In the presented study, 60 cases of KD patients were involved in this retrospective analysis and divided into 5 groups according to the children’s different age interval. There are no obvious differences in the sex distribution as to the incidence of KD in all groups, which has a discrepancy with other studies showing the disease more frequently occurs in boys [13, 14] but is consistent with the data recently reported in Australia [15]. A larger number of patients enrolled will have more convictive conclusion in this issue.
Neutrophil recruitment is an early event induced by the bacteria infection except for severe trauma, massive haemorrhage, malignancy and chemical poisoning [16, 17] and lymphocyte loss is usually looked as early biomarkers for systemic spread of severe infection [18]. Results revealed that in the acute phase of KD the abnormal increase of mean percentage of neutrophil and decreased percentage of the lymphocyte before IVIg constituted predominant changeable feature according to KD children’s laboratory parameters. The concurrence of increased neutrophil, decreased lymphocyte and the obvious correlations between the two indexes indicate that the bacteria infection existing in the KD process most likely acts as an inducer and therefore, the viral infection can be excluded. Particularly, the extent of reduced percentage of neutrophil and that of increased percentage of lymphocyte after the administration of IVIg correlated positively in all age patients in this study indicating a feature of this disease in patients, which showed the possible infection was controlled and the immune function was regulated directing to the normal level by the IVIg. It also suggests the use of antibiotics during the acute phase of KD may contribute to the improvement in KD and have a synergistic effect with the IVIg. Even if the therapeutic course of antibiotics has finished or stopped, post antibiotic effect may still exist.
As it known, a child’s immunologic maturity will not reach until adolescence. Not until 2 years old, adaptive and immune responses could not largely approach those of healthy adult levels and full immune competence will not truly reach until the teenage years [19]. Therefore, it is understandable that the coefficient of correlation (r) shows higher correlation in the scope changed between the neutrophil and the lymphocyte as the children grow older. As the mean of age rises, the correlated coefficient becomes higher. These results also suggest better immune-modulatory effect of IVIg usually obtained on children older than 4 years including the rapid balance of cell count changing to normal between neutrophil and lymphocyte.
Therefore, in the following investigation, the relationship between special subset of lymphocyte and the cytokines was in focus to study further. It found that among the patients aged 4 and over old, before the treatment of IVIg, both the means of absolute CD19+ B cell count and the level of IL-10 were significantly higher than normal level. The more CD19+ B cell absolute count was, the higher level of IL-10 was detected in the child. After the use of IVIg in the acute phase of KD, the inflammatory response regulated effectively manifesting as the anti-inflammatory cytokines IL-10 reduced obviously and the attenuated extent of IL-10 correlated positively well with the absolute count of increased CD19+ B cells assessed prior to the IVIg. CD19+ B cells is immunoglobulin production cells and play major role in the humoral immune response. The obviously higher count of CD19+ B cells before IVIg shows its activation and proliferation under the stimulation of antigen and indicates the predominant role of activated humoral immunity in the acute phase of KD children. The abnormal activation of humoral immunity response is also consistent to the speculation of the bacteria infection as an inducer of the KD. It has been reported previously that in mice CD19 enhances the B cell receptor (BCR) induced signaling which is crucial for the activation and proliferation of B cells and the following enhanced response of humoral immune [20, 21]. This finding may be a new evidence as to our knowledge that proliferating CD19+ B cell plays dominant role in the active humoral immune in KD child older than 4 years.
What’s more, the positive line correlation between the IL-10 and the absolute cell count of CD19+ suggests the possible ability of IL-10 secretion function of CD19+ B cells. IL-10 known as an anti-inflammatory cytokine was first described that it could be secreted in Th2 cells accompanied by other Th2-type cytokines such as IL-4, IL-5 and so on in the classic immunology theory. Later, IL-10 was found to be secreted by other cell types including macrophages, DCs, mast cells even neutrophils [22]. Recently, the concept that B cell can inhibit immunity by the provision of IL-10 has been confirmed in mice. In mouse model, CD19-deficient mice displayed a reduced production of IL-10 by B cells and developed an exacerbated disease [23]. At the same time, another study has demonstrated that in mice reactive oxygen species could suppress humoral immune response through the reduction of CD19 expression and resultant BCR signaling [20]. In some patients treated with rituximab the depletion of B cell leaded to immunopathology [24]. It is also reported that B cell from multiple sclerosis patients secreted markedly less IL-10 than B cells from healthy donors [25] suggesting the possible IL-10 secretion of B cells in some diseases of human. According to the presented result that the positive line correlation relationship between the CD19+ cell count and the reduced extent of IL-10, it is reasonable to speculate the proliferating CD19+ B cell may have the function of the IL-10 secretion as a compensatory response to balance the overactive humoral immune and regulatory effect on the immune response.
It is noticeable that after the treatment of IVIg, IL-10 reduced significantly suggesting the CD19+ cell absolute count may have a tendency of decrease wherever, the mean percentage of lymphocyte increased accompanied with the neutrophil decreased. As it is known the functions of immune globin are complex involving modulation of the expression and function of Fc receptors, interference with the activation of complement and the cytokine network, provision of antiidiotypic antibodies, and effector functions of T cells and B cells and so on [26]. Specially, IVIg could bind to the siglecs expressed on the surface of neutrophils and result in the cell death [27]. In addition, IVIg containing Fc has the ability to stimulate the expression of a population of natural regulatory T cells (nTreg) which could not be detected before IVIg treatment [28]. The phenomenon of decreased percentage of neutrophil and that of increased lymphocyte after IVIg is consistent to these conclusions. It could be reasoned that although the CD19+ B cell count decreases, another major constitution of lymphocyte T cells that also acting as an antigen presentation cell may be acted to increase after the use of IVIg and thus lead to the increase of percentage of lymphocyte as a whole in the KD pediatric patients. Therefore, by IVIg therapy the balance of the disordered immune responses tend to recover rapidly including the inhibition of hyper-humoral immune and the boost of cellular immune in KD children older than 4 years old whose immune system are approaching to maturation.
Taken together, CD19+ B cell absolute count may looked as an index to evaluate the seriousness of the humoral response in KD children. The combination of CD19+ B cell absolute count and the extent of reduced IL-10 post the IVIg could be considered as a marker to evaluate the sensitivity and therapeutic effect of IVIg on the inflammatory immune response in older KD children.
In this study, we put forward data in KD children aged 4 and over as we know for the first time that CD19+ B cell is stimulated to be active and proliferate directing to the enhanced function of humoral immune response, which has been verified in mice but have not clearly confirmed in human. Further, according to the results in this study combined the previous data reported, we proposes the speculation that CD19+ B cell in KD children may have the function of secreting the cytokine IL-10. However, more laboratory data need to be collected to confirm the conclusion.
From the strong positive correlation between the decreased neutrophil and the increased lymphocyte we come to the conclusion that the bacteria infection may be as an important inducer of KD and thus lead to the enhanced humoral immune response which is also consistent with the following change in peripheral blood white cell subgroup and the cytokine.
However, there are still some limitations in this retrospective analysis including the number of cases involved in are not fully enough and the data collected just from single one centre so that it could not completely reveal the overall perspective epidemiology of KD accurately.
Secondly, in the presented study the absolute cell count of subsets in lymphocyte especially the CD19+ cell should be traced to monitor dynamically after the IVIg. Therefore, the change tendency in the number of subset cell in lymphocyte could be analyzed more clear, the mechanism of IVIg and the inflammatory response in the special patients group could be demonstrated more definitely.
Another limitation is the infeasibility of function testify or cell deletion of CD19+ B cells in patients so that the following response can be observed. However, it can tried in mice and some cell subset could be collected and cultured in vitro to verify the IL-10 secretion function of CD19+ B cell.
In the following study, more cases of KD children older than 4 years will be enrolled and more detailed examinations during the course of KD both before and after the IVIg therapy still have potential to refine in-depth the mechanism under the relationship between CD19+ B cell count and the expression of IL-10.