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Original article
Characterisation of lymphocyte subpopulations in infantile haemangioma Elysia M S Tan,1 Tinte Itinteang,1 Daria A Chudakova,1 Jonathan C Dunne,1 Reginald Marsh,1,2 Helen D Brasch,1 Paul F Davis,1 Swee T Tan1,3 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2015-203073). 1
Gillies McIndoe Research Institute, Wellington, New Zealand 2 University of Auckland, Auckland, New Zealand 3 Centre for the Study & Treatment of Vascular Birthmarks, Wellington Regional Plastic, Maxillofacial & Burns Unit, Hutt Hospital, Wellington, New Zealand Correspondence to Dr Swee T Tan, Gillies McIndoe Research Institute, P.O. Box 7184, Newtown, Wellington 6242, New Zealand; [email protected] TI and STT contributed equally. Received 14 April 2015 Revised 20 May 2015 Accepted 25 May 2015 Published Online First 11 June 2015
ABSTRACT Aims Interstitial CD45+ cells and T lymphocytes have previously been demonstrated within infantile haemangioma (IH). This study investigated the expression of B and T lymphocyte markers by the CD45+ population, and the expression of Thy-1, a marker of thymocyte progenitors, which have the ability to give rise to both B and T cells. Methods Immunohistochemical (IHC) staining was performed on proliferating and involuted IHs for the expression of CD45, CD3, CD20, CD79a, Thy-1 and CD34. The presence of mRNA corresponding to CD45, CD3G, CD20 and Thy-1 was confirmed by reverse transcriptase-polymerase chain reaction in snap-frozen IH tissues. Cell counting of 3,3-diaminobenzidine IHCstained slides was performed on CD45+ only cells and dually stained CD45+/CD3+ cells or CD45+/CD20+ cells and analysed statistically. In situ hybridisation and mass spectrometry were also performed to confirm the presence and abundance of Thy-1, respectively. Results IHC staining showed a subpopulation of CD45+ interstitial cells that expressed the T lymphocyte marker, CD3, and another subpopulation that expressed the B lymphocyte marker, CD20, in proliferating and diminished in involuted IHs. The abundant expression of Thy-1 on the endothelium of proliferating, but not involuted IH, was demonstrated by IHC staining and confirmed by in situ hybridisation and mass spectrometry. Conclusions Both B and T lymphocytes are present within the interstitium of proliferating and involuted IH. The expression of Thy-1 by the endothelium suggests that B and T cells in IH may have originated from within the lesion, rather than migrating from the peripheral circulation.
INTRODUCTION
To cite: Tan EMS, Itinteang T, Chudakova DA, et al. J Clin Pathol 2015;68:812–818. 812
Infantile haemangioma (IH), the most common tumour of infancy, typically undergoes rapid proliferation during infancy ( proliferative phase) characterised by excessive vasculogenesis, followed by slow spontaneous involution over 1–5 years of age (involuting phase) with fibro-fatty deposition that replaces the cellular elements up to 10 years of age (involuted phase).1–3 Cells of multiple lineages have been demonstrated within IH including endothelial, mesenchymal and haematopoietic cells.4 5 The haematopoietic cells are divided into the lymphoid and myeloid lineages.6 We have recently identified and characterised the myeloid cell subpopulations within IH.7 Postnatally, all haematopoietic cells originate in the bone marrow and are derived from a pluripotent haematopoietic stem cell.6 These cells then diverge in the bone marrow, into the lymphoid and myeloid lineages.6 8 The common lymphoid progenitor,
which gives rise to all lymphocyte progeny, then differentiates into B and T cells.6 Ritter et al9 report the presence of a population of CD3+/CD8+/CD4− cells within IH, indicating a cytotoxic T cell phenotype, thus inferring their role in inducing apoptosis in target cells. However, they do not find a strong correlation between the number of T cells and the developmental phases of IH, which would be expected if the T cells were to play a role in the involution of IH. It is unclear what role these T cells play in IH or whether they contribute to the proliferation and/or involution of this tumour. The authors suggest three possible explanations for the presence of these cells: (1) CD8+ T cells may be recruited to the IH via a non-specific inflammatory mechanism, possibly mediated by secretion of cytokines from endothelial cells or other cells within IH; (2) CD8+ T cells may promote proliferation of IH by secreting factors that stimulate endothelial proliferation; and (3) CD8+ T cells may be specifically recruited to the IH and contribute to its regression by inducing apoptosis of cells in IH. There is growing evidence suggesting that IH represents an embryonic developmental anomaly1 10–13 derived from a haemogenic endothelium, with mesenchymal5 and erythropoietic14 capacity. It has also been recently suggested that this haemogenic endothelium has the capability to produce myeloid cells.4 We hypothesise that B and T cells in IH originate from within the lesion, possibly arising from the haemogenic endothelium, rather than migrating from outside, as previously proposed.9 We have previously reported the presence of an interstitial cellular population within IH expressing the pan-haematopoietic cell marker, CD45.4 The presence of T lymphocytes within IH,9 and the previous demonstration of the exclusive expression of CD45 by the myeloid population,7 led us to speculate that these CD45+ cells represent a putative lymphocyte subpopulation within the interstitium of IH. In this study, we investigated the expression of thymocyte differentiation antigen-1 (Thy-1, also known as CD90), a marker of thymocyte progenitors,15–17 which possesses the ability to give rise to both B and T lymphocytes.13 We also characterised the CD45+ interstitial cellular population within IH and investigated the expression of both B and T lymphocyte lineage markers.
MATERIALS AND METHODS Tissue samples Proliferating (n=6) and involuted (n=6) IH samples from 12 patients aged 4–7 (mean, 5.5) months and 6–12 (mean, 9) years were used for this study.
Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
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Original article Immunohistochemical staining
Mass spectrometry
Four μm-thick formalin-fixed paraffin-embedded sections of proliferating (n=6) and involuted (n=6) IH from 12 patients were used for immunohistochemical (IHC) staining. 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, GLUT-1, 1:200 (Cell Marque, Rocklin, California, USA); CD45, 1:600 (Abcam, Cambridge, Massachusetts, USA); Thy-1, 1:100 (Thermo Scientific, Rockford, Illinois, USA) and Ready-to-Use CD3, CD20 and CD79a (all from Leica) with detection using the Bond polymer refine detection kit (Leica). To confirm coexpression of two proteins, selected representative slides of each phase of IH underwent immunofluorescent (IF) IHC staining with the same primary antibodies at the same concentrations, but using an appropriate secondary antibody (goat anti-mouse Alexa-488 or goat anti-rabbit Alexa 594) for detection. All antibodies were diluted in Bond primary antibody diluent (Leica), and all DAB IHC and IF IHC staining were performed on the Leica Bond Rx auto-stainer (Leica). The DAB and IF IHC slides were mounted using either Surgipath micromount mounting media (Leica) or Vectashield hardset mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, California, USA), respectively. Human tonsillar tissue was used as positive control for CD45, CD3, CD20, CD79a and Thy-1.
Total protein samples extracted from proliferating (n=2) and involuted (n=2) IH tissue, from the cohort of six patients used for RT-PCR, were trypsin-digested and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tissue pieces were homogenised by grinding with sand (Sigma-Aldrich) in 150 μL of ice cold radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing 1× Complete Protease inhibitor cocktail EDTA-free (Roche Life Science). After protein quantitation (Qubit 2.0 Fluorometer, Life Technologies, San Diego, California, USA), 100 mg of total protein per sample was precipitated overnight (ProteoExtract Protein Precipitation Kit, Merck Millipore), and the washed protein pellets were resuspended in 50 μL of 5% sodium deoxycholate (SDC), 10 mM dithiothreitol, 100 mM triethylammonium bicarbonate (TEAB) buffer (pH 8.5; Sigma Aldrich) and incubated for 30 min at 80°C. Samples were then alkylated for 60 min by the addition of 40 mM iodoacteamide and diluted in 100 mM TEAB buffer (pH 8.5) to a 500 μL final volume. Protein digestion was performed overnight at 37°C using 4 mg trypsin per sample (modified sequencing grade trypsin from bovine pancreas; Roche Life Science). SDC was precipitated from each sample by the addition of 1% formic acid, pelleted by centrifugation for 30 min at 13 000×g, and the peptide supernatants transferred to fresh microcentrifuge tubes. The SDC pellets were washed with 200 μL of 1% formic acid to remove coprecipitated peptides, and after centrifugation for 30 min at 13 000×g, each wash solution was combined with the previously recovered peptide supernatant. Peptide samples were lyophilised to ∼10 μL, supplemented with 500 μL 0.1% formic acid, purified using OMIX C18 pipette tips (Agilent Technologies) and reconstituted in 200 μL of 4% acetonitrile:0.1% formic acid. LC-MS/MS analysis was performed using an UltiMate 3000 HPLC system (Dionex) interfaced with a LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Thirty-five microlitres of each peptide sample was loaded onto an Acclaim PepMap100, C18 column (3 mm, 100 Å, 75 mm i.d. ×15 cm; Thermo Scientific; 0.3 μL/min flow rate), and peptides were eluted and analysed using data-dependent MS/MS acquisition. Four technical replicates were performed for each sample. Raw MS/MS data files were searched against a complete human protein database (SwissProt KB, 22 October 2014, 69 689 sequences) using Proteome Discoverer V1.4 (Thermo Scientific). Scaffold 4.0 (Proteome Software) was used to establish relative protein abundance in proliferating and involuted IH extracts using the spectral counting algorithm.
Cell counting For cell counting the same six proliferating and six involuted lesions used for IHC were stained sequentially for CD45 and either CD3 or CD20, using the Bond polymer refine red (Leica) for CD45 and the polymer refine brown (Leica) for either CD3 or CD20 using the Leica Bond RX auto-stainer. Cell counting was performed on CD45+ only cells, and dually stained CD45+ /CD3+ cells, or CD45+/CD20+ cells, in six fields of view at 40× magnification, with averages taken of the counts.
Real-time polymerase chain reaction Proliferating (n=3) and involuted (n=3) IH samples from six patients, of the original cohort of 12 patients, were used with placenta as a positive control. Total RNA was extracted and quantitated, and reverse transcriptase PCR (RT-PCR) was performed as previously described.7 The following TaqMan Gene Expression Assays were used: CD3G hs00962186_m1, CD20 hs00544819_m1, CD90 hs00174816_m1, CD45 hs04189704_m1 and GUSB hs00939627_m1 (Life Technologies). Data were presented as mean±SD.
In situ hybridisation
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 and captured using an Olympus BX53 light microscope (Tokyo, Japan).
Four μm-thick formalin-fixed paraffin-embedded sections of proliferating (n=3) and involuted (n=3) IH samples from the original cohort of 12 patients were used for mRNA in situ hybridisation (ISH) staining. Pretreatment of the sections was performed by incubating them with sodium citrate (Leica) at 95°C for 15 min followed with 1:1000 proteinase K (Leica) for 10 min at room temperature. Denaturation of the probe was performed at 40°C for 1 h with hybridisation performed for 3 h, using CD90 probe (NM_006288, Affymetrix, California, USA). The CD90 probe was detected using the ViewRNA red stain kit (Affymetrix) followed by staining with haematoxylin. The ISH staining protocol was performed on the Leica Bond Rx autostainer (Leica). All slides were mounted with Histomount RTU (Invitrogen, Auckland, New Zealand). A human tonsillar tissue section was run in parallel as an appropriate positive control.
All tissue samples used in the experiments were confirmed as IH lesions by their expression of GLUT-1 18 (data not shown).
Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
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Statistical analysis Statistical analysis was performed using a t test for independent samples using IBM SPSS (V.22), which included calculating for Levene’s test for equality of variances.
RESULTS IHC staining
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Original article
Figure 1 Representative immunofluorescent immunohistochemical-stained sections of proliferating infantile haemangioma showing (A) a subpopulation of cells costaining for CD45 (red) and CD3 (green) (arrows), and (B) another subpopulation costaining for CD45 (red) and CD20 (green; arrows). Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (blue).
IF IHC staining of proliferating IH demonstrated subpopulations of interstitial cells expressing CD45 (figure 1A, B, red) and either the T lymphocyte marker, CD3 (figure 1A, green), or the B lymphocyte marker, CD20 (figure 1B, green). These subpopulations of CD45+ (figure 2A, B, red) cells that costained with CD3 (figure 2A, green) or CD20 (figure 2B, green) diminished in involuted IH samples. The B lymphocytes that expressed CD20 (see online supplementary figure S1A, B, red) also expressed another B cell marker, CD79a (see online supplementary figure 1A,B, brown), in proliferating (see online supplementary figure 1A) and diminished in involuted (see online supplementary figure 1B) IH lesions. To demonstrate that the B and T lymphocytes are two distinct subpopulations, we performed dual staining for both CD3 (see online
supplementary figure 1C, brown) and CD20 (see online supplementary figure 1C, red) confirming the mutually exclusive expression patterns. The expression of markers of both B and T lymphocytes by cells in the interstitium of IH led us to hypothesise that these cells were potentially derived de novo within the putative haemogenic endothelium of IH.12 To demonstrate this, we investigated the expression of the primitive lymphocyte marker, Thy-1,15 in IH. Intriguingly Thy-1 was only expressed on the CD34+ endothelium (figure 3A, B, green) of proliferating (figure 3A, red), but not in involuted (figure 3B, red) IH lesions. Specificity for CD3, CD20, CD79a and Thy-1 was confirmed with positive staining on human tonsillar tissue (data not shown).
Figure 2 Representative immunofluorescent immunohistochemical-stained sections of involuted infantile haemangioma (IH) showing (A) the subpopulations of CD45+ (A and B, red) cells that coexpressed CD3 (A, green) or CD20 (B, green) diminished in involuted IH. Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (blue). 814
Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
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Original article
Figure 3 Representative immunofluorescent immunohistochemical-stained section of proliferating (A) and involuted (B) infantile haemangioma (IH) showing Thy-1 (red) was abundantly expressed by the CD34+ endothelium (green) in proliferating (A) but not in involuted (B) IH. Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (blue).
Cell counting To determine the relative abundance of CD45+ cells and those costaining with either the B or the T lymphocyte markers, we performed DAB IHC dual staining for CD45 (figure 4A–D, red) with either CD3 (figure 4A, C, brown) or CD20 (figure 4B, D, brown) in both proliferating (figure 4A, B) and involuted (figure 4C, D) IH lesions. The cell count averages for the dual-staining performed
on the CD45+ cells, CD45+/CD3+ cells and CD45+/CD20+ cells on the same six proliferating and six involuted IH lesions are shown in figure 5.
Total CD45+ cells (in CD45/CD3 stain) The number of CD45+ cells in the involuted IH group was significantly and substantially lower than that in the proliferating
Figure 4 Immunohistochemical 3,3-diaminobenzidine showing dual staining for CD45 in both proliferating (2A, B, red) and involuted (2C, D, red) infantile haemangioma lesions with either CD3 (2A, C, brown) or CD20 (2B, D, brown). Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
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Original article proliferating and involuted phases of IH (figure 6). Thy-1 mRNA levels were significantly higher (p=0.017) within proliferating IH than within involuted IH.
In situ hybridisation To confirm that the expression of CD90 at both the IHC and mRNA levels was localised to the same cells, we performed mRNA ISH for CD90, which revealed the expression of CD90 mRNA on the cells of the endothelial layer of proliferating IH lesions (figure 7A, red). This was in contrast to the involuted lesions which did not demonstrate any staining (figure 7B). The tonsillar tissue, as a positive control demonstrated the specificity of the CD90 probe (see online supplementary figure 2, red).
Mass spectrometry Figure 5 Bar graph showing the average total number of CD45+ cells within proliferating and involuted infantile haemangioma, and the proportion of these which were either CD3+/CD45+ or CD20+/CD45+. IH group (t=4.576 at df 10, p0.05, effect size ώ2=8%, mean ( proliferating)=8.5, mean (involuted)=5.2).
We initially confirmed identification of Thy-1 in proliferating IH by matching four peptides, three of which were uniquely matched, to the full length protein (E9PIM6_HUMAN). All four peptides matched to the functional domain and the sequence coverage was 27.9%. In the involuted IH, two peptides were matched to Thy-1, both of which were detected in the proliferating sample, and represented 19.1% sequence coverage. This was followed by determining the relative abundance of the Thy-1 protein by performing quantitation using spectral counting. This demonstrated the abundance of Thy-1 which was 11.5-fold higher in proliferating, relative to involuted IH tissue ( p=0.01; figure 8).
DISCUSSION
Figure 6 Gene expression for CD3G, CD45, CD20 and Thy-1 in proliferating (n=3) and involuted (n=3) infantile haemangioma tissues is presented as ΔCt relative to the housekeeping gene, GUSB. Results are presented as mean±SD.
There has been growing evidence of the presence of a haemogenic endothelium12 within IH with a functional capacity to give rise to erythrocytes14 and myeloid cells.4 19 A population of CD45+ cells has been previously demonstrated within the interstitium of proliferating and involuted IH.4 7 Additionally, Ritter et al9 has reported the presence of T lymphocytes within IH, positive for both CD3 and CD8. In this study, we have demonstrated a lymphocytic phenotype for some of these CD45+ interstitial cells, with a subpopulation expressing CD3 within both proliferating and involuted IH, confirming the presence of a T cells within IH. We have found another subpopulation of the CD45+ cells coexpressing CD20 and CD79a within proliferating IH, inferring a B cell phenotype. To the best of our knowledge, this is the first report showing the presence of B cells within IH. It is intriguing that the number of CD20+ cells was found to be significantly decreased in involuted compared with proliferating IH lesions, yet the mRNA levels for both phases did not reveal any significant change. This may be accounted for, by the inclusion of circulating CD20+ B lymphocytes in the mRNA samples analysed. The ability for haemogenic endothelial phenotypic cells expressing Thy-1 to differentiate into T lymphocytes has been previously reported by Kennedy et al.20 The novel finding of the expression of the primitive lymphocyte marker, Thy-1, both at the protein and mRNA levels by the haemogenic endothelium12 of proliferating but not involuted IH, suggests a potential source of these interstitial lymphocytes from within the IH. The contribution of circulating blood lymphocytes to this population of lymphocytes within IH requires more definitive functional studies and is subject to further investigation. Statistical analysis of cell counting on DAB IHC-stained slides for CD45 and either CD3 or CD20 revealed that the size of the overall CD45+ only subpopulation and the CD45+/CD20+ B cell subpopulation decreases significantly from proliferating to involuted phases of IH, with no significant difference between
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Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
CD45+/CD20+ cells The number of CD45+ cells that are also CD20+ in involuted IH group was significantly lower than that of the proliferating IH group (t=3.386 at df 10, p
Original article
Characterisation of lymphocyte subpopulations in infantile haemangioma Elysia M S Tan,1 Tinte Itinteang,1 Daria A Chudakova,1 Jonathan C Dunne,1 Reginald Marsh,1,2 Helen D Brasch,1 Paul F Davis,1 Swee T Tan1,3 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2015-203073). 1
Gillies McIndoe Research Institute, Wellington, New Zealand 2 University of Auckland, Auckland, New Zealand 3 Centre for the Study & Treatment of Vascular Birthmarks, Wellington Regional Plastic, Maxillofacial & Burns Unit, Hutt Hospital, Wellington, New Zealand Correspondence to Dr Swee T Tan, Gillies McIndoe Research Institute, P.O. Box 7184, Newtown, Wellington 6242, New Zealand; [email protected] TI and STT contributed equally. Received 14 April 2015 Revised 20 May 2015 Accepted 25 May 2015 Published Online First 11 June 2015
ABSTRACT Aims Interstitial CD45+ cells and T lymphocytes have previously been demonstrated within infantile haemangioma (IH). This study investigated the expression of B and T lymphocyte markers by the CD45+ population, and the expression of Thy-1, a marker of thymocyte progenitors, which have the ability to give rise to both B and T cells. Methods Immunohistochemical (IHC) staining was performed on proliferating and involuted IHs for the expression of CD45, CD3, CD20, CD79a, Thy-1 and CD34. The presence of mRNA corresponding to CD45, CD3G, CD20 and Thy-1 was confirmed by reverse transcriptase-polymerase chain reaction in snap-frozen IH tissues. Cell counting of 3,3-diaminobenzidine IHCstained slides was performed on CD45+ only cells and dually stained CD45+/CD3+ cells or CD45+/CD20+ cells and analysed statistically. In situ hybridisation and mass spectrometry were also performed to confirm the presence and abundance of Thy-1, respectively. Results IHC staining showed a subpopulation of CD45+ interstitial cells that expressed the T lymphocyte marker, CD3, and another subpopulation that expressed the B lymphocyte marker, CD20, in proliferating and diminished in involuted IHs. The abundant expression of Thy-1 on the endothelium of proliferating, but not involuted IH, was demonstrated by IHC staining and confirmed by in situ hybridisation and mass spectrometry. Conclusions Both B and T lymphocytes are present within the interstitium of proliferating and involuted IH. The expression of Thy-1 by the endothelium suggests that B and T cells in IH may have originated from within the lesion, rather than migrating from the peripheral circulation.
INTRODUCTION
To cite: Tan EMS, Itinteang T, Chudakova DA, et al. J Clin Pathol 2015;68:812–818. 812
Infantile haemangioma (IH), the most common tumour of infancy, typically undergoes rapid proliferation during infancy ( proliferative phase) characterised by excessive vasculogenesis, followed by slow spontaneous involution over 1–5 years of age (involuting phase) with fibro-fatty deposition that replaces the cellular elements up to 10 years of age (involuted phase).1–3 Cells of multiple lineages have been demonstrated within IH including endothelial, mesenchymal and haematopoietic cells.4 5 The haematopoietic cells are divided into the lymphoid and myeloid lineages.6 We have recently identified and characterised the myeloid cell subpopulations within IH.7 Postnatally, all haematopoietic cells originate in the bone marrow and are derived from a pluripotent haematopoietic stem cell.6 These cells then diverge in the bone marrow, into the lymphoid and myeloid lineages.6 8 The common lymphoid progenitor,
which gives rise to all lymphocyte progeny, then differentiates into B and T cells.6 Ritter et al9 report the presence of a population of CD3+/CD8+/CD4− cells within IH, indicating a cytotoxic T cell phenotype, thus inferring their role in inducing apoptosis in target cells. However, they do not find a strong correlation between the number of T cells and the developmental phases of IH, which would be expected if the T cells were to play a role in the involution of IH. It is unclear what role these T cells play in IH or whether they contribute to the proliferation and/or involution of this tumour. The authors suggest three possible explanations for the presence of these cells: (1) CD8+ T cells may be recruited to the IH via a non-specific inflammatory mechanism, possibly mediated by secretion of cytokines from endothelial cells or other cells within IH; (2) CD8+ T cells may promote proliferation of IH by secreting factors that stimulate endothelial proliferation; and (3) CD8+ T cells may be specifically recruited to the IH and contribute to its regression by inducing apoptosis of cells in IH. There is growing evidence suggesting that IH represents an embryonic developmental anomaly1 10–13 derived from a haemogenic endothelium, with mesenchymal5 and erythropoietic14 capacity. It has also been recently suggested that this haemogenic endothelium has the capability to produce myeloid cells.4 We hypothesise that B and T cells in IH originate from within the lesion, possibly arising from the haemogenic endothelium, rather than migrating from outside, as previously proposed.9 We have previously reported the presence of an interstitial cellular population within IH expressing the pan-haematopoietic cell marker, CD45.4 The presence of T lymphocytes within IH,9 and the previous demonstration of the exclusive expression of CD45 by the myeloid population,7 led us to speculate that these CD45+ cells represent a putative lymphocyte subpopulation within the interstitium of IH. In this study, we investigated the expression of thymocyte differentiation antigen-1 (Thy-1, also known as CD90), a marker of thymocyte progenitors,15–17 which possesses the ability to give rise to both B and T lymphocytes.13 We also characterised the CD45+ interstitial cellular population within IH and investigated the expression of both B and T lymphocyte lineage markers.
MATERIALS AND METHODS Tissue samples Proliferating (n=6) and involuted (n=6) IH samples from 12 patients aged 4–7 (mean, 5.5) months and 6–12 (mean, 9) years were used for this study.
Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
Downloaded from http://jcp.bmj.com/ on November 17, 2015 - Published by group.bmj.com
Original article Immunohistochemical staining
Mass spectrometry
Four μm-thick formalin-fixed paraffin-embedded sections of proliferating (n=6) and involuted (n=6) IH from 12 patients were used for immunohistochemical (IHC) staining. 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, GLUT-1, 1:200 (Cell Marque, Rocklin, California, USA); CD45, 1:600 (Abcam, Cambridge, Massachusetts, USA); Thy-1, 1:100 (Thermo Scientific, Rockford, Illinois, USA) and Ready-to-Use CD3, CD20 and CD79a (all from Leica) with detection using the Bond polymer refine detection kit (Leica). To confirm coexpression of two proteins, selected representative slides of each phase of IH underwent immunofluorescent (IF) IHC staining with the same primary antibodies at the same concentrations, but using an appropriate secondary antibody (goat anti-mouse Alexa-488 or goat anti-rabbit Alexa 594) for detection. All antibodies were diluted in Bond primary antibody diluent (Leica), and all DAB IHC and IF IHC staining were performed on the Leica Bond Rx auto-stainer (Leica). The DAB and IF IHC slides were mounted using either Surgipath micromount mounting media (Leica) or Vectashield hardset mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, California, USA), respectively. Human tonsillar tissue was used as positive control for CD45, CD3, CD20, CD79a and Thy-1.
Total protein samples extracted from proliferating (n=2) and involuted (n=2) IH tissue, from the cohort of six patients used for RT-PCR, were trypsin-digested and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tissue pieces were homogenised by grinding with sand (Sigma-Aldrich) in 150 μL of ice cold radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing 1× Complete Protease inhibitor cocktail EDTA-free (Roche Life Science). After protein quantitation (Qubit 2.0 Fluorometer, Life Technologies, San Diego, California, USA), 100 mg of total protein per sample was precipitated overnight (ProteoExtract Protein Precipitation Kit, Merck Millipore), and the washed protein pellets were resuspended in 50 μL of 5% sodium deoxycholate (SDC), 10 mM dithiothreitol, 100 mM triethylammonium bicarbonate (TEAB) buffer (pH 8.5; Sigma Aldrich) and incubated for 30 min at 80°C. Samples were then alkylated for 60 min by the addition of 40 mM iodoacteamide and diluted in 100 mM TEAB buffer (pH 8.5) to a 500 μL final volume. Protein digestion was performed overnight at 37°C using 4 mg trypsin per sample (modified sequencing grade trypsin from bovine pancreas; Roche Life Science). SDC was precipitated from each sample by the addition of 1% formic acid, pelleted by centrifugation for 30 min at 13 000×g, and the peptide supernatants transferred to fresh microcentrifuge tubes. The SDC pellets were washed with 200 μL of 1% formic acid to remove coprecipitated peptides, and after centrifugation for 30 min at 13 000×g, each wash solution was combined with the previously recovered peptide supernatant. Peptide samples were lyophilised to ∼10 μL, supplemented with 500 μL 0.1% formic acid, purified using OMIX C18 pipette tips (Agilent Technologies) and reconstituted in 200 μL of 4% acetonitrile:0.1% formic acid. LC-MS/MS analysis was performed using an UltiMate 3000 HPLC system (Dionex) interfaced with a LTQ Orbitrap XL mass spectrometer (Thermo Scientific). Thirty-five microlitres of each peptide sample was loaded onto an Acclaim PepMap100, C18 column (3 mm, 100 Å, 75 mm i.d. ×15 cm; Thermo Scientific; 0.3 μL/min flow rate), and peptides were eluted and analysed using data-dependent MS/MS acquisition. Four technical replicates were performed for each sample. Raw MS/MS data files were searched against a complete human protein database (SwissProt KB, 22 October 2014, 69 689 sequences) using Proteome Discoverer V1.4 (Thermo Scientific). Scaffold 4.0 (Proteome Software) was used to establish relative protein abundance in proliferating and involuted IH extracts using the spectral counting algorithm.
Cell counting For cell counting the same six proliferating and six involuted lesions used for IHC were stained sequentially for CD45 and either CD3 or CD20, using the Bond polymer refine red (Leica) for CD45 and the polymer refine brown (Leica) for either CD3 or CD20 using the Leica Bond RX auto-stainer. Cell counting was performed on CD45+ only cells, and dually stained CD45+ /CD3+ cells, or CD45+/CD20+ cells, in six fields of view at 40× magnification, with averages taken of the counts.
Real-time polymerase chain reaction Proliferating (n=3) and involuted (n=3) IH samples from six patients, of the original cohort of 12 patients, were used with placenta as a positive control. Total RNA was extracted and quantitated, and reverse transcriptase PCR (RT-PCR) was performed as previously described.7 The following TaqMan Gene Expression Assays were used: CD3G hs00962186_m1, CD20 hs00544819_m1, CD90 hs00174816_m1, CD45 hs04189704_m1 and GUSB hs00939627_m1 (Life Technologies). Data were presented as mean±SD.
In situ hybridisation
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 and captured using an Olympus BX53 light microscope (Tokyo, Japan).
Four μm-thick formalin-fixed paraffin-embedded sections of proliferating (n=3) and involuted (n=3) IH samples from the original cohort of 12 patients were used for mRNA in situ hybridisation (ISH) staining. Pretreatment of the sections was performed by incubating them with sodium citrate (Leica) at 95°C for 15 min followed with 1:1000 proteinase K (Leica) for 10 min at room temperature. Denaturation of the probe was performed at 40°C for 1 h with hybridisation performed for 3 h, using CD90 probe (NM_006288, Affymetrix, California, USA). The CD90 probe was detected using the ViewRNA red stain kit (Affymetrix) followed by staining with haematoxylin. The ISH staining protocol was performed on the Leica Bond Rx autostainer (Leica). All slides were mounted with Histomount RTU (Invitrogen, Auckland, New Zealand). A human tonsillar tissue section was run in parallel as an appropriate positive control.
All tissue samples used in the experiments were confirmed as IH lesions by their expression of GLUT-1 18 (data not shown).
Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
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Statistical analysis Statistical analysis was performed using a t test for independent samples using IBM SPSS (V.22), which included calculating for Levene’s test for equality of variances.
RESULTS IHC staining
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Original article
Figure 1 Representative immunofluorescent immunohistochemical-stained sections of proliferating infantile haemangioma showing (A) a subpopulation of cells costaining for CD45 (red) and CD3 (green) (arrows), and (B) another subpopulation costaining for CD45 (red) and CD20 (green; arrows). Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (blue).
IF IHC staining of proliferating IH demonstrated subpopulations of interstitial cells expressing CD45 (figure 1A, B, red) and either the T lymphocyte marker, CD3 (figure 1A, green), or the B lymphocyte marker, CD20 (figure 1B, green). These subpopulations of CD45+ (figure 2A, B, red) cells that costained with CD3 (figure 2A, green) or CD20 (figure 2B, green) diminished in involuted IH samples. The B lymphocytes that expressed CD20 (see online supplementary figure S1A, B, red) also expressed another B cell marker, CD79a (see online supplementary figure 1A,B, brown), in proliferating (see online supplementary figure 1A) and diminished in involuted (see online supplementary figure 1B) IH lesions. To demonstrate that the B and T lymphocytes are two distinct subpopulations, we performed dual staining for both CD3 (see online
supplementary figure 1C, brown) and CD20 (see online supplementary figure 1C, red) confirming the mutually exclusive expression patterns. The expression of markers of both B and T lymphocytes by cells in the interstitium of IH led us to hypothesise that these cells were potentially derived de novo within the putative haemogenic endothelium of IH.12 To demonstrate this, we investigated the expression of the primitive lymphocyte marker, Thy-1,15 in IH. Intriguingly Thy-1 was only expressed on the CD34+ endothelium (figure 3A, B, green) of proliferating (figure 3A, red), but not in involuted (figure 3B, red) IH lesions. Specificity for CD3, CD20, CD79a and Thy-1 was confirmed with positive staining on human tonsillar tissue (data not shown).
Figure 2 Representative immunofluorescent immunohistochemical-stained sections of involuted infantile haemangioma (IH) showing (A) the subpopulations of CD45+ (A and B, red) cells that coexpressed CD3 (A, green) or CD20 (B, green) diminished in involuted IH. Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (blue). 814
Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
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Original article
Figure 3 Representative immunofluorescent immunohistochemical-stained section of proliferating (A) and involuted (B) infantile haemangioma (IH) showing Thy-1 (red) was abundantly expressed by the CD34+ endothelium (green) in proliferating (A) but not in involuted (B) IH. Cell nuclei were counterstained with 40 ,6-diamidino-2-phenylindole (blue).
Cell counting To determine the relative abundance of CD45+ cells and those costaining with either the B or the T lymphocyte markers, we performed DAB IHC dual staining for CD45 (figure 4A–D, red) with either CD3 (figure 4A, C, brown) or CD20 (figure 4B, D, brown) in both proliferating (figure 4A, B) and involuted (figure 4C, D) IH lesions. The cell count averages for the dual-staining performed
on the CD45+ cells, CD45+/CD3+ cells and CD45+/CD20+ cells on the same six proliferating and six involuted IH lesions are shown in figure 5.
Total CD45+ cells (in CD45/CD3 stain) The number of CD45+ cells in the involuted IH group was significantly and substantially lower than that in the proliferating
Figure 4 Immunohistochemical 3,3-diaminobenzidine showing dual staining for CD45 in both proliferating (2A, B, red) and involuted (2C, D, red) infantile haemangioma lesions with either CD3 (2A, C, brown) or CD20 (2B, D, brown). Tan EMS, et al. J Clin Pathol 2015;68:812–818. doi:10.1136/jclinpath-2015-203073
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Original article proliferating and involuted phases of IH (figure 6). Thy-1 mRNA levels were significantly higher (p=0.017) within proliferating IH than within involuted IH.
In situ hybridisation To confirm that the expression of CD90 at both the IHC and mRNA levels was localised to the same cells, we performed mRNA ISH for CD90, which revealed the expression of CD90 mRNA on the cells of the endothelial layer of proliferating IH lesions (figure 7A, red). This was in contrast to the involuted lesions which did not demonstrate any staining (figure 7B). The tonsillar tissue, as a positive control demonstrated the specificity of the CD90 probe (see online supplementary figure 2, red).
Mass spectrometry Figure 5 Bar graph showing the average total number of CD45+ cells within proliferating and involuted infantile haemangioma, and the proportion of these which were either CD3+/CD45+ or CD20+/CD45+. IH group (t=4.576 at df 10, p0.05, effect size ώ2=8%, mean ( proliferating)=8.5, mean (involuted)=5.2).
We initially confirmed identification of Thy-1 in proliferating IH by matching four peptides, three of which were uniquely matched, to the full length protein (E9PIM6_HUMAN). All four peptides matched to the functional domain and the sequence coverage was 27.9%. In the involuted IH, two peptides were matched to Thy-1, both of which were detected in the proliferating sample, and represented 19.1% sequence coverage. This was followed by determining the relative abundance of the Thy-1 protein by performing quantitation using spectral counting. This demonstrated the abundance of Thy-1 which was 11.5-fold higher in proliferating, relative to involuted IH tissue ( p=0.01; figure 8).
DISCUSSION
Figure 6 Gene expression for CD3G, CD45, CD20 and Thy-1 in proliferating (n=3) and involuted (n=3) infantile haemangioma tissues is presented as ΔCt relative to the housekeeping gene, GUSB. Results are presented as mean±SD.
There has been growing evidence of the presence of a haemogenic endothelium12 within IH with a functional capacity to give rise to erythrocytes14 and myeloid cells.4 19 A population of CD45+ cells has been previously demonstrated within the interstitium of proliferating and involuted IH.4 7 Additionally, Ritter et al9 has reported the presence of T lymphocytes within IH, positive for both CD3 and CD8. In this study, we have demonstrated a lymphocytic phenotype for some of these CD45+ interstitial cells, with a subpopulation expressing CD3 within both proliferating and involuted IH, confirming the presence of a T cells within IH. We have found another subpopulation of the CD45+ cells coexpressing CD20 and CD79a within proliferating IH, inferring a B cell phenotype. To the best of our knowledge, this is the first report showing the presence of B cells within IH. It is intriguing that the number of CD20+ cells was found to be significantly decreased in involuted compared with proliferating IH lesions, yet the mRNA levels for both phases did not reveal any significant change. This may be accounted for, by the inclusion of circulating CD20+ B lymphocytes in the mRNA samples analysed. The ability for haemogenic endothelial phenotypic cells expressing Thy-1 to differentiate into T lymphocytes has been previously reported by Kennedy et al.20 The novel finding of the expression of the primitive lymphocyte marker, Thy-1, both at the protein and mRNA levels by the haemogenic endothelium12 of proliferating but not involuted IH, suggests a potential source of these interstitial lymphocytes from within the IH. The contribution of circulating blood lymphocytes to this population of lymphocytes within IH requires more definitive functional studies and is subject to further investigation. Statistical analysis of cell counting on DAB IHC-stained slides for CD45 and either CD3 or CD20 revealed that the size of the overall CD45+ only subpopulation and the CD45+/CD20+ B cell subpopulation decreases significantly from proliferating to involuted phases of IH, with no significant difference between
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CD45+/CD20+ cells The number of CD45+ cells that are also CD20+ in involuted IH group was significantly lower than that of the proliferating IH group (t=3.386 at df 10, p
