Impact of cytokines on the pathomechanism of diabetic and alcoholic neuropathies

Introduction

Diabetes and alcohol abuse are the most frequent causes of polyneuropathy in adults. These polyneuro-pathies are morphologically heterogeneous with a va-riable degree of lesion in axons or myelin. Their patho-genesis is complex and it is not clear why in some patients the lesion is limited to axonal degeneration only while in others it develops into demyelinating polyneuropathy [13,15,16]. The main problem of the latter concerns the possible impact of immunological factors on this type of myelin lesion [18,19]. To elucidate these questions we evaluated the expression of some cytokines, i.e. tumour necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1) and growth-regulated oncogene alpha (GRO-α), known also under the term cytokine-induced neutrophil chemoattractant-1 (CXCL1) in the blood serum of patients with diabetic or alcoholic polyneuropathy.
TNF-α is a pleiotrophic cytokine that plays a crucial role in immunological and defence reactions [11,21]. It has an impact on cell infiltration by promoting adhesion molecules and chemokine agents. MCP-1, as a strong monocyte chemoattractant factor, exerts an effect on differentiation of ThO lymphocytes into Th2 cells, by playing the role of a key factor in inflammatory processes [7]. GRO-α is another chemokine acting as a growth stimulator involved in inflammatory reactions and tumorigenesis [4,6,20].


Material and methods

The studied population of polyneuropathy consisted of 29 patients with type-2 diabetes mellitus and 31 with an alcohol abuse history. All study subjects presented with signs and symptoms of polyneuropathy. The diabetic polyneuropathy group consisted of 8 females and 21 males with a mean age of 48.3 years (range 20-70 years). In the alcoholic polyneuropathy group there were 8 females and 23 males with a mean age of 54.5 (range 28-60 years). In both types of polyneuropathy the typical motor and sensory disturbances in upper and lower extremities, with a predominance of the latter ones, were present. The type of polyneuropathy, i.e. the involvement of either axon or myelin, or both, was evaluated by means of electrophysiological methods (electromyo-graphy and velocity of nerve conduction). Cases of diabetic polyneuropathy were divided according to the clinical course and picture into two groups, one with a short duration (2-8 years) and another one with a longer than 9 years duration of the clinical signs of polyneuropathy. The subjects with alcoholic polyneuropathy were divided into two groups, one with a case history of less than 9 years and another one with a clinical course longer than 10 years. The control group consisted of 20 healthy subjects (mean age 49.1; range 23-78 years).
TNF-α, MCP-1 and GRO-α levels in blood serum were measured in duplicate with the ELISA immunoassay test, with the aid of Quantikine human TNF-α, MCP-1 and GRO-α kits (R&D System, USA). For statistical comparison of differences between the polyneuropathy groups and control subjects, the nonparametric Mann-Whitney U-test was used.

Results

The expression of TNF-α in serum of both types of polyneuropathy did not differ from that seen in the control material. The level of MCP-1 in serum was higher (though insignificantly) in patients with the demyelinating form of alcoholic and diabetic polyneuropathy than presented by control patients. The serum level of GRO-α was significantly higher in both patients with alcoholic polyneuropathy and patients of the subgroup presenting the demyelina-ting form of diabetic polyneuropathy than the levels seen in the sera of control subjects. Detailed results are presented in Tables I and II.


Discussion

The pathogenesis of diabetic polyneuropathy is
a complex one and involves multiple pathways. Clinically, it is heterogeneous within a range of abnormalities involving both the proximal and/or distal peripheral motor and sensory nerves as well as the autonomous nervous system. Sima [14], in his critical review on the metabolic and molecular basis for diabetic polyneuropathy, distinctly emphasized that besides hyperglycaemia, there are also deficiencies of insulin and of C-peptide that are responsible for the perturbation of neurotrophic factors and subsequently for the resulting polyneuro-pathy. The author stressed also the possible involvement of an apoptotic mechanism. These causative factors should play a decisive role in the development of microvascular insufficiency, neuro-degeneration and in the autoimmune-mediated reactions in nerve fibres [19]. Cameron et al. [2] emphasises the decisive role of vascular factors provoked by metabolic changes. The thesis was supported by results of studies made on biopsy material, which demonstrated structural changes in the nerve microvasculature, evident thickening of the basement membrane, degeneration of pericytes and hyperplasia of endothelial cells, all of them eventually leading to a reduction of perfusion and to endoneural hypoxia. Another, though not entirely controversial view was expressed by Haslbeck et al. [3], who presented evidence that activation of the RAGE (receptor for advanced glycation end product) pathway may at least be one of the first steps in the mechanism of diabetic polyneuropathy that might become involved even before chronic hyperglycaemia develops.
The pathogenesis of alcoholic polyneuropathy seems to be somewhat less complex than the diabetic one. It includes a direct toxic effect of alcohol abuse, indirect by metabolic and exotoxic changes in parenchymous organs, mainly in the liver, as well as malabsorption and maldigestion [5,9].
In both studied types of polyneuropathy, there is one question that still remains open: why in some cases with this disease there are only some axons that display typical signs of polyneuropathy, while other ones develop the demyelinating form of changes. The next question deals with the impact of immunological events on this process. Skundric and Lisak [17] underline the clinical aspects of the problem, emphasizing that lack of success in preventing polyneuropathy, even after normalisation of the former, impaired glucose metabolism. This observation suggests an involvement of immunological mediators, which once activated may act notwithstanding the favourable effect of hyperglycaemia treatment.
Some authors point to the role of TNF-α in the mechanism of diabetic polyneuropathy. Satoh et al. [11] claim that under conditions of chronic hyperglycaemia, the production or expression of TNF-α is increased in vascular and neural tissues. This in turn leads to increased microvascular permeability and hypercoagulability, resulting in the development of polyneuropathy. Another author [1] found in his material of patients with diabetes mellitus an elevated level of TNF-α. However, we were unable to confirm these findings. In our study material, the expression of TNF-α in blood serum of either type of polyneuropathy study did not differ from that found in control subjects.
The other cytokine investigated in our study, MCP-1, presented only minor changes, i.e. an insignificant increase in serum level in patients with alcoholic polyneuropathy. However, these results cannot suggest an impact of MCP-1 in the studied polyneuropathies.
Adverse results of GRO-α expression were found in our study. Surprisingly enough, the GRO-α levels were higher in the total group of alcoholic and in one subgroup of diabetic polyneuropathy with demyelinating changes evidenced in electrophysio-logical studies. In the literature, there are only a few studies available that deal with the role of GRO-α in primary and secondary inflammatory processes. GRO-, neutrophil-activating peptide-2 (NAP-2) and interleukin-8 are potent chemotactic agents for human neutrophils. Although these chemokines bind to similar, but not identical, receptors, the mechanism governing their signal transduction is very similar [4].
GRO-α, the growth-regulated oncogene acting in some neoplastic and inflammatory processes [6,8,10,20], promotes tumour growth, metastases and infiltration by leukocytes. A host CXC receptor-2 dependent pathway is involved in this mechanism. An interesting point concerning the biological activity of GRO-α was emphasised by Wang et al. [20]. The authors found in their experimental studies that proteinase-activated receptors 1 and 2 induced the release of GRO-α from rat astrocytes, and this action was capable of protecting nerve cells from apoptotic death.
The role of GRO-α in the studied polyneuropathies is by no means entirely clear. However, the presented results do imply that GRO-α a may contribute to unravelling the mechanism leading to alcoholic polyneuropathy and is at least partially responsible for the development of myelin lesion in diabetic polyneuropathy.


References

1. Abdel-Aziz MT, Fouad HH, Mohsen GA, Mansour M, Abdel Ghaffar S. TNF-alpha and homocysteine levels in type 1 diabetes mellitus. East Mediterr Health J 2001; 7: 679-688.
2. Cameron NE, Eaton SE, Cotter MA, Tesfaye S. Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropathy. Diabetologia 2001; 44: 1973-1988.
3. Haslbeck KM, Schleicher E, Bierhaus A, Nawroth P, Haslbeck M, Neundörfer B, Heuss D. The AGE/RAGE/NF-(kappa)B pathway may contribute to the pathogenesis of polyneuropathy in impaired glucose tolerance (IGT). Exp Clin Endocrinol Diabetes 2005; 113: 288-291.
4. l`Heureux GP, Bourgoin S, Jean N, McColl SR, Naccache PH. Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity. Blood 1995; 85: 522-531.
5. Kucera P, Balaz M, Varsik P, Kurca E. Pathogenesis of alcoholic neuropathy. Bratisl Lek Listy 2002; 103: 26-29.
6. Liu S, Wang X, Xu X. Expression of GRO-1 and its relationship with VEGF in squamous cell carcinoma of larynx. Lin Chuang Er Bi Yan Hou Ke Za Zhi 2006; 20: 541-544.
7. Losy J, Zaremba J. Monocyte chemoattractant protein-1 is increased in the cerebrospinal fluid of patients with ischemic stroke. Stroke 2001; 32: 2695-2696.
8. Losy J, Zaremba J, Skrobański P. CXCL1 (GRO-alpha) chemokine in acute ischaemic stroke patients. Folia Neuropathol 2005; 43: 97-102.
9. Neundorfer B. Alcohol polyneuropathy. Fortschr Neurol Psychiatr 2001; 69: 341-345.
10. Rudack C, Sachse F, Alberty J. Primary role of growth-related oncogene-alpha and granulocyte chemotactic protein-2 as neutrophil chemoattractants in chronic rhinosinusitis. Clin Exp Allergy 2006; 36: 748-759.
11. Satoh J, Yagihashi S, Toyota T. The possible role of tumor necrosis factor-alpha in diabetic polyneuropathy. Exp Diabesity Res 2003; 4: 65-71.
12. Sharma KR, Cross J, Farronay O, Ayyar DR, Shebert RT, Bradley WG. Demyelinating neuropathy in diabetes mellitus. Arch Neurol 2002; 59: 758-765.
13. Siemionow M, Demir Y. Diabetic neuropathy: pathogenesis and treatment. J Reconstr Microsurg 2004; 20: 241-252.
14. Sima AA. New insights into the metabolic and molecular basis for diabetic neuropathy. Cell Mol Life Sci 2003; 60: 2445-2464.
15. Sima AA. Pathological mechanisms involved in diabetic neuropathy: can we slow the process? Curr Opin Investig Drugs 2006; 7: 324-337.
16. Simmons Z, Feldman EL. Update on diabetic neuropathy. Curr Opin Neurol 2002; 15: 595-603.
17. Skundric DS, Lisak RP. Role of neuropoietic cytokines in development and progression of diabetic polyneuropathy: from glucose metabolism to neurodegeneration. Exp Diabesity Res 2003; 4: 303-312.
18. Valls-Canals J, Povedano M, Montero J, Pradas J. Diabetic polyneuropathy. Axonal or demyelinating? Electromyogr Clin Neurophysiol 2002; 42: 3-6.
19. Vinik AI. Advances in diabetes for the millennium: new treatments for diabetic neuropathies. Med Gen Med 2004; 6
(3 Suppl): 13.
20. Wang Y, Luo W, Reiser G. Proteinase-activated receptor-1 and proteinase-activated receptor-2 induce the release of chemokine GRO/CINC-1 from rat astrocytes via differential activation of JNK isoforms, evoking multiple protective pathways in brain. Biochem J 2007; 401: 65-78.
21. Zaremba J, Losy J. Early TNF-alpha levels correlate with ischaemic stroke severity. Acta Neurol Scand 2001; 104: 288-295.