ALBERT

Description: The human neutrophil-specific adhesion antigen CD177 (NB1, HNA-2a or PRV-1) becomes upregulated on the cell surface in a number of inflammatory settings, and human alloantibodies specific for CD177 have been implicated in the development of transfusion related acute lung injury (TRALI). Recent studies have shown that CD177 functions as an important counter-receptor for the endothelial cell-cell junctional protein PECAM-1 (CD31) – a heterophilic adhesive interaction that appears to be mediated by Ig domain 6 of PECAM-1 and a still-to-be-identified site on CD177. Interestingly, a common single nucleotide polymorphism (SNP) exists within the PECAM-1 gene that produces an S536N amino acid substitution within Ig domain 6 that is proximal to the CD177 binding site. Whether this polymorphism influences PECAM-1/CD177 interactions and subsequent neutrophil transendothelial cell migration, however, is not known. To investigate the potential for the S536N PECAM-1 SNP to affect functionally important interactions between PECAM-1 and CD177, recombinant CD177 and PECAM-1 S536 or N536 allelic isoforms were produced in CHO mammalian cells, purified by affinity chromatography, and subjected to surface plasmon resonance analysis. We found that the S536 isoform of PECAM-1 bound with much higher affinity to CD177 than did its N536 counterpart (Ka for PECAM-1S536/CD177 = 5×10−7 versus a Ka for PECAM-1N536/CD177=1×10−7). To analyze the possible functional impact of the S536N polymorphism on neutrophil transendothelial migration, fMLP-stimulated neutrophils were allowed to transmigrate across human umbilical vein endothelial cell (HUVEC) monolayers that had been genotyped to be either PECAM-1 homozygous SS, heterozygous NS, or homozygous NN. We found that neutrophils migrated significantly faster across HUVECs expressing the PECAM-1S536, rather than the PECAM-1N536, allelic isoform. In addition, western blot analysis using a phospho-specific antibody revealed that cytoplasmic PECAM-1 ITIM tyrosines became phosphorylated to a greater extent in PECAM-1S536- versus PECAM-1N536-expressing HUVECs that had been incubated with either soluble recombinant CD177 or an anti-PECAM-1 mAb specific for Ig domain 6. Taken together, these data demonstrate that heterophilic interactions between neutrophil CD177 and endothelial cell PECAM-1 play an important role in neutrophil transendothelial migration, and the naturally-occurring S536N polymorphism within Ig domain 6 likely affects ligand binding-induced downstream PECAM-1 signaling. Allele-specific, PECAM-1-mediated effects on neutrophil transendothelial migration and inflammatory events such as TRALI may represent an important area for future investigation.

Description: Transfusion related lung injury (TRALI) is a leading cause for transfusion related morbidity and mortality in industrialized countries. The majority of cases is induced by antibodies present in donor plasma, and exclusion of antibody carriers from the donor pool has decreased the number of TRALI cases significantly. However, TRALI is still reported. We have identified a new and previously overlooked mechanism in which soluble antigens present in the blood component become the target of pre-existing antibodies present in the recipient. Some of us have recently reported a case of TRALI in a female patient precipitating after the transfusion of 120 mL of leuko-reduced packed red blood cells (PRBC). Immediate serologic work-up revealed no antibody in the donor material; but anti-CD177 was detected in the recipient's blood. The following transfusions with two units of PRBCs from CD177 negative donors were uneventful. CD177 is a glycosylphosphatidylinositol-anchored protein exclusively expressed on neutrophils. Only a small minority of Caucasians (

Description: Autoantibody-opsonized platelets in immune thrombocytopenia (ITP) are thought to be destroyed primarily by macrophage Fc gamma receptor (FcγR)-mediated phagocytosis in the spleen. Blockade of splenic macrophage FcγRs has been proposed as a therapeutic mechanism for ITP intervention. Unfortunately, the contribution of specific FcγRs to disease in ITP remains unknown. Our objective was to determine which FcγRs are responsible for the phagocytosis of ITP autoantibody-opsonized platelets by splenic macrophages. Splenic macrophages were purified by CD14 positive selection from spleens of splenectomized ITP patients, and were treated with blocking antibodies to FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII. Blocking antibodies were deglycosylated to prevent non-specific blocking effects by their Fc region. Two separate ITP sera confirmed positive for anti-GPIIb/IIIa autoantibodies by the monoclonal antibody immobilization of platelet antigens (MAIPA) assay were used to opsonize healthy donor human platelets. Phagocytosis was determined by confocal microscopy and non-phagocytosed (external) platelets were differentiated by an anti-platelet antibody stain following macrophage fixation. Human ITP splenic macrophages were found to express FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII, and expression was not significantly different compared to healthy (trauma) controls (n=5). The two anti-GPIIb/IIIa-positive ITP sera induced a mean 3.7- and 4.2-fold increase of platelet uptake by ITP splenic macrophages relative to normal human serum controls (n=3 each, p

Description: Abstract 40 Transfusion related acute lung injury (TRALI) is a severe side-effect of blood transfusion characterized by the acute onset of non-cardiogenic pulmonary edema. Meanwhile, TRALI turned out to be the leading cause of transfusion related fatalities. Accumulated evidence demonstrates that antibodies against human neutrophil antigens (HNAs) play a major role in the pathomechanism of TRALI. Recent studies show that antibodies against the allelic isoform of choline transporter like protein 2 (CTL-2 Arg154 isoform; also known as HNA-3a) are associated with high TRALI mortality. The mechanism underlying this fatal reaction, however, is not clear. In this study, we aimed to identify the mechanism of fatal TRALI induced by HNA-3 antibody under in vitro as well as in vivo conditions. Analysis of mRNA by real-time PCR in different blood cells and endothelial cells (EC) revealed abundant copies of CTL-2 transcripts in ECs in comparison to neutrophils and platelets. This result was confirmed by immunochemical analysis using rabbit antibody specific for CTL-2 as well human antibodies against HNA-3a. In contrast to treatment of neutrophils, treatment of EC with HNA-3a antibodies, but not with antibodies against the antithetical allelic CTL-2 isoform (Gln154 isoform; HNA-3b), leads to significant production of reactive oxygen species (ROS). When HNA-3aa EC monolayers were treated with human anti-HNA-3a antibodies, a significant increase in albumin-FITC influx in transwell system was observed when compared to controls. In line with this observation, a strong reduction of transendothelial electrical resistance was measured. Microscopically, drastic stress fiber formation and gap formation was visible. Immunoblotting analysis of HNA-3a treated ECs showed a significant degradation of VE-cadherin. This observation indicates that anti-HNA-3a antibodies induce EC barrier disturbance via ROS-mediated destabilization of VE-cadherin in cell junctions. Accordingly, this antibody-mediated barrier leakage can be ameliorated by the vasoactive peptide adrenomedulin (ADM), which prevents cell destruction in response to oxidative stress. In an in vivo murine model of TRALI, with lipopolysaccharide primed C57BL/6 mice upon injection of HNA-3a antibodies a significant increase in lung weight and elevated concentration of albumin and number of neutrophils in the bronchoalveolar lavage was observed, indicating the formation of lung edema in these mice. Neutrophil depletion mitigated this effect in mice, but failed to prevent TRALI. Our data demonstrate the direct influence of transfused HNA-3a antibodies on endothelial barrier integrity in vitro as well as in vivo. This novel mechanism of TRALI may be helpful in defining prevention and treatment strategies in order to decrease transfusion mortality. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1108 CD177, also known as NB1 (or HNA-2) is a GPI-linked glycoprotein, which is exclusively expressed on neutrophils. Approximately 3 to 5% of healthy individuals do not express this antigen on their neutrophils (NBnull). Recent data demonstrated a strong upregulation of NB1 on neutrophils in patients with bacterial, but not viral infections. The mechanism underlying this phenomenon, however, is unknown. Our previous studies showed that NB1 functions as a partner of endothelial PECAM-1 and therefore plays a role on neutrophil diapedesis. Consequently, neutrophils carrying NB1 (NB1plus) migrate faster through endothelial cells than NBnull neutrophils. However, several studies have documented an abrogation of neutrophil migratory abilities in sepsis conditions. In this study, we aim to clarify the impact of neutrophil NB1 expression in bacterial sepsis. To mimic this condition in vitro, we first compared the transendothelial migration ability of NB1 phenotyped neutrophils after stimulation with the bacterial peptide fMLP in a transwell system. Lower transmigration ability (75% vs. 40%) was observed in fMLP-treated NB1plus neutrophils (n = 5) in comparison to untreated neutrophils. In contrast, no significant difference in the migration ability was observed between fMLP-treated and untreated NB1null neutrophils (n = 3). Expression analysis by flow cytometry showed a dose-dependent upregulation of NB1 after stimulation with fMLP (10−6 to 10−8 μM) in NB1plus neutrophils. Interestingly, down regulation of PECAM-1 expression was observed in these treated cells. Contrary, no PECAM-1 downregulation was detected in NBnull fMLP-treated neutrophils. These results could be confirmed by immunoblotting analysis using specific antibodies directed against different epitopes on NB1 (mabs 7D8, MEM166) and against different PECAM-1 Ig domains (mabs PECAM1.1, 1.2 and 1.3). Analysis of the supernatants of fMLP-treated neutrophils demonstrated the shedding of PECAM-1 from NB1plus, but not from NB1null neutrophils. These results indicate that shedding of PECAM-1 from neutrophils during bacterial infections depends on NB1 expression. Recent studies showed that a single nucleotide polymorphism (42C〉G) located in NB1 promoter region is associated with the regulation of NB1 expression. Individuals homozygous for C allele express high NB1 surface density in comparison to individuals homozygous for G allele. Our association study showed higher frequency of C allele in patients with bacterial sepsis (n=98) compared with healthy cohort (n = 132) (8.16% vs. 12.88%; P

Description: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality in the United States and other countries. In most TRALI cases, human leukocyte antigen (HLA) class II antibodies are detected in implicated donors. However, the corresponding antigens are not present on the cellular key players in TRALI: neutrophils and endothelium. In this study, we identify monocytes as a primary target in HLA class II–induced TRALI. Monocytes become activated when incubated with matched HLA class II antibodies and are capable of activating neutrophils, which, in turn, can induce disturbance of an endothelial barrier. In an ex vivo rodent model, HLA class II antibody–dependent monocyte activation leads to severe pulmonary edema in a relevant period of time, whenever neutrophils are present and the endothelium is preactivated. Our data suggest that in most TRALI cases, monocytes are cellular key players, because HLA class II antibodies induce TRALI by a reaction cascade initiated by monocyte activation. Furthermore, our data support the previous assumption that TRALI pathogenesis follows a threshold model. Having identified the biologic mechanism of HLA class II antibody–induced TRALI, strategies to avoid plasma from immunized donors, such as women with a history of pregnancy, appear to be justified preventive measures.

Description: Human neutrophil-specific antigen CD177 (NB1; HNA-2a) belongs to the member of the cyctein-rich LY-6 superfamily. Transcriptional analysis of this family revealed intron-retaining mechanism, which produce many spliced forms of gene. In some cases, this mis-spliced isoform was the most abundant form suggesting a control mechanism of gene expression. CD177 glycoprotein carries neutrophil antigen HNA-2a, and antibodies to HNA-2a frequently causes transfusion related acute lung injury (TRALI). Recently, CD177 has been found to be associated with the expression of proteinase-3 (PR3) which plays a role in autoantibody mediated Wegener’s granulomatosis. Neutrophils from some people lack CD177 (termed HNA-2a negative), however, the mechanism responsible for this deficiency is not fully understood. In this study, we investigated whether mis-spliced form of CD177 exist in neutrophils. Neutrophil from blood donors were phenotyped for the presence or absence of CD177 by flow cytometry using two mabs 7D8, MEM166 recognising different epitopes on CD177. HNA-2a positive individuals carrying high surface CD177 density and HNA-2a negative individuals were selected. mRNA was isolated from CD177 phenotyped individuals and were analyzed by PCR using different sets of primer set. Amplification of CD177 transcript (bases 98–1401) showed the expected ~1300 bp band encoding for the entire CD177 (isoform 1), and a smaller band (~417bp) with similar intensity in HNA-2a positive as well as in HNA-2a negative individuals. Sequencing analysis of the short transcript showed the presence of intronic sequence between exon 3 and 4, which produces premature stop codon. Transfection of insect cells with related transcript resulted in production of a ~23 kDa protein which is detectable in immunoblot using rabbit polyclonal antibody against synthetic CD177 peptide. This ~23 kDa protein (isoform 4) did not react with mabs 7D8 and MEM166. Furthermore, immunoblotting analysis of other blood cells showed that isoform 4 was exclusively found on neutrophils. In contrast to the isoform 1, isoform 4 was not upregulated on neutrophils of individuals receiving GCSF. Neutrophils treated with fMLP in vitro showed down-regulation of isoform 4 transcript. This phenomenon however, has not been observed in neutrophils treated with LPS and IL-8. In summary, we characterized a new CD177 isoform in neutrophils. The presence of this isoform in both HNA-2a positive and negative CD177 phenotyped individuals and its regulation with fMLP indicate the role of this protein in inflammatory process.