J. Bux

Neutrophil CD177 (NB1 gp, HNA-2a) expression is increased in severe bacterial infections and polycythaemia vera

The NB1 glycoprotein (CD177, HNA‐2a antigen) is exclusively expressed on human neutrophils. As the clinical significance of CD177 expression is unknown, we investigated its expression in healthy individuals before and after stimulation with granulocyte colony‐stimulating factor (G‐CSF), in patients with rheumatoid arthritis, viral hepatitis, severe bacterial infections and polycythaemia vera. Expression was quantitatively determined by flow cytometry and by real time polymerase chain reaction. Only G‐CSF‐stimulated individuals and patients with severe bacterial infections and polycythaemia showed a significantly (P < 0·001) increased CD177 expression compared with healthy individuals, indicating that neutrophil CD177 expression can increase significantly in certain clinical conditions.

Ouantitation of granulocyte antibodies in sera and determination of their binding sites

Summary An enzyme immunoassay using eluates was developed for the quantitation of granulocyte antibodies in sera. Incubation of donor granulocytes with sera containing granulocyte alloantibodies (NA1, NA2, NB1, 5b. HLA) or autoantibodies resulted in a significantly higher amount of immunoglobulin G (IgG) per cell than did incubation with control sera. Ultracentrifugation of the sera prior to testing reduced unspecific binding of IgG to granulocytes. In eight of 10 assessed sera from patients with Feltys syndrome eluted IgG decreased from elevated to normal levels. Ultracentrifugation further allowed determination of the binding sites of granulocyte‐reactive alloantibodies. Using sera containing NA1‐and NA2‐specific alloantibodies 173000–188000 binding sites on homozygous and 84000–110000 on heterozygous cells were determined. A similar number was found using a Fc gamma receptor III (FcRIII)‐speciflc monoclonal antibody. Granulocytes from patients with paroxysmal nocturnal haemoglobinuria showed greatly reduced binding sites for NA‐specific alloantibodies. The binding sites for the human alloantibody NB1 ranged from 36000 to 318000 and for the 5b alloantibody from 44000 to 65000. Mean values of 139000 binding sites for HLA antigens and 27000 binding sites for the FcRII were determined using monoclonal antibodies.

B‐lymphoblastoid cell lines as a source of reference DNA for human platelet and neutrophil antigen genotyping

BACKGROUND: Human platelet and neutrophil antigens (HPAs, HNAs) are targets for platelet or granulocyte antibodies causing immune thrombocytopenia or neutropenia, respectively. Currently, genotyping is replacing phenotyping as the preferred method of diagnosis of immune cytopenia. To establish a reliable genotyping analysis, however, the availability as reference DNA of genomic DNA from persons of known genotype is essential.

Molecular nature of antigens implicated in immune neutropenias.

Granulocyte (neutrophil) antibodies can cause autoimmune neutropenia, drug-induced neutropenia, immune neutropenia after bone marrow transplantation, neonatal immune neutropenia, refractoriness to granulocyte transfusions as well as febrile and pulmonary transfusion reactions. In the last decade, considerable progress has been made in the characterization of the implicated antigens. In 1998, the Granulocyte Antigen Working Party of the ISBT introduced a new nomenclature for human neutrophil alloantigens (HNA), which is based on the antigens’ glycoprotein location. In the HNA nomenclature the immunogenic (glyco-) proteins are indicated by arabic numbers followed by a letter of the alphabet which identify the (glyco-) proteins’ polymorphisms, i.e. the specific antigens. Currently, seven HNA antigens are assigned to five systems. The HNA-1a, HNA-1b and HNA-1c antigens, the former NA1, NA2, and SH antigens, have been identified as polymorphic forms of the neutrophil Fcγreceptor IIIb (CD16b) encoded by three alleles. Recently, we could elucidate the primary structure of the HNA-2a antigen, the former NB1. We could identify the HNA-2a-bearing glycoprotein as a novel member of the Ly-6/uPAR superfamily which has been clustered meanwhile as CD177. The HNA-3a antigen, the former 5b, is located on a 70–95 kDa glycoprotein. However, its molecular basis is still unknown. Finally, the HNA-4a and HNA-5a antigens, the former MART and OND, were found to be caused by single nucleotide mutations in the αM (CD11b) and αL (CD11a) subunits of the leucocyte adhesion molecules (β3 integrins). The glycoproteins CD11b, CD16b, and CD177 have been found to be also frequent targets ofautoantibodies — approximately 30% of neutrophil autoantibodies are directed against CD16b. Characterization of granulocyte antigens have expanded our diagnostic tools by the introduction of genotyping techniques and immunoassays for antibody identification. In addition, it allowed new insights in the pathophysiology of immune neutropenias and transfusion reactions. Ongoing studies will further improve the prevention and management of granulocyte antibody-mediated diseases.

HNA-1d: a new human neutrophil antigen located on Fcγ receptor IIIb associated with neonatal immune neutropenia.

Neonatal immune neutropenia (NIN) is a rare, but potentially life‐threatening, disorder caused by maternal alloantibodies recognizing paternal neutrophil antigens on fetal cells. Alloantibodies directed against the human neutrophil alloantigen system (HNA)‐1 located on Fcγ receptor IIIb (FcγRIIIb) are most frequently implicated in NIN. In this report, we describe two cases of NIN with alloantibodies against FcγRIIIb, which did not match one of the known HNA‐1a, ‐1b, or ‐1c specificities, but define a new antigen, HNA‐1d.

Alloimmune neonatal neutropenia resulting from immunization to a high- frequency antigen on the granulocyte Fc gamma receptor III

BACKGROUND: Alloimmune neonatal neutropenia is mainly caused by NA‐ or NB1‐specific alloantibodies. An antibody in the serum of a Turkish mother who had given birth to an infant with alloimmune neonatal neutropenia showed no NA or NB specificity and was therefore investigated further.

Neutropenia and anaemia due to carbimazole‐dependent antibodies

Carbimazole‐dependent antibodies to erythrocytes were detected in the sera of three anaemic patients who had been treated with carbimazole for hyperthyroidism. By the use of Rhnull‐typed erythrocytes, we could show that some of these were directed against the proteins of the Rh complex. Carbimazole‐dependent antibodies eluted from erythrocytes showed no binding to other blood cells. One patient also presented with neutropenia and mild thrombocytopenia. Additional carbimazole‐dependent antibodies against the neutrophil‐specific Fcγ receptor IIIb (FcγRIIIb, CD16b) and the broadly expressed platelet endothelial cell adhesion molecule 1 (PECAM‐1; CD31) were detected in this patients serum. Surprisingly, the PECAM‐1‐reactive drug‐dependent antibodies were also detectable in the sera of the other two patients with normal leucocyte and platelet counts. We assume that carbimazole can induce cell lineage‐specific drug‐dependent antibodies that cause cytopenia and also drug‐dependent antibodies against the broadly expressed PECAM‐1 molecule that may cause mild but not severe cytopenia.

Workshop report on the genotyping of blood cell alloantigens.

The immunization against alloantigens present on platelets, granulocytes and red blood cells (RBCs) is responsible for various clinical syndromes. Since the molecular basis of these antigens has become clear during the last decade, genotyping is nowadays used in several laboratories. However, many DNA‐based techniques still have to be evaluated. We therefore organized a workshop on the genotyping of the most relevant alloantigens on platelets and granulocytes as well as on selected RBC alleles.

Serum G-CSF levels are not increased in patients with antibody-induced neutropenia unless they are suffering from infectious diseases

By the use of a G‐CSF‐specific ELISA we determined the serum granulocyte‐colony stimulating factor (G‐CSF) levels in 63 patients with antibody‐induced neutropenia including neonatal immune neutropenia, autoimmune neutropenia, and drug‐induced immune neutropenia. In the sera of 20 patients, elevated G‐CSF levels of 60–1006 pg/ml (normal <39 pg/ml) were observed. These patients suffered from infectious diseases at the time of blood collection. G‐CSF levels normalized after successful antibiotic treatment, indicating that increased G‐CSF production in patients with immune neutropenia may be primarily the result of infection and not of neutropenia.

Typing of the granulocyte-specific NA antigens by restriction fragment length polymorphism analysis.

SUMMARY. An RNA‐based method has been developed to genotype donors for the granulocyte‐specific alloantigens NA1 and NA2. mRNA was isolated from granulocytes, reversely transcribed into cDNA and amplified using an Fcgamma‐receptor III‐1 sequence‐specific primer in the polymerase chain reaction (PCR). PCR products were analysed by restriction fragment length polymorphism (RFLP) using the restriction endonuclease Taq I, which provided a distinct restriction fragment pattern corresponding to the NA alleles. 17 donors were typed by PCR‐RFLP and the results were in close accordance with those obtained by serological phenotyping by granulocyte immunofluorescence and the antigen capture assay MAIGA.