In the present study, we measured inflammatory secreted and cytosolic cytokines levels in eAPS and adjuvant mice brains during a 24-week period. Secreted TNF-α
level was higher in eAPS mice compared to adjuvant mice for the whole period. Higher secreted TNF-α
levels in eAPS mice are in line with previous studies that found higher TNF-α
levels in eAPS mice and APS patients [15
]. On the other hand, cytosolic TNF-α
levels were somewhat lower in eAPS mice compared to adjuvant mice at 6 and 24 weeks and similar at 15 weeks after immunization.
Explanation for the lower cytosolic TNF-α
level in eAPS mice at 6 weeks after immunization might be a high secretion of TNF-α
during the pathological processes, resulting in emptying the cell reserves. As mentioned above, the cytosolic cytokine content of brain homogenate consists of a stored fraction and a secreted fraction. The first fraction is stored within immune and endothelial cells as a reserve and has less immediate effect on the immune response. The second fraction is secreted from the activated immune cells or the activated endothelial cells and participates in the modulation the immune response. We hypothesize the levels of secreted cytokines reflect, as closely as possible, the condition of cytokines levels in vivo
, since the tissue and the cells structures remain unbroken. We have previously measured TNF-α
level in mice whole brain homogenate. TNF-α
level was found to be significantly higher in eAPS mice compared to adjuvant mice [16
]. We hypothesize that the TNF-α
level measured in mice whole brain homogenate is similar to the secreted TNF-α
level rather than to the cytosolic TNF-α
level measured in mice cytosolic brain fraction. In comparison to the clean cytosolic fraction, whole brain homogenate contains other components which may mask the cytokine fraction stored within the immune and endothelial cells.
Other important cytokines in the development of the inflammatory process are IL-10 and IFN-γ
. Cytosolic and secreted IL-10 and IFN-γ
levels in eAPS mice were lower at 6 and 15 weeks and higher at 24 weeks after immunization compared to adjuvant mice. The low stable secreted and cytosolic IFN-γ
levels in the eAPS group is in agreement with previous study which showed a rise in IFN-γ
in eAPS mice after treatment with anti-idiotypic monoclonal antibody (MoAb) [17
is a proinflammatory cytokine and has a major role in the inflammatory process. Low secreted and cytosolic IFN-γ
levels in eAPS mice might be a result of upegulation of other cytokines which inhibit its production. Proinflammatory cytokines have an important role in regulation of haemostatic balance in both physiological and pathologic states. TNF-α
influences endothelial cells function by upregulating tissue factor (TF) levels [18
]. High levels of TF trigger endothelial cells to change their antithrombotic properties into procoagulant state. Beside its ability to induce procoagulant activity, TNF-α
also inhibits the thrombomodulin/protein C anticoagulation pathway and affects fibrinolysis by upregulating both urokinase-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) [20
contributes to the inflammatory process, IL-10 has an important role in modulating the inflammatory response and autoimmune disease. Impairment of the balance between the inflammatory process and the anti-inflammatory response may lead to disproportionate pathology or immunosuppression. Initially, IL-10 was considered as typical Th2 cytokine. It was identified as a product of activated Th2 cells. Recent studies have shown that IL-10 is also produced by Th1, Th0, regulatory T (Tr1) cells, and in mice also by activated macrophages and B cells [21
]. IL-10 downregulates the inflammatory response by blocking the production of a number of cytokines, including IL-2, IFN-γ
, and TNF-α
]. Therefore, reduced IL-10 levels in eAPS mice may down modulate the immunosuppressive effects, resulting in a proinflammatory process. In addition, low IL-10 levels enable TNF-α
unregulated production, resulting in procoagulant state. Moreover, during the B-cell activation, IL-10 delivers negative signals that promote the apoptosis of B cells [23
]. Decreased IL-10 levels can be associated with lymphocyte activation, which leads to the continuation of the autoimmune response. At 24 weeks, both total and secreted IL-10 levels in eAPS mice increased. This is compatible with an anti-inflammatory stage of disease occurring in conjunction with the drop in the levels of antibodies and TNF-α
. Explanation for the increased IL-10 levels might be their positive stimulation on B cells. IL-10 has a biphasic effect on B cells. On activated B cells, IL-10 promotes both their proliferation and differentiation into antibody secreting cells.
The importance of immune mediators in APS was demonstrated in previous studies. Several studies in murine model of APS showed that in pregnancy loss, some of the damage is caused by aPL-induced complement activation [24
]. Heparin, an anticoagulant has long been known to inhibit complement activity [26
]. Girardi et al. found that heparin, but neither fondaparinux nor hirudin, inhibited the generation of complement split products and protected mice from fetal loss caused by aPL antibodies [28
]. Some CNS manifestations of APS are caused by inflammation, cytokines or antibody-mediated tissue damage, and, therefore, antithrombotic therapy in APS is not sufficient.
The results suggest that course of the inflammatory process in eAPS depends upon the balance between proinflammatory and anti-inflammatory cytokines. Today, the accepted treatment for APS is antithrombotic therapy. Our results in line with previous cytokine studies may indicate a new therapeutic approach to APS. Drugs against proinflammatory cytokines may rebalance the cytokines levels and moderate the inflammatory response.