Increased numbers of peripheral blood CD14+ monocytes/macrophages and activated CD14+/CD23+ monocytes/macrophages are seen during the acute stage of KD. They secrete high levels of TNFα and IL-1 in acute KD. In our study, we documented increase in CD14+ monocytes, classical (CD14+CD16-) and intermediate (CD14++CD16+) monocytes during acute phase of KD that normalised in follow-up. Both early (CD69) and late (HLA-DR) activation markers in monocytes were also elevated in acute phase of KD that reduced in follow-up. There were significantly elevated levels of CCL2 in acute KD, as compared with post-IVIg and convalescent phase. We observed higher levels of CCL2 in patients with KD and febrile controls as compared to healthy controls.
In our cohort, we found significantly increased AMC in acute stages of KD as compared to healthy and febrile controls. Majority of the previous studies in KD have focussed on mononuclear cell counts rather than just monocyte counts. Matsubara et al. found no significant difference in mononuclear cell count between KD patients and control but monocyte count (CD14+) was significantly higher in patients with acute KD as compared to control subjects . Several other studies have documented elevated levels of different serum cytokines (TNF-alpha, IL-1, and IL-6) that are reckoned to be produced by monocytes in the acute stage of KD  Matsuguma C et al studied the difference in absolute monocyte counts between IVIg responsive and IVIg resistant groups. They found AMC to be significantly higher in IVIg responsive group as compared to IVIg resistant group .
Classical monocytes [CD14+ CD16-], which constitute the majority of monocytes, act as scavengers toward apoptotic cells and assist in resolving inflammation . Our study showed a significant increase in absolute counts of classical monocytes [CD14+CD16-] in acute KD as compared to febrile and healthy controls. Intermediate monocytes [CD14++CD16+] express chemokine and TNF receptors and they are considered as pro-inflammatory form of monocytes . Katayama et al. studied 28 patients with KD along with other febrile healthy controls. They found a significant increase in percentage as well as absolute numbers of CD14+CD16+ monocytes in acute KD as compared with convalescent KD and control subjects . In our study, we have studied both intermediate monocytes [CD14++CD16+] and non-classical [CD14+CD16++] monocytes separately. On comparing acute KD with post-IVIg and follow-up samples, we also found a significant decrease in the absolute counts of intermediate monocytes [CD14++CD16+]. Non-classical monocytes [CD14+CD16++] are a minor subset of monocytes that have a proinflammatory role. These monocytes also have a role in surveillance of endothelial integrity and are thus referred to as patrolling monocytes . In our study, we noted no difference in either the percentage or absolute counts of non-classical monocytes [CD14+CD16++] between acute KD, healthy and febrile controls. Burbano et al. found decreased percentage of non-classical monocytes [CD14+CD16++] in active SLE as compared to inactive SLE and healthy controls . To the best of our knowledge, no study has studied nonclassical monocytes [CD14+CD16++] in KD and this area requires more research.
CD69 is an early activation marker that is expressed on all hematopoietic cells except RBCs. Crosslinking of this molecule leads to calcium influx and formation of both cyclooxygenase and lipoxygenase metabolites . Many studies have been done on CD69 expression on T-lymphocytes in KD. However, studies on CD69 expression on monocytes in KD are lacking. CD69 expression on monocytes has been studied in various other diseases like Alzheimer's disease and sarcoidosis. In our cohort of KD patients, we found an increased percentage expression of CD69 in total monocytes (CD14+) in KD cases and febrile controls as compared to healthy controls suggesting the role of early monocyte activation in KD. HLA-DR is a late monocyte activation marker and is mainly seen on antigen presenting cells (APCs). HLA-DR is a surrogate marker for immune system stimulation and the expression of HLA-DR on monocytes has been shown to determine the efficacy of antigen presentation to T-helper cells . Increased numbers of monocytes with high expression of HLA-DR have been shown in other inflammatory disorders like rheumatoid arthritis and inflammatory bowel disease [20–21].
On comparing acute KD with post-IVIg and convalescent stage we found a significantly higher percentage of total monocytes (CD14+) expressing the late activation marker (HLA-DR) in acute stage and post-IVIg as compared to convalescent stage. These findings indicate increased antigen presentation by monocytes in acute stage. One of the well-known mechanisms of IVIg is to decrease antigen presentation by downregulating MHC-II and dendritic cells and also to decrease T-lymphocyte expansion and to increase apoptosis of T-lymphocytes. Hence, decrease in HLA-DR expression could be related to immunomodulatory action of IVIg .
Several studies have documented changes in the levels of different activation markers of monocytes like sCD14, sCD163, CCL2/MCP-1 etc in patients with KD (Table 4). [5–8] In the present study, we documented significantly elevated levels of CCL2 in acute KD, as compared with post-IVIg and convalescent phase, and MCP-1/CCL2 levels were higher in patients with KD and febrile controls in comparison to healthy controls. These findings were consistent with the results of Asano et al. and Terai et al who observed significantly elevated circulating levels of MCP-1 in the acute phase of KD that drastically reduced after IVIg therapy. [7, 23] In our study, there was no significant rise in sCD14 and sCD163 levels in children with acute KD when compared with febrile and healthy controls. This is in contrast to the results obtained by Takeshita et al and Azuma et al who reported significant elevation in sCD14 levels and sCD163 levels respectively in children with acute KD compared with febrile and healthy controls. [5, 6] Apart from variation in ethnicity, the difference in duration of fever in children with KD enrolled in those studies can also possibly explain the contradictory results. While Takeshita et al and Azuma et al enrolled children with KD who had median fever duration of 7.5 days and 4 days respectively, we enrolled cases with KD with the median duration of fever of 9 days. It is possible that elevation of sCD14 and sCD163 levels occurs mostly in the first week of illness in KD that subsides in the second week.
Azuma et al also noted significant elevation in levels of sCD163 following IVIg therapy in initial IVIg responder group, however, the same observation was not noted in IVIg non-responder group. Authors speculated that IVIg could possibly detach CD163 from the surface of macrophages due to cross-linking of Fc-gamma receptors.  We also observed increase in sCD163 levels following IVIg therapy. However, the difference was not statistically significant. Small sample size of our cohort could have possibly resulted in not achieving the statistical significance.
Cheung et al. (2005) showed significant overexpression of CCL2/MCP1 genes in THP1 macrophages in children with CAAs who had past history of KD . But this study was carried out in children with KD at mean 7 years after acute illness. These findings suggest that MCP-1 expression persisted to remain elevated in CAAs in KD.
Our study was unique as multiple monocyte activation markers were assayed in study population using methods like flow cytometry and ELISA. Another strength of this study was inclusion of different subsets of monocytes (classical, intermediate and non-classical) in analysis. Limitation of our study include a small sample size. However, this is understandable given that our study has to be carried out in a limited time span. Studying monocyte activation markers in a large multicentric cohort of patients would provide more convincing data on monocyte activation status in children with KD.
In conclusion, our study showed that both classical and intermediate monocytes along with early and late monocyte activation markers were elevated in acute phase of KD that subsided in follow-up. We also documented elevated levels of CCL2/MCP1 in acute stage of KD implying monocyte activation in the acute phase of KD. Our results suggest IVIg may decrease monocyte activation in KD, thereby controlling systemic inflammation. However, results of our study must bereplicated in larger cohorts with inclusion of more severe forms of KD to assess whether thedegree of monocyte activation could predict IVIg resistance or formation of CAAs.