Platelet Proteins Cause Allergic Transfusion Reactions through an Immunoglobulin-Dependent Mechanism

Background: A general understanding of allergic transfusion reaction (ATR) mechanisms remains elusive. Various hypotheses invoke proteins, small molecules, mitochondria, or microparticles that may be plasma or platelet derived and suggest antibody dependent or independent mechanisms. There has been no systematic comparison of these proposed mechanisms. The aim of this study is to characterize the mechanistic determinants of ATRs. Methods: Basophil enriched cell suspensions were collected from healthy donors (n=8). Basophil histamine release was measured in response to platelet-derived components: platelet component supernatant (plasma), platelet lysate, and manipulated platelet lysates were examined to characterize the unknown allergic stimuli. Lysate manipulations were: 1) dialysis against a 3,500 molecular weight cutoff membrane, 2) butanol/DIPE delipidation, 3) trypsinization with immobilized trypsin beads, 4) mild heat denaturation at 47˚C for 20 minutes, and 5) ultracentrifugation at 200,000 gfor 1hr. Immunoglobulin-dependent mechanisms were investigated through lactic acid immunoglobulin depletion from the basophil cell surface and signaling inhibition with ibrutinib. Histamine release from magnetic bead isolated platelet mitochondria was compared to platelet lysate with or without DNase treatment. Platelet lysate histamine content represented an average of <4% of total basophil histamine content. The residual histamine originating from platelet lysate was subtracted from post-reaction histamine concentrations. Atopic histories and grass, tree, and weed-specific IgE were measured in basophil donors. Results: Robust, dose-responsive histamine release to platelet lysate was observed in two of eight healthy donors. No histamine release was observed with plasma. Reactivity did not correlate with the clinical allergic phenotype, and reactive donors were nulliparous and had no prior transfusion. Trypsin treatment of platelet lysate reduced histamine release by 39% (p=0.008). Delipidation of platelet lysate decreased histamine release by 20% (p=0.051). Dialysis, ultracentrifugation, and mild heat denaturation of platelet lysate did not significantly affect histamine release, compared to unmanipulated platelet lysate. To investigate the immunoglobulin dependence of basophil activation, in separate experiments we 1) depleted immunoglobulins, including IgE, from the basophil cell surface, and 2) inhibited immunoglobulin-mediated intracellular signaling with ibrutinib. Histamine release in response to platelet lysate significantly decreased in both cases. Finally, isolated platelet mitochondria induced minimal basophil histamine release, and DNase treatment did not inhibit activation of basophils by platelet lysate. Conclusion: Type I immediate hypersensitivity to platelet, not plasma, proteins is a primary mechanism for ATRs. Small molecules, microparticles, and mitochondria are not significant contributors to ATRs in this human basophil model. Prior sensitization to human proteins is not required for basophil responses to platelet proteins. Donor variability and storage conditions that promote accumulation of soluble platelet-derived proteins may contribute to ATRs. Disclosures No relevant conflicts of interest to declare.

The CML-related oncoprotein BCR/ABL induces expression of histidine decarboxylase (HDC) and the synthesis of histamine in leukemic cells

Basophil numbers are typically elevated in chronic myeloid leukemia (CML) and increase during disease progression. Histamine is an essential mediator and marker of basophils and is highly up-regulated in CML. We examined the biochemical basis of histamine synthesis in CML cells. The CML-specific oncoprotein BCR/ABL was found to promote expression of histidine decarboxylase (HDC) and synthesis of histamine in Ba/F3 cells. Moreover, the BCR/ABL tyrosine kinase inhibitors imatinib (STI571) and nilotinib (AMN107) decreased histamine levels and HDC mRNA expression in BCR/ABL-transformed Ba/F3 cells, in the CML-derived basophil cell line KU812, and in primary CML cells. Synthesis of histamine was found to be restricted to the basophil compartment of the CML clone and to depend on signaling through the PI3-kinase pathway. CML cells also expressed histamine receptors (HRs), including HR-1, HR-2, HR-4, and histamine-binding CYP450 isoenzymes which also serve as targets of HR antagonists. The HR-1 antagonists loratadine and terfenadine, which bind to CYP450, were found to counteract proliferation of CML cells, whereas no growth inhibition was observed with the HR-1 antagonist fexofenadine which is not targeted or metabolized by CYP450. Moreover, DPPE, an inhibitor of histamine-binding CYP450 isoenzymes, produced growth inhibition in CML cells. Together, these data show that BCR/ABL promotes histamine production in CML cells and that certain HR-targeting drugs exert antileukemic effects on CML cells.

The basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatase/phosphodiesterase 3

It has recently been shown that monoclonal antibody (MoAb) 97A6 detects a surface antigen expressed on basophils and their CD34+ precursor cells, as well as the basophil cell line KU-812. In this report the partial amino acid sequence of affinity chromatography– and sodium dodecyl sulfate–polyacrylamide gel electrophoresis–separated 97A6 antigen(s) from KU-812 lysates was determined. Sequence alignment of high-performance liquid chromatography–selected tryptic peptides from the resulting 130- and 150-kd bands revealed a 100% identity with amino acids 393 to 405 of ectonucleotide pyrophosphatase/phosphodiesterase-3 (E-NPP3; CD203c) but not of the related ectoenzyme PC-1 (E-NPP1). Moreover, MoAb 97A6 selectively recognized 293 cells transfected with human E-NPP3, but did not react with cells transfected with PC-1 or parental 293 cells. In addition, E-NPP3 messenger RNA expression was detected in basophils but not other peripheral blood cells. Finally, MoAb 97A6 immunoprecipitated phosphodiesterase activity from KU-812 cells and peripheral blood basophils, but not from other cell populations. These data demonstrate that MoAb 97A6 recognizes the functionally active type II transmembrane ectoenzyme E-NPP3.