**2.2 Adaptive immune system**

As innate immunity, adaptive immunity has a role in ALS (Sta et al., 2011). Unlike innate immune responses, the adaptive responses are highly specific and they consist of antibodies, lymphocytes activation and cell mediated response. The cells of the adaptive immune system are B and T lymphocytes: B cells, which are derived from the bone marrow, become the cells that produce antibodies. T cells can cross-talk with neurons and microglia, and either damage or protect neurons from stressful stimuli (Alexaniu et al., 2001), also in spinal cord and brain (Chiu et al., 2008).

T-helper cells have been observed in proximity of degenerating corticospinal tracts; T-helper and T-suppressor cells, with a variable number of macrophages, have been found in ventral horns of the spinal cord (Troost et al., 1990).

Infiltration of T cells compatible with adaptive response have been found in the areas of motor neuron destruction in the CNS but no correlation was found between clinical

The Role of TNF-Alpha in ALS: New Hypotheses for Future Therapeutic Approaches 417

and IFN-γ, (Harman et al., 1991; Hemnani et al., 1998). Furthermore, high cytokine levels have been described in plasma, serum and cerebral fluid (CSF) from ALS patients and sometimes correlate with the clinical status (Kelly et al., 1994; Lee et al., 2005; Khule et al.,

As concern inflammatory cytokines (IL-7, 9, 12, 17 and IL-1) levels were found higher in CSF of sporadic ALS patients (Tateishi et al., 2010). IL-15 and IL-12 serum levels, they also have been found higher in patients with ALS (Rentzos et al., 2010). The same authors measured IL-17 and IL-23 levels in serum and CSF from ALS patients that were found increased compared to controls (Rentzos et al., 2010). TGF-1 concentrations in the serum and CSF of ALS patients did not differ from controls, but TGF-1 serum concentration was significantly higher in ALS patients at the terminal clinical status (Ilzecka et al., 2002). Higher amount of IL-6 has been found in sera and CSF from sporadic ALS patients and it has been related to hypoxemia severity rather than pathological condition (Moreau et al.,

Plasma concentration of TNF-, TNF-R1 and TNF-R2 and their time course during disease progression were studied in ALS patients in order to assess the TNF- system implication in ALS pathogenesis. In all plasma patients soluble forms of the TNF- and its receptors are found increased already at disease onset and remain over the normal range during the disease progression time (Cereda et al., 2008). In addition TNF- amounts have been found higher in sera from sporadic ALS patients but no correlation was found with the clinical criteria (Poloni et al., 2000; Cereda et al., 2008). TNF- role in neurodegeneration will be

The Tumour Necrosis Factor Alpha (TNF-) is a pro-inflammatory cytokine produced by monocytes/macrophages and activated by mast cells, endothelial cells, fibroblasts, neurons and glial cells during acute inflammation and it is responsible for a wide range of cell signals about cell viability, gene expression, homeostasis control and synaptic

TNF- was described for the first time by Carswell et al. in 1975 as a protein component of serum of mice stimulated with bacterial antigens, and was brought to light the ability to induce death in cancer cell lines in vitro and in vivo to destroy transplanted sarcomas. Characteristically, this cytokine was able to cause tumor cells death without compromising the viability of healthy cells. The subsequent isolation and molecular characterization of the gene have provided information on the structure and functioning

The gene coding for TNF- is located on chromosome 6 within the region encoding the Major Histocompatibility Complex (MCH), HLA in human, between the HLA-DR class II and HLA-B class I genes (Fig. 3). Its location and strict linkage disequilibrium present between some alleles of class I and class II genes has permitted to hypothesize associations between TNF- alleles and some diseases. The gene for TNF- include about 3 Kb and contains four exons (almost 80% of the protein is codified by the exon four) and three

2009; Tateishi et al., 2010).

further highlight in the next paragraph.

**3. Tumor necrosis factor-alpha (TNF-)** 

2005).

integrity.

of this molecule.

**3.1 TNF- gene** 

introns.

parameters and infiltrating T cells (Holmoy et al., 2008). The majority of T cells characterized in the infiltration were CD8+ cytotoxic T cells, but a substantial number of T CD4+ cells were also present (Beers et al., 2008).

Alterations of total lymphocyte count (Provinciali et al., 1988; Tavolato et al., 1975) and T subset distribution in peripheral system of ALS patients (Westall et al., 1983) have been reported. Low T cells numbers and decreased proliferative capacity in T cells are found in the blood of ALS patients (Holmoy et al., 2008).

As concerned CD8+ and natural killer T cells, they were found increased in ALS patients compared to control cohort (Rentzos et al., 2011).

Interestingly, ALS patients showed a reduction of CD4+/CD25+ regulatory T cells that are known to interact with the local microglia, reinforcing the hypothesis of the involvement of the adaptive immune system associated with neuroinflammatory process in ALS (Mantovani et al., 2009). Beers and colleagues (Beers et al., 2011) observed that regulatory T cells as CD4+/CD25+/FoxP3 correlated with disease progression; in fact, the number of T cells were found inversely correlated with disease progression rate.

Animal studies showed that in ALS model T cells deficiency decreases microglia reactivity and accelerates ALS disease progression; specific and progressive accumulation of monocytes/macrophages was observed along the length of degenerating nerve fibers and activated microglia was detected in spinal cord of ALS model mice (Chiu et al., 2009).

No infiltrating B-cells have been found even if a role of B lymphocytes in the pathogenesis of ALS has been hypothesized, as secreted autoantibodies by B cells identified in CSF and serum from ALS patients (Naor et al., 2009).

As concern antibodies, since the eighties the presence of IgG in serum or tissues of ALS patients has been documented (anti-ganglioside GM1, anti-sulfoglucuronyl paragloboside, anti-neurofilaments and anti-Fas) (Sengun & Appel, 2003; Yi et al., 2000). Indeed, IgG deposits have been demonstrated in motor cortex, spinal cord and in motor neurons from ALS patients (Donnenfeld et al., 1984; Engelhardt & Appel, 1990; Fishman & Drachman, 1995). Serum immunoglobulins from ALS patients showed enhanced binding to rat spinal cord cells *in vitro* (Digby et al., 1985), demonstrating cytotoxic effects when they were added to a motor neuron cell cultures (Alexianu et al., 1994; Demestre et al., 2005) and that the presence of an immune response to spinal cord cell membrane components in patients with motor neuron disease was a damaging event.

IgG from ALS patients reacts with the skeletal muscle DHP (bisognerebbe spiegare cosa è) sensitive Ca2+ channels reducing the peak of the Ca2+ current and the charge movement in single cut fibres from the rat extensor muscle (Delbono et al., 1991). About 60% of ALS sera contained different monoclonal immunoglobulins: in particular IgG (72.7%) and IgM (27.3%) have been found (Duarte et al., 1991).

### **2.3 Cytokines**

Different interactions have been found between innate and adaptive immunity in ALS, as concern cytokines involvement. Cytokines have an effect on the expression of other inflammatory factors and on each other, and these functional relationships are non-linear: the causal relationships of cytokines and disease are complex and difficult to prove (Marklund et al., 1992).

Several studies regarding immune system changes in sporadic ALS reveal that there are increased levels of circulating monocytes and macrophages, producing cytokines as IL-1 and IFN-γ, (Harman et al., 1991; Hemnani et al., 1998). Furthermore, high cytokine levels have been described in plasma, serum and cerebral fluid (CSF) from ALS patients and sometimes correlate with the clinical status (Kelly et al., 1994; Lee et al., 2005; Khule et al., 2009; Tateishi et al., 2010).

As concern inflammatory cytokines (IL-7, 9, 12, 17 and IL-1) levels were found higher in CSF of sporadic ALS patients (Tateishi et al., 2010). IL-15 and IL-12 serum levels, they also have been found higher in patients with ALS (Rentzos et al., 2010). The same authors measured IL-17 and IL-23 levels in serum and CSF from ALS patients that were found increased compared to controls (Rentzos et al., 2010). TGF-1 concentrations in the serum and CSF of ALS patients did not differ from controls, but TGF-1 serum concentration was significantly higher in ALS patients at the terminal clinical status (Ilzecka et al., 2002). Higher amount of IL-6 has been found in sera and CSF from sporadic ALS patients and it has been related to hypoxemia severity rather than pathological condition (Moreau et al., 2005).

Plasma concentration of TNF-, TNF-R1 and TNF-R2 and their time course during disease progression were studied in ALS patients in order to assess the TNF- system implication in ALS pathogenesis. In all plasma patients soluble forms of the TNF- and its receptors are found increased already at disease onset and remain over the normal range during the disease progression time (Cereda et al., 2008). In addition TNF- amounts have been found higher in sera from sporadic ALS patients but no correlation was found with the clinical criteria (Poloni et al., 2000; Cereda et al., 2008). TNF- role in neurodegeneration will be further highlight in the next paragraph.
