The aim of the present study was to investigate the role of IL-6, VEGF and TGF-β1 in the pathogenesis of SRMA.
VEGF concentrations were measured, since this parameter has been widely studied in human patients affected with Kawasaki Disease (KD), an acute febrile systemic vasculitis of children. SRMA has been proposed as an animal model for KD [30, 31]. In KD patients, concentrations of VEGF were found significantly elevated [16, 17, 32]. The histopathology of meningeal arteries of dogs euthanised during the acute stage of SRMA typically revealed endothelial and subendothelial oedema, hyaline degeneration and a mild to moderate periarteritis . Therefore, we hypothesized that elevated VEGF might cause these early vascular changes. Indeed, our results showed that VEGF was increased both systemically and intrathecally in SRMA patients during the clinical phases of the disease (‘SRMA’ and ‘SRMA R’). At these time points the mentioned vascular changes can be observed .
In other different inflammatory processes within the CNS (“ME” group), VEGF was found to be increased only in single cases, but overall the CSF VEGF concentrations in this group was significantly lower than in the SRMA group. We might conclude that VEGF does not play an important role in other meningoencephalomyelitides. However, due to the heterogeneity of the diseases included in the ME group (e.g. granulomatous meningoencephalomyelitis, necrotising encephalitides and meningoencephalomyelitis of unknown origin) we can not exclude from this study a role for VEGF in some of these diseases.
The values of VEGF in cases with systemic inflammation were lower than in dogs with SRMA (median 3.9 pg/mL and 43.92 pg/mL, respectively). However, the difference was not statistically significant. This is in accordance with a recent study in which VEGF values were elevated in 64% of dogs with systemic inflammatory response syndrome. However, a correlation with clinical signs or increased permeability could not be proved .
VEGF is involved in the proposed pathogenesis of vasculitis in KD. After the permeability of the vessels is increased under to the influence of VEGF, platelets might adhere to the vascular wall and inflammatory cells cross the loose endothelium, accumulating in the intima and becoming a source of proinflammatory cytokines. As final consequence there is a thickening of the intima as found in both diseases, KD and SRMA. If coronary artery lesions occur, this might even lead to life-threatening complications . In KD patients VEGF was correlated with the development of coronary artery lesions .
Interestingly in our study VEGF was not only found increased in the acute stage of SRMA, but also during relapses of SRMA (median in CSF 20.80 pg/mL; median in serum 33.71 pg/mL). Therefore, VEGF in SRMA may enhance vascular wall destruction in the acute phase of the disease before immune complexes appear . However, further in vitro studies evaluating directly the effect of VEGF on canine vasculature (e.g. using endothelial cells culture or cultured vessels) are needed to confirm the role of this protein in the pathogenesis of canine arteritis.
Since VEGF concentrations were increased during relapses, consequently, VEGF may be also involved in the development of the arterial lesions found during the chronic phase, such as increased wall thickness, stenosis and fibrosis . On the other hand, VEGF might indicate simply vascular damage. A limitation of this study is the lack of comparison to other pure vasculitides. Experimental in vivo studies are probably necessary to elucidate the long-term effect of VEGF on canine vessels. Experimental studies on SRMA dogs are feasible, due to the natural occurrence and the favourable prognosis of the disease.
The recruitment and activation of different lymphocytes subsets after alteration of the CNS tissue by an environmental factor are caused by multiple mechanisms . These include chemotactic agents , probably additional mechanisms such as changes of the blood–brain barrier [36, 37], and altered expression of selectins and integrins [38, 39]. To add more information to these previous studies and to investigate the hypothesis that IL-6 and TGF-β1 are correlated to and eventually involved in the pathogenesis of fever, pleocytosis and increased IgA production in SRMA, these proteins were determined in CSF and serum samples. Previous studies on cytokine expression in SRMA patients, showed an up-regulation of IL-4 and IL-8, while IL-2 and IFN-γ were found in low concentrations [12, 35]. Hogenesch et al.  investigated IL-6 in serum of dogs with juvenile polyarteritis syndrome and detected increased IL-6 serum values. In preliminary studies, measurement of IL-6 in CSF was considered to be a valuable biomarker for the diagnosis of SRMA . Qualitative studies containing information about the bioactivity of IL-6 in CSF were missing.
In the current study IL-6 values were increased intrathecally and systemically in SRMA patients, the highest concentrations were found in CSF samples (median 1582 pg/mL in ‘SRMA’ and median 637.7 pg/mL in the ‘SRMA R’ groups). In case of other inflammatory diseases of the CNS (‘ME’ group) the concentrations of CSF IL-6 were significantly lower (median 3 pg/mL), leading to the conclusion that IL-6 is an important biomarker for disease activity in SRMA. The exact role of IL-6 in the pathogenesis of the disease could be investigated in experimental studies. Further, IL-6 in SRMA strongly correlated with the degree of pleocytosis. This fact might also suggests, that IL-6 values might be the result of the severe pleocytosis because of its production by macrophages . Lowrie et al.  also detected elevated IL-6 CSF values in samples of dogs with a putative relapse and a normal CSF cell count, making the latter hypothesis less likely, nevertheless further studies should be addressed to clarify causes and consequences.
IL-6 has long-range effects, indeed it is one of the most important endogenous pyrogens, induces hepatocytes to synthesize acute-phase proteins, stimulates neutrophil mobilization from bone marrow and stimulates terminal differentiation of B cells to secret immunoglobulins [23, 42, 43]. Therefore it is very likely that an overproduction of IL-6 is a major mediator of the most peculiar findings, such as fever, increased acute-phase proteins, CSF neutrophilic pleocytosis and peripheral leukocytosis as well as increased IgA production during the acute phase of SRMA. The extreme high values of IL-6 in CSF also during relapses suggest that IL-6 exerts its major functions intrathecally and throughout the waxing and waning course of the disease. As previously mentioned, the cell population in CSF of dogs during the acute phase of SRMA is predominantly composed of neutrophils, during the chronic form macrophages tend to prevail. Upregulation of CD11a on neutrophils , increased IL-8 levels in CSF  and the destruction of the blood–brain barrier  have all been shown to be involved in neutrophil migration into CSF. Factors involved in the accumulation of monocytes in CSF of SRMA patients during the protracted form have not been investigated. Interestingly, IL-6 has been recently proposed to be a regulator of the transition from a neutrophil-dominated to a macrophage-dominated process . We therefore propose a leading role for IL-6 in both the acute and protracted forms of the disease.
As expected, serum IL-6 concentrations were similar to the group of systemic inflammatory diseases supporting other studies, where IL-6 has been used not only as a general marker of inflammation , but in particular as a prognostic factor in canine systemic inflammatory response syndrome and sepsis . Also in KD IL-6 is increased in serum, but contrary to VEGF, the increase was not correlated with the development of coronary artery aneurysms and dilatation [46, 47].
In SRMA dogs both VEGF and IL-6 were much higher in CSF compared to serum values. This might reflect a more severe inflammatory process affecting meninges and meningeal vessels compared to peripheral vessels, or a main intrathecal production of these signalling proteins, followed by a secondary leakage into the systemic circulation. Further studies including protein associated gene expression and immunohistochemistry of meningeal and peripheral vessels might be necessary to elucidate the site of production.
TGF-β1 in SRMA patients was decreased in serum (median 13.57 ng/mL) and elevated in CSF (median 90 pg/mL). The increased concentration in CSF was not specific for SRMA, indeed similar values have been found in other meningoencephalomyelitides (median 58 pg/mL), while the reduced concentration in serum statistically differed from the other groups.
The serum concentrations of TGF-β1 were found to be decreased also in patients with KD , but to the authors knowledge data concerning concentrations of TGF-β1 in CSF of patients with KD are lacking. The hypothesis that TGF-β1 might be the most important pathogenetic factor for the excessive IgA production in SRMA could be partially rejected in the current study. Our results support the suggestion that TGF-β1 has a minor role in systemic production of IgA, whereas it is highly likely that it might still play a certain role in the intrathecal production of IgA. Indeed, TGF-β1 was positively correlated with IgA concentrations in CSF (rSpear = 0.3549; P = 0.0337). However, CSF IgA concentrations remain high during teatment [5, 8, 10] and concentration of TGF-β1 decline. Therefore, this rather unspecific elevation of TGF-β1 values in CSF samples supports a more immunoregulatory function of this cytokine in inflammatory CNS diseases [49, 50]. Further experimental in vivo and in vitro studies are needed to confirm this hypothesis.
Our findings indicate that the CSF cytokine profile of SRMA dogs during the acute phase is characterized by increased values of IL-6 and TGF-β1. Recent progress in immunology led to the discovery of Th17 cells, a new subset of T helper cells [51, 52]. According to one study, the combined influence of both IL-6 and TGF-ß1 is necessary for the Th17 lineage to develop, while TGF-β1 alone shifts the development of naïve T-cells towards T regulatory cells, a T-cell subset that restrains inflammation and maintains tolerance .
The detected combined high intrathecal production of TGF-β1 and IL-6 in SRMA could possibly lead to an increase of Th17 lymphocyte subset and subsequently enhance the development of an autoimmune response. IL-17, the main product of this lymphocyte subset, plays an active role in inflammatory response and in autoimmune diseases  and experimental studies displayed a neutrophil-mobilizing mechanism of IL-17A . The massive invasion of neutrophils into the subarachnoidal space in SRMA dogs might be the results of a Th17 immune response. Further studies to prove direct evidence of Th17 cells and its products in SRMA patients have to be conducted.
To date, SRMA has been believed to be a Th2-mediated immune disorder , our results suggest indirectly that in SRMA a Th17 skewed immune response might play a major role, particularly in the development of the meningitis.