Cytokines in sural nerve biopsies from inflammatory and non-inflammatory neuropathies

Proinflammatory cytokines are supposed to play a major role in the pathophysiology of vasculitis and in the development of neuropathic pain. Here we studied the cytokine expression in sural nerve biopsy specimens from patients with vasculitic and other inflammatory and non-inflammatory neuropathies, and investigated whether an increased cytokine expression was correlated with the presence of neuropathic pain. We used immunohistochemistry including double labeling and morphometry to localize and quantify the expression of interleukin-1 beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF) in sural nerve biopsy samples of 41 patients with vasculitic neuropathy (VANP), chronic inflammatory demyelinating neuropathy (CIDP), non-inflammatory chronic axonal neuropathy (CANP), and 3 controls. Overall cytokine immunoreactivity was highest in VANP, less strong in CIDP and lowest in CANP. Cytokine immunoreactivity was directly correlated with the degree of axonal degeneration, endoneurial macrophages and epineurial T cells. In VANP and CANP, a higher cytokine content was associated with neuropathic pain.     

Chronic inflammatory demyelinating polyneuropathy: decreased claudin-5 and relocated ZO-1

OBJECTIVES: To clarify the dynamics of molecules composing the blood-nerve barrier (BNB) in inflammatory neuropathies. METHODS: The expression of four tight junction (TJ) proteins-claudin-1, claudin-5, occludin, and ZO-1-was analysed immunohistochemically in sural nerve biopsy specimens obtained from patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). RESULTS: Claudin-1 was detected only in perineurial cells, whereas claudin-5 was present in endothelial cells, irrespective of vessel location or size. Occludin and ZO-1 were found in perineurial cells, in addition to some epineurial and endoneurial endothelial cells. In CIDP, percentages of endoneurial small vessels immunoreactive for claudin-5 were significantly decreased, as were ZO-1 immunoreactive endoneurial small vessels, with staining localised to interfaces between cells. Claudin-1 and occludin immunoreactivity did not differ appreciably between the neuropathies examined. CONCLUSIONS: The downregulation of claudin-5 and altered localisation of ZO-1 seen in CIDP specimens may indicate that BNB derangement occurs in inflammatory neuropathies. Further investigation of TJ molecules may suggest new treatments based on properties of the BNB.     

<Emphasis Type="Italic">N</Emphasis><Superscript>ε</Superscript>-Carboxymethyllysine in diabetic and non-diabetic polyneuropathies

Increased oxidative stress and advanced glycosylation are important factors in the development of diabetic neuropathy. In non-diabetic neuropathies their influence has not been investigated in detail so far. We studied the localisation of N(epsilon)-carboxymethyllysine (CML) - a biomarker for oxidative stress - by immunohistochemistry in sural nerve biopsies of 31 patients with different polyneuropathies [diabetic polyneuropathy (n=5), alcohol-associated polyneuropathy (n=4), vitamin B12-deficient polyneuropathy (n=6), chronic inflammatory demyelinating polyneuropathy (CIDP) (n=6), vasculitic neuropathy (n=6), Charcot-Marie-Tooth disease type I (CMT I) (n=4)] and 4 normal controls. CML was detected in the perineurium of patients with diabetic, alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. Epineurial, perineurial and endoneurial vessels were CML positive in diabetic, vitamin B12-deficient and vasculitic polyneuropathies. CML was also found in mononuclear inflammatory cells in vasculitic neuropathy. In CIDP and normal controls there was only marginal perineurial CML deposition in 2/6 and 1/4 cases. In CMT I no CML was detected. Immunohistochemical results were confirmed by immunoblot. Our data suggest a role of oxidative stress in the pathogenesis not only of diabetic but also of alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathies. It may be a minor pathogenetic factor in CIDP and may not be involved in CMT I. Underlying causes for increased oxidative stress may be an elevated production of reactive oxygen species and an impairment of antioxidative defences. Therefore, an antioxidative treatment should be considered in alcohol-associated, vitamin B12-deficient and vasculitic polyneuropathy.     

Pattern of cytokine secretion by peripheral blood cells of patients with multiple sclerosis in Brazil

Autoimmune T cells play a key role as regulators and effectors of organ-specific autoimmune disease. In multiple sclerosis (MS), activated T cells specific for myelin components produce a plethora of inflammatory cytokines and mediators that contribute to myelin damage. The production of proinflammatory and regulatory cytokines by peripheral blood cells from patients with active and stable MS and healthy controls were examined. The results show that TNF alpha production was somewhat elevated in active MS with no significant increase in the level IFN gamma, whereas in the chronic phase the anti-inflammatory cytokines IL-10 and TGF beta increased, accompanied by a reduction in IFN gamma when stimulated by myelin basic protein. Multiple Sclerosis (2000) 6 293 - 299     

Interferon-gamma but not TNF alpha promotes neuronal differentiation and neurite outgrowth of murine adult neural stem cells

Neural trauma, such as traumatic brain injury or stroke, results in a vigorous inflammatory response at and near the site of injury, with cytokine production by endogenous glial cells and invading immune cells. Little is known of the effect that these cytokines have on neural stem cell function. Here we examine the effects of two inflammatory cytokines, interferon-gamma (IFN gamma) and tumour necrosis factor-alpha (TNFalpha), on adult neural stem cells. Neural stem cells grown in the presence of either cytokine failed to generate neurospheres. Cytotoxicity assays showed that TNF alpha but not IFN gamma was toxic to the neural stem cells under proliferative conditions. Under differentiating conditions, neither cytokine was toxic; however, IFN gamma enhanced neuronal differentiation, rapidly increasing beta III-tubulin positive cell numbers 3-4 fold and inhibiting astrocyte generation. Furthermore, neurite outgrowth and the number of neurites per neuron was enhanced in cells differentiated in the presence of IFN gamma. Therefore, both inflammatory cytokines examined have substantial, but different effects on neural stem cell function and suggests that regulation of the inflammatory environment following brain injury may influence the ability of neural stem cells to repair the damage.     

Restless Legs Syndrome In Peripheral Neuropathy

The role of extracellular matrix (ECM) in the development and maintenance of the normal nervous tissue structure has been demonstrated. However, little is known about ECM modifications taking place in peripheral nerve pathology. This gap has prompted us to study the expression of several components of the peripheral nerve ECM in the major categories of human peripheral neuropathies. Normal and pathological sural nerve biopsies, including cases of CMT1, HNPP, CIDP and axonal neuropathies were studied by immunohistochemistry for the expression of collagen (COL) types I, III, IV, V, VI, laminin (LN), tenascin (TN), fibronectin (FN) and vitronectin (VN). The immunoreactivity of ECM proteins in pathologic nerves showed different patterns in relation to their distribution in normal nerves. Indeed, COL I and III, which are the main stromal components of the epineurium and endoneurium, respectively, showed increased expression in these compartments, generally in chronic neuropathies. COL IV, V, VI and LN localized mainly to basement membranes (BM) of Schwann cells, perineurial cells and blood vessels, showing increased deposition in the presence of BM redundancy. FN localized diffusely to the endoneurium and perineurium in control nerves and its expression increased in CIDP nerves. VN immunoreactivity was limited to several spots along the perineurium and epineurial vessels in normal nerve. However, in CIDP and in some cases of axonal neuropathy the VN signal was increased in the perineurium and appeared also in endoneurial vessels. Finally, TN was detected in the perineurium and the nodal/paranodal region in control nerve, while in some cases of CMT1 and CIDP it appeared to mark several internodes. These data, showing specific changes of the ECM components in peripheral neuropathies, suggest that a complex ECM remodeling is associated with mechanisms of nerve damage and repair. Supported by MURST-COFIN 98 (n°86) and by CNR 98 (n°03081 CT04).     

Isolation and characterisation of T lymphocytes from sural nerve biopsies in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.

OBJECTIVES: To characterise cultured T lymphocytes from nerve biopsies in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy (CIDP). METHODS: Sural nerve biopsies, obtained from six patients with Guillain-Barré syndrome, four with CIDP, and six controls with other neuropathies, were cultured with 20 U/ml recombinant interleukin-2 (IL-2) for eight weeks. Flow cytometry was used to determine the phenotype of cultured T lymphocytes. Their proliferative responses to a range of bacterial antigens were also examined. RESULTS: T cell lines were established from four of six patients with Guillain-Barré syndrome, one of four with CIDP, one patient with peripheral nerve vasculitis, and none of five controls with non-inflammatory neuropathies. One of these T cell lines from a patient with Guillain-Barré syndrome, preceded by Campylobacter jejuni infection, consisted entirely of gamma delta TCR+ T lymphocytes. The peripheral blood of this patient also contained an increased frequency of gamma delta T cells when stimulated with C jejuni. The nerve derived T cell lines failed to show a proliferative response to bacterial antigens or to a preparation of myelin proteins. CONCLUSIONS: A new technique to isolate T cells from nerve biopsies in patients with Guillain-Barré syndrome and CIDP is reported. This technique may prove to be a useful tool in the investigation of the pathogenesis of other inflammatory neuropathies such as peripheral nerve vasculitis. The isolation of a gamma delta TCR+ nerve T cell line is of interest because of the possibility that these cells might respond to glycolipid epitopes common to C jejuni and peripheral nerve gangliosides.     

Nerve biopsy in chronic inflammatory neuropathies: in situ biomarkers

We collected the evidence for potential biomarkers in nerve biopsies that might be of use in diagnosis, assessment, or treatment response in chronic inflammatory demyelinating polyneuropathies (CIDPs). We performed a literature search in PubMed from 1965 to May 2010 using the key words (["chronic inflammatory polyneuropathy" or "polyradiculoneuritis" or {"chronic and neuritis"}] and "nerve biopsy") and searched manually within these references for relevant publications related to the subject. Twenty references gave information about potential biomarkers for CIDP. Evidence of demyelination alone is not specific for CIDP, but may support the diagnosis in the context of a typical clinical pattern. Although the total numbers of inflammatory cells do not distinguish well between CIDP and non-inflammatory neuropathies, the pattern of macrophage clusters around endoneurial vessels may be a simple marker of inflammation with good sensitivity and specificity. Immunohistochemistry for matrix metalloproteinase-9 may be useful for the distinction of inflammatory and non-inflammatory neuropathies. Microarrays which give a complex pattern of up- and downregulated genes also show promise for developing a biomarker. Immunohistochemistry on sural nerve biopsies has the potential to distinguish inflammatory from non-inflammatory neuropathies. More research is needed to establish the diagnostic validity of specific markers and of gene expression studies and to test whether they can distinguish between subtypes of inflammatory neuropathies.     

Plasma proteins in human peripheral nerve.

Using the immunofluorescent technique, the distribution of nine plasma proteins, differing in molecular weight, was studied in sural nerve biopsies from 25 neurological patients. The results of this study show that endoneurial vessels are normally permeable to all plasma proteins, though not to an equal degree. Permeability of the nerve blood vessels may be increased in pathological conditions. Proteins of molecular weight equal to or greater than 340,000 tend to accumulate at the site of the perineurium, indicating a slow removal from this structure. A marked accumulation of plasma proteins at the site of the perineurium was observed in hypertrophic neuropathy.     

Immunoelectronmicroscopical demonstration of major histocompatibility class II antigen: expression on endothelial and perivascular cells but not Schwann cells in human neuropathy

A pre-embedding technique for identifying major histocompatibility (MHC) class II antigen with monoclonal antibody LN3 at the electron microscope level by immunogold silver enhancement was applied to sections from 12 human nerve biopsies. Many perivascular mononuclear cells in the epineurium, perineurium and endoneurium expressed MHC class II antigen and had the morphological appearance of macrophages. Cell processes expressing MHC class II antigen extended throughout the endoneurium, often close to Schwann cells. No MHC class II expression was identified on myelinating or non-myelinating Schwann cells. The endothelial cells of epineurial blood vessels expressed MHC class II antigen on their luminal surfaces more often and more strongly than those of the endoneurial vessels. These observations indicate that perivascular cells in both the endoneurium and perineurium commonly express the molecules necessary to present antigen to CD4⁺ T lymphocytes, but Schwann cells do not.     

T-cell cytotoxicity of human Schwann cells: TNFalpha promotes fasL-mediated apoptosis and IFN gamma perforin-mediated lysis

The ability of resident cells to induce apoptosis of invading immune cells is a major regulatory factor operating in immune-privileged tissues, including the nervous system. We investigated the cellular and molecular factors participating in modulation of immune response in peripheral nerves, focusing on two cytotoxic pathways: fas ligand (fasL) and perforin. fasL and perforin expression was found by immunochemistry on Schwann cells (Sc) in nerve biopsies from patients with chronic inflammatory demyelinating polyneuritis and on human Sc cultures. Treatment of Sc with tumor necrosis factor (TNF) alpha and interferon (IFN) gamma upregulated the expression of both molecules. In a coculture model, Sc exposed to TNFalpha or IFN gamma were able to induce both apoptotic and lytic injury of T-lymphocytes. Inactivation of fasL with the neutralizing antibody NOK-2 abolished T-cell apoptosis induced by Sc treated with TNFalpha, but not by Sc treated with IFN gamma. Conversely, T-cell lysis was significantly decreased when IFN gamma-activated Sc were treated with concanamycin A, which inhibited perforin release. At variance with T-lymphocytes, B-cells were less sensitive to cytokine-treated Sc toxicity. Thus, Sc exposed to inflammatory cytokines have the capacity of inducing selective damage of T-lymphocytes and have the potential of regulating the immune response in the peripheral nervous system.     

Inflammatory infiltrates in sural nerve biopsies in Guillain-Barré syndrome and chronic inflammatory demyelinating neuropathy

Prompted by observations in experimental autoimmune neuritis we reanalyzed immunohistochemically the inflammatory infiltrates in sural nerve biopsies of 22 cases with Guillain-Barré syndrome (GBS) and 13 cases with chronic inflammatory demyelinating polyneuropathy (CIDP). Endoneurial infiltration of CD3+ T cells was found in 20 cases of GBS (median 5.5 cells/mm²) and in 10 cases of CIDP (5 cells). Epineurial T cells were present in all GBS cases (19.5 cells) and in 11 CIDP cases (21 cells). CD68+ macrophages were abundant in these neuropathies and often occurred in endoneurial perivascular clusters. In GBS subgroups the number of endoneurial T cells was significantly higher in patients with hypoesthesia and abnormal electrophysiological findings in the sural nerve. In CIDP hypoesthesia was associated with significantly higher numbers of macrophages. Our study also indicates that other factors including the time point of biopsy or previous corticosteroid treatment may influence the inflammatory cell profile. Quantifying cell infiltration may aid in establishing the diagnosis of an immunoneuropathy in patients with mild and noncharacteristic pathology. © 1996 John Wiley & Sons, Inc.     

Enhanced B7 costimulatory molecule expression in inflammatory human sural nerve biopsies

OBJECTIVES: To define the role of the costimulatory molecules B7-1 and B7-2 in inflammatory disorders of the peripheral nervous system. B7 molecules are essential for effective antigen presentation and may determine the differentiation of T cells into a Th-1 or Th-2 phenotype, thus modulating immune response and disease course. METHODS: Forty nine sural nerve biopsies from patients with neuroborreliosis, Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), CIDP variants and hereditary neuropathies, and those with no detectable abnormality were investigated. The expression of B7-1 and B7-2 mRNA and protein was investigated by polymerase chain reaction (PCR) and immunocytochemistry. RESULTS: B7-1 mRNA was strongly upregulated in both cases of neuroborreliosis, in two cases of GBS and one case of variant CIDP. Moderate to low levels were detected in the remaining GBS and CIDP biopsies and were rarely found in a non-inflammatory control group consisting of hereditary neuropathy and normal nerves. At the immunocytochemical level, strong expression of B7-1 protein was found in both neuroborreliosis cases, and moderate or low expression in six of eight GBS cases and seven of 17 CIDP cases investigated, whereas only one of five non-inflammatory control nerves showed staining, which was very weak. In neuroborreliosis, B7-1 protein was found very pronounced in epineurial infiltrates, whereas in GBS and CIDP, labelling was predominantly endoneurial and localised to putative macrophages. B7-2 mRNA and protein were expressed only at low levels in neuroborreliosis and selected autoimmune neuropathy cases, and were essentially absent from non-inflammatory controls. CONCLUSIONS: B7 molecules are expressed in the peripheral nervous system and regulated during disease, and their presence in macrophages underlines the putative function of endoneurial macrophages as local antigen presenting cells in the immunopathology of peripheral nerve. B7-1 rather than B7-2 is preferentially upregulated, possibly promoting the induction of a Th-1-type T cell response within the nerve.     

Parallel expression of neurotrophic factors and their receptors in chronic inflammatory demyelinating polyneuropathy

The mRNA levels of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and interleukin-6 (IL-6) were examined in sural nerves of 22 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). The mRNAs for NGF, GDNF, LIF, and IL-6 were upregulated, whereas CNTF mRNA was downregulated significantly in the nerves. The NGF, GDNF, and CNTF, but not LIF mRNA expressions were parallel to those of the cognate receptors, suggesting that these cognate soluble receptors effectively present these factors to maintain and regenerate the axons. Furthermore, IL-6 mRNA expression was significantly parallel to both binding and signal-transducing receptor expression, implying a role of the IL-6 signal for non-neuronal cells in CIDP. These findings indicate that multiple neurotrophic growth factors and cytokines are expressed cooperatively with their concomitant receptors in the nerve lesions of CIDP and play an important role particularly in nerve repair.     

THE BLOOD-NERVE BARRIER AND RECONSTITUTION OF THE PERINEURIUM FOLLOWING NERVE GRAFTING

Sural nerve autografts were performed on intact rat sural nerves and on sural nerves excised proximal to the site of grafting. The effect of the presence or absence of regenerating axons upon reconstitution of the perineurium at the graft junctions and upon re-establishment of the blood-nerve barrier to horseradish peroxidase (HRP) were studied over the succeeding 3--24 weeks. Compartmentation of the nerve fascicle occurred at the graft junctions where the perineurium was damaged. Each compartment contained Schwann cells with or without axons and was surrounded by elongated fibroblast-like cells which resembled perineurial cells in the longer surviving animals. It was concluded that, (a) compartments form in a nerve at the site of perineurial damage even in the absence of axons; (b) although compartmentation may be a mechanism for perineurial regeneration and reconstitution of the blood-nerve barrier, blood vessels and the cell layers forming compartments at graft junctions remain permeable to HRP for at least 6 months; and (c) the intact perineurium around the distal stump of a denervated nerve is permeable to HRP but the endoneurial blood vessels are not.