Based on our observations, it can be concluded that ASCs were involved in the acute stage of KD. In this stage, we found a significantly increased percentage of ASCs. ASCs could be anti-inflammatory via the secretion of immunoglobulins, alternatively through the production of IL-10 . So next, we investigated the correlations between the percentage of ASCs and laboratory findings including the levels of serum immunoglobulins and the inflammatory indicators. The percentage of ASCs positively correlated with the level of serum IgM, but not IgG or IgA, indicating that the ASCs played their roles in the acute stage of KD predominantly through secreting IgM. However, individuals with a higher percentage of ASCs did not show relatively lower levels of inflammatory indicators, suggesting that their anti-inflammatory role in the acute stage of KD was likely to be less satisfactory. Hence, it might be reasonable to speculate that these increased ASCs in the acute stage of KD developed from extrafollicular B cells [19, 30]. The increased percentage of ASCs may be as a result of the elevation of stimulative factors. One of the advantages for the differentiation of B cells into ASCs may be the elevation of B-cell-activating factor (BAFF), which can effectively promote the proliferation, differentiation, and survival of B cells . Another superiority for ASCs is the increased levels of cytokines, such as IL-6, IL-17 and IL-21 [13, 32, 33]. Furthermore, increased expression of CD138 is capable of promoting the maturation, accumulation and particularly, survival of ASCs upon IL-6 signaling . Therefore, elevated BAFF and CD138 in concert with those increased cytokines can directly enhance the differentiation of B cells into ASCs. Meanwhile, the expression of IgG in the cytoplasm was enhanced. In contrast to our results, Shingadia et al. have reported a decreased absolute number of cytoplastic IgG+ plasma cells . The precise reason causing the contrary results is unclear. Perhaps, it is due to the difference of the definition of the ASCs. Recent research found that CD19 negative ASCs would emerge in circulation at the early stage plasmablasts to plasma cell transition . Hence, the category of ASCs based on CD19 expression might leave out a part of B cell capable of producing antibodies. The enhanced expression of cytoplasmic IgG positive plasmablasts was demonstrated in ulcerative colitis likewise . Their results also found positive correlations between the level of IgG+ plasmablasts and indicators of disease activity, thereby suggesting this subset could be pro-inflammatory in the pathogenesis of ulcerative colitis. In KD, the overall levels of both cytoplasmic IgG and inflammatory indicators were increased, apparently insisting the pro-inflammatory role of IgG+ ASCs. However, our data did not find any significant positive correlations between IgG+ ASCs and inflammatory indicators including CRP, ESR. Additionally, although the percentage of IgG+ ASCs in the acute stage was significantly increased, it was not correlated with the level of serum IgG, and the median level of serum IgG in KD patients was equivalent to that in HCs, suggesting the increased cytoplasmic IgG was not completely excreted. Indeed, it was reported that cytoplasmic IgG was likely to be beneficial for the elimination of kinds of intracellular virus, which were indicated as pathogenic candidate agents in KD, possibly via intracellular antibody-mediated degradation [37,38,39]. Thus, we hypothesize that the roles of cytoplasmic IgG in KD are heterogeneous and may be related to the kind of invading pathogen.
The precise mechanism of IVIG in the treatment of KD remains unknown. Potential mechanisms of action include the neutralization of toxin, modulation of the activity of monocyte/macrophage and neutrophils, provision of anti-idiotypic IgG, regulation of T cell differentiation and cytokine release . To date, the study regarding the action of IVIG on B cells in KD was limited. A previous study found a significant decrease in B cells after IVIG treatment, suggesting that IVIG could restore B-cell abnormalities . In the present study, we found the percentage of IgG+ ASCs, which was increased in the acute stage, was significantly reduced after IVIG administration. The results provided strong evidence demonstrating the involvement of IgG+ ASCs in KD inflammation and implied a regulatory effect of IVIG on IgG+ ASCs. The decrease of IgG in ASCs cytoplasm may be as a result of the increased level of serum IgG caused by the application of high dose IVIG, which contributes to the neutralization of toxin and antigens, and thereby negatively regulates the synthesis of cytoplasmic IgG. Those cell-penetrating ingredient antibodies in IVIG may be responsible for the inhibition of cell activation and the clearance of intracellular pathogens . It also could be associated with the presence of anti-BAFF antibodies in IVIG preparation and the triggering of Fas apoptotic pathway by IVIG . Moreover, it was shown that IVIG promoted the expression of Fc-gamma Receptor (FcγR)-IIB on B cells, which could bind to the Fc segment of IgG and subsequently induced inhibitory signal . Thus, these mechanisms would eventually attenuate the activities of IgG+ ASCs and enhance their sensitivity to apoptosis. Importantly, our data did not find a definitely inhibitory effect of IVIG on ASCs or CD138+ ASCs, because they presented a heterogeneous variation after IVIG administration. Accordingly, the action of IVIG on ASCs should include other regulatory mechanisms. It was reported that the expression of A Proliferation-inducing Ligand (APRIL), which is advantageous for development and survival of B cells, was increased after IVIG administration, opposing to the variation of BAFF . In addition, in vitro study on the patients with SLE found increased plasma cell differentiation in the presence of IVIG . Consequently, the specific role of IVIG in regulating ASCs remains to be further elucidated. Another valuable matter was that in comparison with other ASCs, the overall level of CD138+ ASCs in remission was higher, despite not significantly, than that in acute, suggesting their distinct role in the remission stage of KD. The latest researches demonstrated that CD138+ plasma cells in bone marrow were inclusive of a group of long-lived plasma cells, which present a memory nature through persistent secretion of specific antibodies even though the patients had not exposed to the pathogens for decades [46,47,48]. Hence, it can be speculated that those increased CD138+ ASCs may be an explanation for the low recurrent rate of KD, as well as for the self-antibodies lasting for years .
Besides ASCs, memory B cells were also believed to be essential for maintaining humoral immunity. In the acute stage, the percentage of DN B cells was lower, but not significantly, than that in HCs. By contrast, the percentages of Sm and MZ B cells were significantly decreased. The data demonstrated that the patients with KD underwent profound variations and imbalances of memory B-cell subsets. Intriguingly, the variation of memory B cells was contrary to the variation of ASCs; however, there were no definite correlations among the subsets of memory B cells and ASCs. Thus, it may be hard to decide whether the reduction in memory B cells is simply due to their switching into ASCs. Among others, MZ B cells shown correlations with multiple laboratory findings, suggesting that MZ B cells contributed to both innate and adaptive responses, more likely, to the alleviation of inflammation via positive effects on immunoglobulins secretion and complements activation. A systemic review of the distinct features of MZ B cells insisted on their importance in inflammation . When patients entered into remission, the overall levels of memory B cells were significantly lower than those in HC. It seemed that suppressed memory response was throughout the course of KD and IVIG failed to modulate the memory immunity. However, it may be partial to draw this conclusion only upon the analysis of circulating memory B cells because in some conditions, memory B cells are abundant in the spleen [51, 52] or the mucosa . Therefore, in order to present a more integrated memory immunity, it may be necessary to investigate the status of memory B cells in the organ or tissue.
In our current study, we described a general picture of the status of ASCs and memory B cells during the course of KD. However, we also realized the limitations of our study. First, it may be worthy of analyzing the function of ASCs, particularly IgG+ ASCs, as well as those long-lived ASCs in bone marrow, if accessible. Second, investigation on the memory response in such as spleen and mucosa lymphoid tissue may be necessary. Third, the sample size in the remission stage should be enlarged. We will focus on these issues in subsequent studies.