IgE-Mediated Immune Response and Antibody-Mediated Rejection

Abstract

Background and objectives: Active antibody-mediated rejection is the main cause of kidney transplant loss, sharing with SLE the alloimmune response and the systemic activation of the IFN-α pathway. IgE-mediated immune response plays a key role in the development of SLE nephritis and is associated with IFN-α secretion. The aim of our study was to investigate IgE-mediated immune response in antibody-mediated rejection.

Design, setting, participants, & measurements: This was a cross-sectional study of 56 biopsy-proven antibody-mediated rejection study participants, 80 recipients with normal graft function/histology (control), 16 study participants with interstitial fibrosis/tubular atrophy, and six participants with SLE. We evaluated graft IgE deposition, tryptase (a mast cell marker), and CD203 (a specific marker of activated basophils) by immunofluorescence/confocal microscopy. In addition, we measured serum concentration of human myxovirus resistance protein 1, an IFN-α-induced protein, and anti-HLA IgE.

Results: We observed a significantly higher IgE deposition in tubules and glomeruli in antibody-mediated rejection (1766±79 pixels) and SLE (1495±43 pixels) compared with interstitial fibrosis/tubular atrophy (582±122 pixels) and control (253±50 pixels). Patients with antibody-mediated rejection, but not control patients and patients with interstitial fibrosis/tubular atrophy, presented circulating anti-HLA IgE antibodies, although with a low mean fluorescence intensity. In addition, immunofluorescence revealed the presence of both mast cells and activated basophils in antibody-mediated rejection but not in control and interstitial fibrosis/tubular atrophy. The concentration of circulating basophils was significantly higher in antibody-mediated rejection compared with control and interstitial fibrosis/tubular atrophy. MxA serum levels were significantly higher in antibody-mediated rejection compared with control and correlated with the extent of IgE deposition.

Conclusions: Our data suggest that IgE deposition and the subsequent recruitment of basophils and mast cells within the kidney transplant might play a role in antibody-mediated rejection.

Keywords: IgE; basophils; chronic rejection; interferon alpha; mast cells.

Graphical abstract

Figure 1.

Deposition of IgE antibodies in graft biopsies. IgE deposition (green) in tubulointerstitial (A–D) and glomerular areas (E–H) was investigated by confocal laser microscopy in antibody-mediated rejection (ABMR; n=30), control (n=40), and interstitial fibrosis/tubular atrophy (IFTA) graft biopsies (n=16). Patients with SLE (n=6) were used as positive controls for IgE tissue immunofluorescence. Nuclei are highlighted with To-pro-3 in blue. Negative control was obtained as described in Materials and Methods (K). Overall quantification of mean florescence intensity, performed as described in Materials and Methods and expressed as percentage of pixels per area fraction, demonstrated a statistically significant higher IgE deposition in ABMR biopsies compared with IFTA and control graft tissues (I). Serum total IgE levels, expressed in units per milliliter, were significantly lower in patients with ABMR compared with control graft recipients (J).

Figure 2.

Evaluation of graft-infiltrating and circulating basophils. The number of graft-infiltrating activated basophils cells (CD203c expression; red), along with IgE deposition (green), was investigated by confocal microscopy in ABMR (n=30), control (n=40), and IFTA (n=16) biopsies; patients with SLE (n=6) were used as positive controls for tissue immunofluorescence (A–D). Nuclei are highlighted with To-pro-3 in blue. The arrows indicate colocalization of IgE (green) and CD203c (red). Double-positive cells (yellow) are expressed as mean n cells per field ± SD (E). The absolute number of circulating basophils subset was detected on blood samples obtained from patients with ABMR (n=40), control patients (n=80), and IFTA graft recipients (n=16) and expressed as mean n 10e3/µl ± SD (F).

Figure 3.

Analysis of graft-infiltrating mast cells. Graft-infiltrating mast cells (tryptase expression; red), along with IgE deposition (green), were visualized by confocal microscopy in ABMR (n=30), IFTA (n=16), and control (n=35) biopsies; patients with SLE (n=6) were used as positive controls for tissue immunofluorescence (A–D). Nuclei are highlighted with To-pro-3 in blue. The white rectangles indicate colocalization of IgE (green) and tryptase (red; B). The number of tryptase-positive cells (yellow), counted by two observers blinded to the origin of the slides and expressed as mean n cells per field ± SD, was significantly higher in ABMR biopsies compared with IFTA and control graft tissues (E).

Figure 4.

Analysis of MxA (human myxovirus resistance protein 1) serum levels. MxA serum levels were evaluated by ELISA test in patients with ABMR (n=52) and in an independent set of anti-HLA⁺ (n=12) and anti-HLA⁻ control transplant recipients (n=63; A). Receiver operating characteristic curve analysis was used to evaluate the sensitivity and specificity of MxA as a marker of ABMR (B). AUC, area under the curve.