Abstract

AIHA is characterized by a high erythrocyte destruction rate associated to autoantibodies directed against blood cell antigens and is usually treated with glucocorticoid (GC). SIRP-a (Signal Regulatory Protein-a)) is an inhibitory receptor in phagocytes and a direct substrate for the phosphatase SHP-1, an important regulator of macrophage proliferation and activation. SIRP-a activation and consequent phosphorylation of immunoreceptor tyrosine-based inhibitory motifs, occur by the binding to CD47 on the erythrocyte membrane, and allow SHP-1, SHP-2 and SHIP recruitment, which in turn dephosphorylate specific protein substrates involved in the mediation of several physiologic effects. The aim of this study was to evaluate the “in vitro” and “in vivo” effects of GC on SIRPa and SHP-1 expression. Peripheral blood monocytes (PBM) were isolated from AIHA patients with and without GC therapy. For the “in vitro” studies PBM from healthy donors and U937 cells were cultured for 48 hours with Dexamethasone (Dexa-1mM) and/or IFNg (100U/ml) and TNFa (1000U/ml). SIRPa and SHP-1 mRNA expression was determined by Real Time PCR, using b-actin expression as an internal control and PBM from healthy donor as a calibrator. AIHA patients underwent clinical and immunohematological investigation. SIRPa and SHP-1 mRNA expression was significantly increased in U937 cells and normal PBM treated with IFN/TNF alone (Mean±SD, SIRPa U937: 4.0±3.2, p=0.009; PBM: 14.9±9.0, p=0.004)(SHP- 1 U937:8.1±6.9, p=0.0002; PBM 12.3±7.1, p=0.006) or associated with Dexa (Mean SIRPa U937:2.7±1.3, p=0.02; PBM 36.1±34.5, p=0.0001)(SHP-1 U937 :5.7±4.2, p=0.002; PBM: 18.9±16.0, p=0.001) compared to basal conditions (Mean SIRPa U937 :1.8±2.1; PBM 3.8±3.9)(SHP 1 U937 :1.7±2.2; PBM 3.9±4.0). Regarding treatment with Dexa alone, SIRPa and SHP-1 were significantly increased in normal PBM but not in U937 (SIRPa 24.8±12, p=0.0004, SHP-1 19.2±6.2, p=0.0001). SIRP-a and SHP-1 expression was significantly higher in PBM from AIHA patients (SIRPa 5.6±1.9, SHP-1 6.1±1.8 n=6) compared to normal (SIRPa 1.8±1.40; p=0.008, SHP-1 2.4±2.1, n=10; p=0.01). After GC therapy, SIRP-a and SHP-1 expression was similar in PBM of AIHA patients (SIRPa 0.6±0.3, SHP-1 0.9±0.18, n=5) compared to healthy donors. AIHA patients studied before GC therapy showed positive direct antiglobulin test (DAT) with anti-IgG and C3d, low hemoglobin (7.7±3.3g/dl) and hematocrit (23.6±11.6%), and reticulocytosis (258.1±194.5). AIHA patients studied after GC therapy showed DAT with only anti-IgG, normal level of hemoglobin (12.1±1.6), hematocrit (35.1±4.8) and reticulocytes (71.8±42.3). In the present study, SIRPa and SHP1 mRNA were higher in mature monocytes compared to U937 and upregulated by dexametasone and IFN /TNF. This result could suggest that this pathway is involved in the reduction of phagocytosis by GC. However, patients with AIHA showed upregulation of these proteins in basal conditions and after GC treatment and hemolysis reduction, SIRPa and SHP1 mRNA expression decreased to normal levels. A balance between inhibition and activation signals determined the macrophage phagocytic activity. Increased SIRP-a and SHP-1 expression in AIHA patients before GC therapy could be a consequence of a homeostatic mechanism, activated by massive phagocytosis, with the purpose of inhibiting the predominant activating signals.

Author notes

Disclosure:Research Funding: This study was supported by FAPESP.