Rhnull red blood cells with reduced CD47 do not show increased interactions with peripheral blood monocytes

Authors


The Rh complex on human red blood cells (RBCs) is thought to consist of a tetramer of two Rh and two Rh-associated glycoprotein (RhAG) subunits to which CD47, LW and glycophorin  B are non-covalently linked (Colin, 2002). Studies suggest that the Rh complex might participate in the association between the membrane lipid bilayer and the RBC skeleton (Colin, 2002). RBCs from Rhnull patients (with mutations in the RH or RHAG genes) are known to be associated with stomatocytosis and haemolytic anaemia (HA) (Colin, 2002).

CD47 functions as a marker of ‘self’ on RBCs (Oldenborg et al, 2000). It binds to signal regulatory protein alpha (SIRPα) on splenic red pulp macrophages, inhibiting RBC elimination in the spleen (Oldenborg et al, 2000). It has been shown that murine CD47 null RBCs, when transfused into normal CD47+ mice, are rapidly cleared due to the absence of inhibitory CD47-SIRPα signalling (Oldenborg et al, 2000). Oldenborg et al (2000) suggested that the HA associated with Rhnull RBCs might be secondary to reduced CD47-SIRPα signalling to splenic macrophages.

Two groups (Bruce et al, 2002; Mouro-Chanteloup et al, 2003) demonstrated that three unrelated patients with protein 4.2 deficient hereditary spherocytosis had an 80–90% reduction in CD47 on their RBCs. Bruce et al (2002) made a similar suggestion as Oldenborg et al (2000) that there may be a relationship to the HA seen in their protein 4.2 deficient patient. Mouro-Chanteloup et al (2003) and others showed a 40–90% decrease in CD47 in RBCs that lack most or all Rh antigens, e.g. RN or D(C)(e), D- -, D•• and Rhnull phenotypes. They stated that as the RN, D- - and D•• RBCs are morphologicaly normal with no signs of HA, then the CD47 deficiency is not responsible for the RBC shape abnormalities and the compensated HA seen in patients with protein 4.2 deficient and Rhnull RBCs (Mouro-Chanteloup et al, 2003).

Our studies support the conclusions of Mouro-Chanteloup et al (2003). We tested RBCs from seven Rhnull individuals with fluorescein isothiocyanate anti-CD47 and flow cytometry, and by a monocyte monolayer assay (MMA) proven to predict the in vivo clinical significance of blood group antibodies (Nance et al, 1987). RBCs of rare phenotypes (e.g. Rhnull, D- -) were stored frozen in liquid nitrogen and thawed at the time of use. Controls for these studies consisted of fresh RBCs (with common Rh phenotype) and frozen/thawed RBCs (age-matched with common Rh phenotype and from one D- - individual). Studies were also performed on heat-treated RBCs [incubated for 20 min at 50°C to artificially ‘age’ the RBCs (Mollison et al, 1993)] and untreated controls. Results are shown in Table I.

Table I.  Results of testing RBCs of common and unusual phenotype with FITC anti-CD47 by flow cytometry and by MMA.
RBCsFITC anti-CD47*MMA†
nMeanRangenMeanRange
  1. RBCs, red blood cells; FITC, fluorescein isothiocyanate; MMA, monocyte monolayer assay; NA, not applicable

  2. *Results expressed as median fluorescence intensity.

  3. †Results expressed as percentage of reactive monocytes; >5% reactivity indicates clinical significance based on monocyte Fc receptor interactions with IgG/C3-coated RBCs (Garratty & Arndt, 1998).

Common Rh (fresh)17141·4107·5–165·50NANA
Common Rh (frozen)7134·7115·5–167·072·30·3–5·4
Rhnull (frozen)725·920·2–31·672·90·7–6·4
D- - (frozen)141·1NA11·9NA
Heat-treated1115·5NA24·63·7–5·5
Untreated1134·6NA22·72·0–3·3

The results confirmed that Rhnull and D- - RBCs have greatly reduced levels of CD47 when compared with control RBCs. The MMA results of the Rhnull RBCs were comparable with those of the age-matched frozen/thawed RBCs of common phenotype. It is of interest to note that the ‘oldest’ frozen RBCs (frozen 16–17 years) gave the highest MMA results (5·4% and 6·4%), while the ‘youngest’ frozen RBC sample (frozen 1 year) gave the lowest MMA result (0·3%). Heated RBCs gave similar results to unheated controls. Thus, we could not find any evidence that RBCs with greatly decreased amounts of CD47 (Rhnull RBCs) are phagocytosed more than RBCs with normal amounts of CD47.

Ancillary