Immunohistochemical characterisation of immune cells in fixed equine endometrial tissue: a diagnostic relevant method

Abstract

Endometrial diseases are the major cause of subfertility in mares. The detection of subclinical diseases requires the histopathological examination of an endometrial biopsy. To prevent autolysis endometrial biopsies have to be fixed prior to their submission to the diagnostic laboratory. The most frequent type of chronic subclinical inflammation is non-suppurative endometritis. An immune mediated pathogenesis is suspected. Type 1 and type 2 adaptive immune responses exist; these are mediated by CD4+ helper T cells, CD8+ cytotoxic T cells, CD20+ B cells, plasma cells as well as polarized macrophages. In equine research, CD172a has been proposed as panmacrophage marker, CD14 and CD206 as markers of M1 and M2 polarisation, respectively; immunostaining for these antigens was only performed on cryostat sections of unfixed equine tissue. The aim of the present study was a) to establish an immunohistochemical method for the detection of macrophages in fixed equine tissue samples and b) to characterize the immune cell populations in fixed endometria with and without chronic endometritis. In regard to the former objective, immunostaining for CD172a, CD14 and CD206 was performed on paraffin embedded equine tissue with resident macrophages (lymph node, liver, small intestines) fixed either with IHC Zinc Fixative or 10 % neutral buffered formalin. In regard to the latter, immune cell populations in zinc fixed paraffin embedded (ZnFPE) endometrial biopsies of mares without endometritis (n = 4) and those with a superficial non-suppurative endometritis (n = 28) were compared. This study established an immunohistochemical method for the detection of the examined macrophage markers within fixed tissue samples, i.e. ZnFPE (CD172a, CD14, CD206) and formalin fixed paraffin embedded (FFPE) tissue after antigen retrieval (CD172a, CD206). It indicated that CD172a likely represents a pan-macrophage marker also in the equine endometrium. Immunohistochemical phenotyping showed that non-suppurative endometritis is mainly a T cell mediated disease with participation of plasma cells. In addition to CD4+ and CD8+ T cells, results indicate the presence of CD4-/CD8-double negative T cells. B cells and/or macrophages were observed in 15 cases. Endometrial macrophages expressed mainly CD172a and only rarely CD206, whereas CD14 was not detected. Notably, examined endometria showed a high variability in the numbers of T cell populations as well as plasma cells. This suggests the existence of subforms of this disease that may even differ in pathogenesis and etiology. The established method of immunophenotyping can be included in the routine diagnostic work-up of equine endometrial biopsies and can be beneficial for the identification of new therapeutic approaches. Its standardized application will likely assist to uncover immunological features that may predispose mares to develop persistent endometritis. The successful future application of this method will likely be beneficial for the genital health of individual mares, it also may help to prevent...

The Healthy and Diseased Equine Endometrium: A Review of Morphological Features and Molecular Analyses

“…Low numbers of resident CD3+ T lymphocytes are present within the luminal and glandular epithelium as well as the stroma of the SC and SP [18,22]. CD3+ cells are more numerous in the SC than in the SP [22].…”

“…CD3+ cells are more numerous in the SC than in the SP [22]. According to Rudolph et al [22], CD3+ T lymphocytes in the SC and SP range from 74 to 96 and from 21 to 44 per five 40× HPFs, respectively. Numbers of CD3+ T lymphocytes in the luminal and glandular epithelium vary from 4 to 17 and from 5 to 26 per five 40× HPFs, respectively [22].…”

“…According to Rudolph et al [22], CD3+ T lymphocytes in the SC and SP range from 74 to 96 and from 21 to 44 per five 40× HPFs, respectively. Numbers of CD3+ T lymphocytes in the luminal and glandular epithelium vary from 4 to 17 and from 5 to 26 per five 40× HPFs, respectively [22]. The SC contains also more resident CD4+ lymphocytes and CD8+ lymphocytes than the SP [22,56].…”

“…Low numbers of resident CD3+ T lymphocytes are present within the luminal and glandular epithelium as well as the stroma of the SC and SP [18,22]. CD3+ cells are more numerous in the SC than in the SP [22]. According to Rudolph et al [22], CD3+ T lymphocytes in the SC and SP range from 74 to 96 and from 21 to 44 per five 40× HPFs, respectively.…”

“…In recent years, a major research focus in equine endometrial pathology was to reveal disease-associated cellular and molecular mechanisms [15][16][17][18][19][20][21][22][23][24][25][26][27][28]. This will help to gain further insights into the pathogenesis of endometrial diseases [15][16][17][18][19][20][21][22][23][24][25][26][27][28], and it may contribute to the development of effective prophylactic regimes and novel treatment options. Some of the identified biological parameters have the potential to be used as biomarkers for endometrial health and disease.…”