Distribution And Phenotype Changes Of Macrophage In Infantile Hemangioma

BackgroundIn 1982, Mulliken and Glowacki divided vascular lesions into two categories: vascular malformation and hemangioma, according to clinical manifestations, histopathological features and biological behavior. There are greatly differences between vascular malformation and hemangioma. The vascular malformation exist when born, with the corresponding growth of physical development, without fading; but hemangioma show a small red spot at birth, growing in 2 weeks, mostly stop proliferation in 1 year and then go to fade,90% in 9 years away. While the latter is common in infants, so the "infantile hemangioma" is named to more easily understand this disease.The endotheliocyte is largely self-cloning and then form microvessel during proliferating stage of the infantile hemangioma; in the other hand, The endotheliocyte scatter, microvessel collapsing and fibrous tissue are accumulating during involuting stage. Therefore, many researchers are committed to explore the origin of endotheliocyte, the formation mechanism of microvessel and the causation of endotheliocyte apoptosis. But recent studies have shown that the infantile hemangioma contain plenty of non-endothelial cells which involved in the evolution. Some data indicated that the dendritic cells were involved in the formation of tumor blood vessels by secreting cytokines VEGF; the mast cells stimulated angiogenesis to promote the proliferation of hemangioma by releasing VEGF andⅧcollagen. Ritter et al found that hypoxia stimulated myeloid cells to secrete vascular endothelial growth factor. However, as immune-related cells, macrophages are also abundant in infantile hemangioma, but its role and distribution are rarely reported.Immunological studies have shown that macrophages are plasticity and pluripotency, they are significant functional differences in different environment. Macrophages are mainly divided into two types:M1 macrophages and M2 macrophages. Available information suggests that Ml macrophages are potent effector cells that kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. In contrast, M2 cells tune inflammatory responses and adaptive Thl immunity, scavenge debris, and promote angiogenesis, tissue remodeling and repair. Since the infantile hemangioma has a clear process of proliferation and fading, while studies have shown that they have plenty of macrophage, then the macrophage which gather around the microvascular during early proliferating stage is M2 macrophage or not? Is there possibility that the M2 macrophage can switch to Ml macrophage following the local microenvironment change?ObjectiveHere, we tested the distribution and phenotype of macrophages in different stages of infantile hemangioma by using immunohistochemical methods, to explore the relationship between changes of macrophages phenotype and evolution of hemangioma. So as to provide evidence about how to early prevent, predict and intervent the hemangioma.Methods1. Collect and test specimensWe collected the 46 specimens which were diagnosed of hemangioma from the Pathology department of the First Affiliated Hospital of Gannan Medical College. The specimens were divided into four stages:early proliferating stage (1 to 3 months, 7 cases), middle proliferating stage (4 to 6 months,17 cases), late proliferating stage (7 to 12 months,16 cases), early involuting stage (1 to 3 years,6 cases). Extract the vascular malformations from the specimens by using Glut-1 immunohistochemistry, and compared hemangiomas with vascular malformations using routine HE staining.2. Detected the proliferation activity of the infantile hemangioma endothelial cellIt was positive when the Ki-67 positive endothelial cells were more than 10%, and then explored the significance in hemangioma about the endothelial cell proliferation activity.3. macrophages labelled with CD68, CD16/32 and CD163Macrophages labelled with CD68, CD16/32 and CD163 were investigated in infantile hemangioma by immunohistochemistry, Observed the distribution and changes in hemangioma.Results1.43 specimens identified as infantile hemangioma which the endothelial cells were positive for Glut-1,3 cases were vascular malformation. The "lobula" structure was found in infantile hemangioma, vascular malformation had not. The "lobula" structure at early proliferating stage could be divided into three "zones" from center to edge:naive area, middle transition region and maturation zone, this structure gradually disappeared at late proliferating stage and early involuting stage.2. It was negative as the positive rate of Ki-67 was less than 10%.3. CD68-positive macrophages appeared at all stages, there were statistical significance among four stages (F=12.815, P=0.000); The fore three stages, had statistical significance compared with the early involuting stage(P<0.05), but no statistical significance among themselves(P> 0.05). The rate of CD163-positive macrophages was 20.93% at early proliferating stage, mainly appeared in the specimens which less than 3 months, and, more than 3 months, weakened rapidly; there were not only statistical significance among four stages because the 95%CI didn't include zero at early and middle proliferating stage, but also statistical significance between early and middle proliferating stage (t=7.778, P=0.000); CD16/32-positive macrophages did not express at early proliferating stage, and began to appear at middle proliferating stage, the positive rate gradually increased to 8.07% at late proliferating stage; the 95%CI didn't include zero at late proliferating stage, there were statistical significance compare with early proliferating stage and early involuting stage, but there weren't statistical significance at middle proliferating stage, because it's 95%CI included zero, at last, there were statistical significance between late and middle proliferating stage (t=-7.855, P=0.000)。Discussion1. The pathological difference between hemangioma and vascular malformation.In our collection, there may had the specimens of vascular malformation, because the diagnosis of vascular disease is still confusion in many hospitals. In 2000, North et al found that all infantile hemangioma expressed Glut-1, while vascular tumor or malformation not, so it was used to distinguish infantile hemangioma from vascular malformation, we confirmed this result again.By observing the pathological differences between infantile hemangioma and vascular malformation, we found that the former were separated by fibrous tissue, while the latter had not, so the structural feature of "lobula" should be able to distinguish infantile hemangioma and vascular malformation in pathology.Further study, it was found that each "lobula" could be divided into three "zones" from center to edge:the center of naive area, transition region and the edge of maturation zone, especially at early proliferating stage. This might help us further understand the proliferating mechanism of microvessel in infantile hemangioma. There are two main mechanisms formed microvessel:angiogenesis and vasculogenesis. At first, the angiogenesis was considered to be main formation mechanism of new vessels in infantile hemangiomas as other tumors. In 2004, Nguyen found microvessel and perivascular cells which had the markers of monocytes or macrophages or dendritic cells appeared in infantile hemangioma, which made him recommended that the microvessel resulted form vasculogenesis, not angiogenesis. The performance of two main mechanisms formed microvessel should be different in pathology. We observed all specimens of the infantile hemangioma, and then imaged the process that the endothelial cell mass, separated by fibrous tissue, extended to surrounding tissue by forming new capillaries. It more clearly confirmed that the microvessel of infantile hemangioma resulted from angiogenesis.2. The prognosis of infantile hemangioma.In 1987, Bauer demonstrated the importance of the proliferating activity in tumor prognosis; followed up,Schutte put forward that tumor cell proliferating activity was an independent prognostic indicator. It has great significance to detect the proliferating activity of the endothelial cells in infantile hemangiomas because it has self-cloning.It was easy to false because PCNA staining was very sensitive, so we selected Ki-67. The Ki-67 positive endothelial cells were persistent low level, less than 10%. This result led us to speculate:1,surgeon, who intentionally or unintentionally, excluded the patients who were difficult to excise.2,In these cases, the speed of self-cloning of endothelial cells couldn't keep up with the forming of microvessel. It might be faded earlier, the residual deformities should also be less obvious, so the prognosis is good.3. The role of macrophage in infantile hemangioma.The infantile hemangiomas have unique natural history, it is important that the endothelial cell and microvessel change in pathology. Previous studies of endothelial cells were committed to explore its origin and how to generate the capillary, but recent studies have shown that the non-endothelial cells of infantile hemangioma were involved in the process. Macrophages, as immune cells, also appeared in infantile hemangioma.Immunological studies have shown that macrophage was plasticity and pluripotency, they are significant functional differences in different environment. Macrophages are mainly divided into two types:M1 macrophages and M2 macrophages. Available information suggests that M1 macrophages are potent effector cells that kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. In contrast, M2 cells tune inflammatory responses and adaptive Thl immunity, scavenge debris, and promote angiogenesis, tissue remodeling and repair.Our study indicated that large numbers of macrophages appeared in periphery of the endotheliocyte mass and gathered around new vessels at early proliferating stage; the number of macrophage was declined at middle proliferating stage and was rare at late proliferating stage and early involuting stage. This observation showed that the macrophage presented at all stages and involved in the evolution of the infantile hemangioma.Using CD 163, we found the macrophages that gathered around new vessels were M2-type macrophages. Studies have shown that it can promote the formation of capillaries by producing a variety of vascular factors, and create favorable conditions for microvascular migration by secreting various proteases to degrade extracellular matrix. Thus, we could believe that the M2 macrophages played an important role at early proliferating stage. While CD 16/32 positive macrophages could be observed in part of the late proliferating and early involuting stage, studies had shown that Ml macrophages could made TNF-a and ET-1 secretion, NO reduction to promote proliferation of fibroblast cells and adipose cell, coinciding with the pathology of this stage that adipose and fibrous tissue began to increase. These indicated that the function of macrophage transformed when the phenotypes had changed in different microenvironments. We speculated that other macrophages of different phenotypes participated in the evolution of infantile hemangioma because fully polarized Ml and M2 macrophages were the extremes of a continuum of functional states.We confirmed the presence of macrophages in the infantile hemangioma by immunohistochemistry and observed the phenotypes changing. So it not only provided a meaningful basis for further studying the regulatory mechanism of microvessel, but also helped us to find out how to change the phenotype of macrophages in infantile hemangioma. Conclusion1. There are totally different pathological performance between hemangioma and vascular malformation, the "lobula" structure can be used as a reference to distinguish them; the "lobula" structure might indicate that the microvessel of hemangioma result mainly from vasculogenesis; Glut-1 can be used as identification of hemangioma and vascular malformation.2. The low Ki-67 expression might indicate that the infantile hemangiomas, represent by the experiment samples, are easy to fade, the prognosis is good, and not to intervent positively.3. CD68 positive macrophages were observed in all of the infantile hemangiomas, but the number decreased gradually. CD163 positive macrophages were observed at the early proliferating stage. CD16/32 positive macrophages could be observed in part of the late proliferating and early involuting stage. Macrophages may be involved in the wholel process of infantile hemangioma. M2 macrophages play an important role at early proliferating stage. M1 macrophages may participate in the involuting process.