A significantly elevated level of tear IL-4 detected by enzyme-linked immunosorbent assay (ELISA) has been reported in patients with SBS with ocular complications in our previous study . IL-4 has been shown to induce the production of extracellular matrix components by fibroblasts , and the major source of IL-4 is thought to be type 2 helper T (Th2) cells. However, it is still unclear whether such increased cytokine production is caused by increased numbers of cytokine-producing cells or enhanced ability of cytokine production. Analysis of cytokine production has been approached by measuring each cytokine by ELISA or by measuring protein or mRNA levels. Flow cytometric analysis of cytokine-producing cells has been reported in several disorders including ocular allergic disease [8, 10–11]. Therefore, we applied this method to detect intracellular cytokines in individual cells and determined the percentage of IL-4- or IFN-g-producing CD4 + T cells in the conjunctiva and peripheral blood from patients with SBS in comparison with ACD. We found that the percentage of IFN-g-producing CD4 + T cells in PBMC did not show a significant difference between patients with SBS and normal controls (Fig. 1); in contrast, the percentage of IL-4-producing CD4 + T cells in PBMC from patients with SBS was significantly higher than that in PBMC from normal controls (Fig. 2). These results indicate that the systemic cytokine production profile observed in CD4 + T cells of patients with SBS had different characteristics to that of normal controls and suggest that increased frequency of IL-4-producing CD4 + T cells may be one reason for increased type 2 cytokine production in patients with SBS. There have been few studies on the immunological features of SBS, whereas a previous study reported that eosinophil count in peripheral blood was a predictor of SBS . This might support the systemic immunological finding of elevated percentage of IL-4-producing CD4 + T cells in PBMC. However, further evaluation is necessary regarding the systemic allergological aspects of SBS.
In this study, the significantly higher percentage of conjunctival IL-4-producing CD4 + T cells in patients with SBS compared with normal controls (Fig. 4) was similar to the finding of our previous report that significant elevation of tear IL-4 was observed in patients with SBS compared to that in normal controls . The percentage of IFN-g-producing CD4 + T cells in the conjunctiva in SBS did not show a significant elevation compared with that in normal controls (Fig. 3). We reported the absence of a significant change in IFN-g level in tears of SBS patients compared with that in normal controls, and this seems to support our present results . The result that the nasal lavage fluid level of IFN-g was significantly lower and the IL-4/IFN-g ratio was significantly higher in allergic than in control children led to the hypothesis that deficient release of type 1 helper (Th1) cytokines, such as IFN-g, plays an important role in the pathogenesis of allergic inflammation . Regardless of whether defective IFN-g secretion is primary or a consequence of suppression by other cytokines, it would enhance the release of Th2 cytokines in allergic subjects, which in turn would facilitate the development of allergic inflammation, since SBS and AC, which are on the mild spectrum of ACD, showed a similar tendency regarding the percentage of IFN-g-producing CD4 + conjunctival T cells in this study (Fig. 3).
The significant elevation of the percentage of conjunctival IL-4-producing CD4 + T cells in SBS compared to normal controls (P < 0.05) may lead to the hypothesis that allergic reaction at least plays a partial role in the development of ocular disorder in SBS patients. However, the significantly lower percentage of conjunctival IL-4-producing CD4 + T cells in SBS compared to that in AKC or VKC might reflect the lower severity of allergic disorder or may suggest that SBS belongs to a different entity from ACD. The significantly lower percentage of conjunctival IFN-g-producing CD4 + T cells in patients with SBS than in those with AKC and VKC, with no significant elevation compared to normal controls (Fig. 3), showed similar results to IL-4-producing CD4 + T cells except for the comparison with controls (Fig. 4). However, the percentage of IFN-g-producing CD4 + T cells in PBMC showed significant differences among patients with ACD (Fig. 1), and this showed a significant opposite tendency to the frequency of IFN-g-producing CD4 + T cells in the conjunctiva among ACD; the reason for this discrepancy is unclear, but it might indicate that Th1 cells are only locally activated in the severe spectrum of ACD, such as AKC and VKC .
It has been revealed that the clinical severity of SBS correlated significantly with the percentage of IL-4-producing T cells in the conjunctiva, whereas the percentage of IFN-g-producing conjunctival T cells did not correlate with clinical score in this study. This seems to be consistent with the tendency towards a switch from a predominantly type 2 response in the cytokine pattern that is observed in allergic disorders ; however, there have been few reports on the levels of IL-4 and IFN-g with regard to the clinical ocular severity of ACD. Leonardi et al. reported that IL-4 tear levels were increased in VKC and AKC compared with controls, but only IFN-g significantly correlated with corneal involvement . They suggested that Th1 cells are locally activated and IFN-g has a role in the pro-inflammatory phase in the active phase of chronic allergic eye diseases, via IFN-g-secreting cells, such as conjunctival fibroblasts other than mononuclear cells [16–17]. As mentioned above, our present study carried out intracellular cytokine assays in CD4 + T cells in the conjunctiva and PBMC; therefore, this may explain the discordance between our results and those of Leonardi et al [15–17]. Thus, from the results that both type 1 and type 2 cytokines are produced in CD4 + T lymphocytes in SBS, with some difference in the percentage observed in the severe end of the spectrum of ACD, the possibility that IFN-g plays a role in the development of allergic disorders in SBS ocular lesions cannot be excluded, regardless of our results.
There are several limitations of this study as follows. Firstly, we have focused on the features of local cytokine production in SBS in comparison with ACD, and other biomarkers, such as chemokines, eosinophil cationic protein, matrix metalloproteinases, periostin etc., were not evaluated in this study. Multifactorial mechanisms should be evaluated in future studies. Secondly, this study was designed from the standpoint of ophthalmological aspects of SBS; therefore, the study population was small, and measurement of environmental parameters using calibrated instruments for each patient was not done because of the restriction of laboratory equipment and considerable diversity in the specific location of each patient. The causal elements might vary in each case, such as volatile organic compounds (VOC), odors, dust and bioaerosols. A VOC exposure study might be meaningful for direct clinical observation of ocular findings, but this type of experimental equipment is still restricted in Japan at present .