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Abstract
Aims Cells expressing markers of mast cells, macrophages and dendritic cells have previously been demonstrated within the interstitium of infantile haemangioma (IH). This study characterised these myeloid cellular subpopulations within IH.
Methods Immunohistochemical staining was performed on proliferating and involuted IHs for the expression of Nanog, tryptase, CD163, DC-SIGN and CD45. The presence of mRNA corresponding to Nanog, tryptase α/β-1, tryptase β-2, CD163 and DC-SIGN was confirmed by NanoString and RT-PCR in snap-frozen IH tissues.
Results Immunohistochemical staining showed expression of Nanog by interstitial phenotypical mast cells within proliferating IH, which were separate from the interstitial M2-polarised macrophages that also expressed DC-SIGN, a dendritic cell marker. These two myeloid cellular subpopulations in IH did not express the pan-haematopoietic marker, CD45.
Conclusions There are two interstitial subpopulations of myeloid cells within IH: phenotypical mast cells which also express Nanog, indicating a primitive phenotype; and M2-polarised macrophages which also express DC-SIGN.
- HISTOPATHOLOGY
- PAEDIATRIC PATHOLOGY
- VASCULAR TUMOURS
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Introduction
Infantile haemangioma (IH) typically undergoes rapid growth during infancy (proliferating phase) followed by spontaneous involution over 1–5 years (involuting phase) with continued improvement up to 10 years (involuted phase).1
Cells expressing markers of mast cells, macrophages and dendritic cells have been previously reported within IH.2–4 Phenotypical mast cells which express tryptase are most abundant during the involuting phase, less in the involuted phase and least in the proliferative phase of IH.5 Phenotypic M2-polarised macrophages, characterised by the expression of CD163,6 have been shown within IH.3 Dendritic cells are specialised antigen-presenting cells, characterised by the expression of DC-SIGN, which may be derived from monocyte precursors.7 The presence of phenotypic DC-SIGN+ cells within IH has also been reported.4
The expression of multiple markers of the myeloid lineage by cells within the interstitium of IH led us to characterise the interstitial subpopulations.
Materials and methods
Tissue samples
Proliferating (n=6) and involuted (n=6) IH samples from patients aged 4–7 (mean, 5.5) months and 6–12 (mean, 9) years, respectively, were used for this study.
Immunohistochemical staining
For immunohistochemical (IHC) staining 4µm-thick formalin-fixed paraffin-embedded sections of proliferating (n=6) and involuted (n=6) IH from 12 patients were used. Antigen retrieval was performed using sodium citrate (Leica, Sydney, Australia) at 95°C for 15 min. All sections underwent single 3,3 diaminobenzidine (DAB) IHC staining for the primary antibodies, Nanog, 1:1000 (Cell Signalling Technology, Danvers, Massachusetts, USA); CD34, Ready to Use (Leica, Newcastle-Upon-Tyne, UK); tryptase, 1:300 (Leica); CD163, 1:50 (Leica); DC-SIGN, 1:500 (Abcam, Cambridge, Massachusetts, USA); GLUT-1, 1:200 (Cell Marque, Rocklin, California, USA); CD45, 1:1500 (Dako, Glostrup, Denmark); CD45, 1:600 (Abcam) using the bond polymer refine detection kit (Leica), all DAB IHC slides were counterstained with haematoxylin (Leica). To confirm dual expression of proteins, selected representative slides of each phase of IH underwent immunofluorescent (IF) IHC staining with the same primary antibodies, but using an appropriate secondary antibody for detection (goat antimouse Alexa-488, goat antirabbit Alexa 594, or chicken antigoat Alexa 647 (Life Technologies, Carlsbad, California, USA)). All antibodies were diluted in Bond primary antibody diluent (Leica), and all DAB and IF IHC staining were performed on the Leica Bond Rx autostainer (Leica). The DAB and IF IHC slides were mounted using either Surgipath Micromount mounting medium (Leica) or Vecta Shield Hardset mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, California, USA).
Gene expression
NanoString nCounter Gene Expression Assay
Snap-frozen proliferating (n=6) and involuted (n=6) IH samples were used to isolate total RNA for NanoString nCounter Gene Expression Assay (NanoString Technologies, Seattle, Washington, USA). RNA was extracted from frozen tissue using RNeasy Mini Kit (Qiagen, Hilden, Germany) and subjected to the NanoString nCounter Gene Expression Assay performed by New Zealand Genomics (Dunedin, New Zealand). Probes for the genes encoding CD163 (NM_004244.4) and Nanog (NM_024865.2) were designed and synthesised by NanoString Technologies. Raw data was analysed by nSolver software (NanoString Technologies) using standard settings and were normalised against the housekeeping genes.
Comparisons of mRNA levels for CD163 and Nanog between proliferating and involuted IH samples were analysed using Student's t test from Microsoft Excel 2013.
Quantitative real-time PCR
Total RNA was extracted from snap-frozen tissue samples from proliferating (n=3) and involuted (n=3) IH using RNeasy Mini Kit (Qiagen). RNA was reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad), qRT-PCR was performed on Rotor-Gene Q Platform (Qiagen). The following TaqMan Gene Expression Assays were used: hs02576518_gH (genes TPSAB1, TPSB2 encoding tryptase α/β-1 and tryptase β-2), hs01588349_m1 (gene CD209 encoding DC-SIGN) (Life Technologies). Data were analysed using the 2−ΔCT method.
Image analysis
IF IHC stained slides were viewed and captured using an Olympus FV1200 biological confocal laser-scanning microscope (Tokyo, Japan). All DAB IHC stained slides were viewed using an Olympus BX53 light microscope (Tokyo, Japan).
Results
IHC staining
All tissue samples used in the experiments expressed GLUT-18 (data not shown). IHC staining confirmed expression of Nanog (figure 1A, red) by the majority of tryptase+ (figure 1A, green) phenotypic mast cells in proliferating IH, as previously reported.2 The presence of phenotypic M2-macrophages expressing CD163 had also been previously reported in IH.3 Staining for the mast cells expressing Nanog (figure 1B, red, arrowheads), and the M2-macrophages, expressing CD163 (figure 1B, green, arrows) demonstrated two distinct myeloid subpopulations within IH.
Immunofluorescent immunohistochemical staining of representative proliferating (A–D) and involuted (E and F) infantile haemangioma (IH) sections. (A) Coexpression of Nanog (red) and tryptase (green) by the phenotypic mast cells, appearing as yellow (arrows). (B) These phenotypic mast cells that express Nanog (red, arrowheads) are distinct from the phenotypic M2-macrophages that express CD163 (green, arrows). (C) The presence of phenotypic dendritic cells, expressing DC-SIGN (red, arrowheads), are also distinct from the mast cells, expressing tryptase (green, arrows). (D) DC-SIGN (red) is expressed the M2-macrophage population, expressing CD163 (green) appearing as yellow (arrows). The distinct subpopulations, one expressing DC-SIGN (E, red, arrowheads) and the other expressing tryptase (E, green, arrows), with the subpopulation expressing DC-SIGN (F, red) and CD163 (F, green), persist in involuted IH. All images were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).
Phenotypic dendritic cells, characterised by DC-SIGN, had been previously reported within IH.4 Staining for tryptase (figure 1C, green, arrows) and DC-SIGN (figure 1C, red, arrowheads) demonstrated the presence of two distinct subpopulations. Staining for CD163 (figure 1D, green) and DC-SIGN (figure 1D, red) showed coexpression of these markers by the same cells (figure 1D, arrows). These distinct subpopulations of tryptase+ (figure 1E, green, arrows) and DC-SIGN− (figure 1E, red, arrowheads) cells; and CD163+ (figure 1F, green) and DC-SIGN+ (figure 1F, red, arrows) cells persisted in involuted lesions.
We have previously reported on the presence of an interstitial cellular population within IH expressing the pan-haematopoietic cell marker, CD45.2 Dual staining for CD45 with either tryptase or DC-SIGN was performed to investigate if CD45 was expressed by the mast cells and/or M2-macrophage subpopulations within proliferating IH. Double staining for CD45 (figure 2A, green, arrows) and DC-SIGN (figure 2A, red, arrowheads), demonstrated two distinct subpopulations within proliferating IH. Double staining for CD45 (figure 2B, red, arrowheads) and tryptase (figure 2B, green, arrows) also demonstrated two distinct subpopulations within proliferating IH.
Immunofluorescent immunohistochemical staining of representative proliferating infantile haemangioma (IH) sections, demonstrating the distinct subpopulations of cells expressing CD45 (A, green, arrows; B, red, arrowheads), and those expressing DC-SIGN (A, red, arrowheads), and those expressing tryptase (B, green, arrows) in IH. All images are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).
Gene expression
NanoString nCounter Gene Expression Assay
CD163 and Nanog mRNA were present within proliferating and involuted IH (figure 3). Statistical analysis of the data using Student's t test showed that Nanog mRNA levels were higher in proliferating compared with involuted lesions (p=0.032). However, there was no statistical difference in CD163 mRNA levels between proliferating and involuted lesions.
CD163 and Nanog mRNA levels in proliferating (n=6) and involuted (n=6) infantile haemangioma (IH) tissues. mRNA level was assessed using NanoString nCounter Gene Expression Assay with specific probes for CD163 and Nanog genes. Results are presented as mean±SD. Nanog mRNA levels were significantly higher in proliferating compared with involuted lesions (p=0.032). No significant (n.s) differences were observed between proliferating and involuted IH samples for CD163.
Quantitative real-time-PCR
Tryptase α/β1, tryptase β2 and DC-SIGN (CD209) mRNA were present in proliferating and involuted phases of IH, with no significant difference between proliferating and involuted samples, when compared with the housekeeping gene, GUSB (figure 4).
qRT-PCR of TPSAB1 (tryptase α/β1), TPSB2 (tryptase β2) and CD209 (DC-SIGN) gene expressions relative to the housekeeping gene, GUSB, are represented as ΔCT values in proliferating (n=3) and involuted (n=3) infantile haemangioma tissues.
Discussion
Expression of myeloid lineage markers by phenotypic mast cells, M2-macrophages and dendritic cells, has been demonstrated previously within IH.2–4 We show here the presence of two distinct interstitial myeloid subpopulations within proliferating IH, one expressing CD163 and DC-SIGN, and the other expressing Nanog and tryptase. Whether these subpopulations of cells have been recruited from the circulation, or arisen from within the lesion, is beyond the scope of this study. However, the presence of tryptase+ cells expressing decreasing levels of Nanog as IH involutes is consistent with the notion that cells only positive for tryptase express a more mature phenotype as IH involutes, and that potentially the more mature myeloid phenotypic mast cells are derived from the primitive tryptase+/Nanog+ population.2 This is more plausible than the alternative concept that proliferating lesions are first infiltrated by a wave of Nanog+ mast cells that are later replaced by a wave of Nanog− phenotypic mast cells with ongoing involution.2
First trimester placental cells have been suggested as the origin of IH.2 It is exciting to speculate that a subtype of placental macrophages known as Hofbauer cells, which coexpress CD163 and DC-SIGN,9 may represent the interstitial M2-macrophages described in this report, persisting in involution.
Wang et al3 recently reported a subpopulation of M2-polarised macrophages coexpressing CD68 and CD163, and a smaller subpopulation of CD68+/CD163− cells. We infer that the latter may be a subpopulation of M1-polarised macrophages, based on their expression pattern.9 M2-polarised macrophages are the alternatively activated macrophages,3 which express CD1636 and possess anti-inflammatory and proangiogenic functions.3 In contrast to M1 macrophages, they are well suited to promote tumour development.6 M2 macrophages may be involved in the proliferation of IH by promoting angiogenesis through secretion of factors such as vascular endothelial growth factor and fibroblast growth factor-2.3 The cytokine niche within IH may promote predominant differentiation towards an M2-macrophage phenotype, although determination of this is beyond the scope of this study. Whether this CD163+/DC-SIGN+ phenotype represents a putative progenitor phenotype for macrophages and dendritic cells, remains a topic for further investigation.
Take home messages
This study confirms the presence of two different interstitial myeloid cellular subpopulations within proliferating and involuted infantile haemangioma (IH).
These two subpopulations of myeloid cells in proliferating IH are primitive mast cells that express tryptase and Nanog, and M2-macrophages that express CD163 and DC-SIGN, the dendritic cell marker.
These two myeloid cellular subpopulations in proliferating IH do not express the pan-haematopoietic marker, CD45.
Acknowledgments
The authors thank Ms Liz Jones for her assistance in immunohistochemical staining, and Dr Andrea Mikulasova for preparing samples for NanoString and qRT-PCR analyses and statistical analysis of the NanoString data. EMST was supported by a summer studentship at the Gillies McIndoe Research Institute.
Footnotes
TI and STT contributed equally.
Correction notice This article has been corrected since it was published Online First. The provenance and peer review statement has been amended.
Handling editor Cheok Soon Lee
Contributors EMST, TI, STT came up with the study hypothesis. EMST, TI, STT, PFD and HDB designed the study. EMST and TI interpreted the IHC results. EMST and DAC interpreted the NanoString data. EMST and DAC performed RT-PCR experiments and interpreted the results. EMST drafted the manuscript. TI and STT revised the manuscript. All authors approved the manuscript.
Competing interests None.
Ethics approval Central Regional Health and Disability Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.