 +/- 0.64 vs. NIND 0.81 +/- 0.35, P = 0.01). Similarly, high serum IgM antibody titres were also detected in NBD (1.83 +/- 0.72 vs. 1.16 +/- 0.49, P = 0.0005) and in MS (1.57 +/- 1.07, P = 0.046), whereas elevated CSF IgM responses were observed only in GBS (2.09 +/- 1.09 vs. 1.41 +/- 0.7, P = 0.007). Humoral responses against alpha B-crystallin are increased in NBD and GBS, which may implicate this central nervous system antigen in the causation and pathogenesis of these inflammatory nervous system disorders.

Late rather than early responses of human dendritic cells highlight selective induction of cytokines, chemokines and growth factors by probiotic bacteria.

The probiotic properties of commensal bacteria including lactobacilli and bifidobacteria are likely to be determined at least in part by their effects on dendritic cells. Like traditional immune stimulants such as lipopolysaccharides (LPS), probiotic bacteria promote maturation of cultured human dendritic cells (DC) by inducing elevated expression of MHC-II and co-stimulatory molecules. Different effects have been reported on cytokine induction, especially of major regulatory cytokines such as TNF-α, IL-12 and IL-10. Yet, these previous analyses have failed to reveal consistent differences between such effects of probiotics on the one hand, and of LPS on the other. Selective response markers for probiotics, however, would be important for our understanding of their biological properties and for a rational selection of strains for in vivo studies. In this study, we compared in detail both early and late effects on cultured human DC of 4 different probiotics with those of LPS. At the early stages of stimulation, all stimuli induced qualitatively very similar responses in DC at the level of surface markers and secretion of cytokines and chemokines. A lower immune stimulatory effect was observed by Bifidobacterium animalis BB-12 as compared to lactobacilli. Late responses, on the other hand, tended to diverge. Microarray transcript profiling for 268 cytokines, chemokines, growth factors and their receptors after 2 days of culture revealed various transcripts to be selectively induced by certain probiotics but not LPS. Our data indicate that late rather than early DC responses may be helpful to clarify the divergent biological effects of probiotics on human innate immune responses.

Stress proteins in CNS inflammation.

Stress proteins or heat shock proteins (HSPs) are ubiquitous cellular components that have long been known to act as molecular chaperones. By assisting proper folding and transport of proteins, and by assisting in the degradation of aberrant proteins, they play key roles in cellular metabolism. The frequent accumulation of insoluble protein aggregates during chronic neurodegenerative disorders suggests failure of HSP functions to be a common denominator among such diseases. Recent developments have clarified that functions of HSPs extend well beyond their role in protein folding and degradation alone. Stress-inducible HSPs also regulate apoptosis, antigen presentation, inflammatory signalling pathways and, intriguingly, also serve as extracellular mediators of inflammation. Several receptors have been identified for extracellular HSPs, which control inflammatory pathways similar to those activated by cytokines and chemokines. In this review, both the traditional and the exciting novel functions of HSPs are discussed, with a focus on their relevance for neurodegeneration and neuroinflammation. Recent advances in this field suggest that HSPs represent attractive novel targets as well as therapeutic entities for CNS disorders.