Another blow to leukocyte antibody-mediated TRALI

Keywords::

• CTL2
• HNA-3a
• neutrophil antibody
• SLC44A2
• TRALI

Transfusion-related acute lung injury (TRALI) is a serious reaction that occurs following a blood transfusion. Although the exact pathophysiologic mechanism has not been fully determined, recent animal models [1,2] confirm that transfusion of various biologic response modifiers (BRMs), including bioactive lipids [3], sCD40L [4] and leukocyte antibodies [5], into a susceptible patient activates primed neutrophils in the lung resulting in lung injury, pulmonary edema and acute respiratory distress – the clinical hallmarks of TRALI.

Transfusion-related acute lung injury continues to be the most frequent cause of transfusion-related death [101]. However, since blood centers in the USA implemented the use of male-only plasma in the fall of 2007 and the screening of blood donors for HLA antibodies in the fall of 2008, TRALI fatalities have steadily decreased from 34 reports in 2007 to 16 reports in 2008 and 13 reports in 2009 [101]. These risk-reduction measures have apparently reduced the transfusion of leukocyte antibodies, which are the most frequently implicated BRMs in TRALI. Although several different leukocyte antibodies have been shown to cause TRALI, antibodies specific for the human neutrophil antigen (HNA)-3a are the most frequent cause of fatal TRALI [5]. Unlike HLA antibodies, blood centers have been unable to screen for anti-HNA-3a because of a lack of a suitable assay.

Despite discovery of the HNA-3 antigens over 45 years ago [6], the protein carrier remained unknown until its recent identification by our group and independently by another group in Germany [7,8]. Using genome-wide association studies, we identified a single nucleotide polymorphism (SNP) 542G→A in the gene SLC44A2 that encodes an arginine (R)→glutamine (Q) substitution at amino acid 154 in choline transporter-like protein 2 (CTL2) [7]. The nucleotide position for the SNP is 461 if numbering from the ATG start site. Further work using mass spectrometry provided additional evidence that this amino acid change in CTL2 determines the HNA-3a and -3b antigens, respectively [7].

CTL2 is a complex multipass protein with ten transmembrane spanning regions and five extracellular loops, which explains why its identification as the carrier for HNA-3a/3b remained undetermined for so long. Once released from the cell membrane, HNA-3a epitopes on the majority of CTL2 molecules are lost, making detection of HNA-3a antibodies using conventional immunoprecipitation methods extremely difficult. The gene SLC44A2, which encodes CTL2, is one member of a superfamily of solute carrier (SLC) proteins consisting of 55 members with over 362 potentially functional genes that encode proteins that transport amino acids, oligopeptides, sugars, inorganic and organic ions, bile salts, carboxylate, acetyl coenzyme A, metals, biogenic amines, neurotransmitters, vitamins, fatty acids, lipids, nucleosides, ammonium, choline, thyroid hormone, urea, and other molecules across cell membranes [9]. The function of CTL2 is as yet unknown, but the presence of a KOG1362 homology domain suggests it might function as a lipid transporter [10–13]. The finding that crosslinking of HNA-3a on neutrophils is a potent trigger for activation [14] suggests that CTL2 is functionally important in this cell type and might explain the severity of TRALI reactions induced by HNA-3a-specific antibodies [5,14–16]. Determination of the function of CTL2 could provide important information helpful for the elucidation of the pathophysiologic mechanism responsible for TRALI and neutrophil function in general. Interestingly, autoantibodies specific for CTL2 on supporting cells of the inner ear have been implicated as a cause of autoimmune deafness [10,11].

Identification of the SNP that determines HNA-3a/3b is important for blood transfusion safety, since it is now possible to genotype blood donors to identify those at risk of producing HNA-3a antibodies. It should also now be possible to develop a blood donor screening assay for antibodies to HNA-3a and, for the first time, HNA-3b, to further reduce the risk of TRALI. One approach to producing a suitable target protein for detection of HNA-3 antibodies would be to use CTL2 proteins isolated from blood leukocytes, but this would require large volumes of blood in order to obtain enough leukocytes for isolation of sufficient protein. Alternatively, using recombinant DNA technology, recombinant CTL2 protein could be expressed in a suitable cell line allowing for production of large quantities of protein from cultured cells. Expression of full-length recombinant human CTL2 in Sf9 insect cells using two different baculovirus constructs was recently reported [10], making this approach a distinct possibility. Greinacher et al. were able to show reactivity of two HNA-3a antibodies with glutathione-S-transferase-fusion proteins by Western blot indicating that specific portions of CTL2 might be suitable for use in antibody detection [8]. Therefore, another approach would be to produce recombinant CTL2 proteins or peptide fragments that could be coupled to a solid support in an ELISA format, which unlike Western blot is amenable to large-scale screening of donor sera. Our group is currently developing such an assay.

The CTL2 protein sequence is highly conserved across different species, being more than 90% identical in humans, guinea pigs, mice, and rats, including the R154 form in both mice and rats [10,11]. It is therefore possible that HNA-3b/3b donors could be immunized by naturally occurring HNA-3a. Additional evidence that HNA-3a might be naturally immunogenic is two fatal cases of TRALI triggered by anti-HNA-3a present in plasma from two different non-transfused male blood donors [Silliman C, Pers. Comm.]. In addition, van Leeuwen et al. made the observation that HNA-3 antibodies maintain their presence in the circulation for a long period of time following the initial immunizing event, if the immunizing event is pregnancy [6]. We have made the same observation. All of the HNA-3a antibodies we have detected in our reference lab were from multiparous women, all of whom were over 50 years of age at the time of antibody detection. Therefore, HNA-3a is not only probably quite immunogenic, but once an individual is immunized, the antibodies remain in circulation for long periods, so the incidence of HNA-3a and HNA-3b antibodies is probably much higher than is known. These characteristics of anti-HNA-3a together with its ability to induce explosive activation of neutrophils, make HNA-3a antibodies especially prone to cause serious TRALI reactions and demand that measures to screen for their presence in the blood donor population be implemented. Identification of the HNA-3a/3b protein carrier will now greatly facilitate implementation of such measures and deliver another blow to leukocyte antibody-mediated TRALI.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.