Role and regulation of sickle red cell interactions with other cells: ICAM-4 and other adhesion receptors.

Erythrocytes containing primarily hemoglobin S (SS RBCs) are abnormally adherent. We now know that SS RBCs express numerous adhesion molecules, and that many of these can undergo activation. SS RBCs exposed briefly to epinephrine show markedly increased adhesion to both laminin and endothelial cells. In vivo, infusion of epinephrine-activated but not unstimulated SS RBCs causes RBC adhesion, vaso-occlusion, organ trapping, and shortened RBC survival in the circulation. Epinephrine treatment of SS RBCs before infusion also induces adhesion of murine leukocytes to vascular walls. Indeed, in vitro, SS RBCs can activate leukocyte adhesion and cytokine production. We now have demonstrated both in vitro and in vivo evidence for the importance of RBC signaling and have also shown that SS RBC adhesion is determined by genetic polymorphisms in the signaling pathway that activates adhesion. These advances will hopefully lead to new therapeutic modalities for sickle cell disease.

Lack of isohemagglutinin production following minor ABO incompatible unrelated HLA mismatched umbilical cord blood transplantation.

While immune hemolysis due to donor isohemagglutinin (IH) production often complicates minor ABO incompatible peripheral blood hematopoietic stem cell transplantation (PBSCT), it is not known if this occurs with umbilical cord blood transplantation (UCBT). We compared IH production and hemolysis following minor ABO allogeneic PBSCT and UCBT. We reviewed 24 ABO minor incompatible allogeneic PBSCTs and 14 ABO minor incompatible UCBTs. Patients were evaluated for donor-derived IH by reverse ABO grouping. Evaluation of hemolysis was based on clinical and laboratory findings of anemia associated with increased RBC transfusion need, concomitant with the appearance of donor-derived IH. Of the 24 ABO minor incompatible allogeneic PBSCTs, 15 produced donor-derived IH from 6 to 88 days following transplantation, with seven of 15 patients exhibiting clinically evident hemolysis. There was no significant difference in days to leukocyte engraftment or infused CD34 cells in patients with or without donor-derived IH. None of the 14 patients receiving ABO incompatible UCBTs showed evidence of donor-derived IH following transplantation with a median follow-up of 60 days. We conclude that donor IHs are not produced in patients undergoing minor ABO incompatible UCBTs suggesting fundamental immunologic differences between allogeneic PBSCT and UCBT.

LW protein: a promiscuous integrin receptor activated by adrenergic signaling.

The LW blood group antigen glycoprotein, although part of the Rh macromolecular complex, is nonetheless a member of the intercellular adhesion molecule (ICAM) family. Thus, while it is only rarely clinically important in the setting of transfusion and pregnancy, LW is likely to contribute to red cell adhesion in a variety of settings, including during hematopoiesis, as well as in vascular disorders. The best documentation of a pathophysiological role for LW in human disease is in sickle cell disease, where it contributes to red cell adhesion to endothelial cells and the development of vaso-occlusion, the hallmark of that disease. LW may also contribute to other intravascular processes, such as both venous and arterial thrombosis, due to its ability to interact with both activated platelets as well as leukocytes. The evidence that LW itself can undergo activation on red cells holds promise that pharmacotherapeutic maneuvers may be found to prevent such pathophysiologic interactions.

Principles and problems of transfusion in sickle cell disease.

Sickle cell disease (SCD) is associated with red blood cell (RBC) abnormalities and moderate to severe anemia, and blood transfusion is naturally a mainstay of treatment. However, transfusion therapy for SCD may incur special and distinctive adverse effects. Thus, it is important to understand the indications for and goals of transfusion therapy and to be aware of the potential side effects of therapy. Years of unsystematic clinical observations, followed by more carefully designed and in some cases randomized studies, have contributed substantially to our knowledge of transfusion therapy in SCD. However, much remains unknown and areas of controversy persist. In addition, serologic barriers pose enduring roadblocks to the optimization of transfusion therapy for patients with SCD, and the syndrome of massive hemolytic transfusion reactions and hyperhemolysis in SCD persists as a life-threatening complication for which appropriate clinical management is not yet defined.

Molecular defects underlying the Kell null phenotype.

Expression of the Kell blood group system is dependent on two proteins, Kell and XK, that are linked by a single disulfide bond. Kell, a type II membrane glycoprotein, is a zinc endopeptidase, while XK, which has 10 transmembrane domains, is a putative membrane transporter. A rare phenotype termed Kell null (Ko) is characterized by the absence of Kell protein and Kell antigens from the red cell membrane and diminished amounts of XK protein. We determined the molecular basis of eight unrelated persons with Ko phenotypes by sequencing the coding and the intron-exon splice regions of KEL and, in some cases, analysis of mRNA transcripts and expression of mutants on the cell surface of transfected cells. Six subjects were homozygous: four with premature stop codons, one with a 5' splice site mutation, G to A, in intron 3, and one with an amino acid substitution (S676N) in exon 18. Two Ko persons with premature stop codons had identical mutations in exon 4 (R128Stop), another had a different mutation in exon 4 (C83Stop), and the fourth had a stop codon in exon 9 (Q348Stop). Two Ko persons were heterozygous for two mutations. One had a 5' splice site mutation (G to A) in intron 3 of one allele that caused aberrant splicing and exon skipping, and the other allele had an amino acid substitution in exon 10 (S363N). The other heterozygote had the same amino acid substitution in exon 10 (S363N) in one allele and a premature stop codon in exon 6 (R192Stop) in the other allele. The S363N and S676N mutants, expressed in 293T cells, were retained in a pre-Golgi compartment and were not transported to the cell surface, indicating that these mutations inhibit trafficking. We conclude that several different molecular defects cause the Kell null phenotype.

Cardiac metastasis from a transitional cell carcinoma: a case report.

We report the case of a patient with a metastatic tumor in the right ventricle, apparently derived from a transitional cell carcinoma. The patient presented with severe hypoxemia as a result of right-to-left shunt due to the position of the tumor and a patent foramen ovale. The clinical course of this case is presented and the pathophysiology of the physiological effects caused by the metastatic tumor is discussed. The literature concerning cardiac metastases is reviewed.

Red blood cell surface adhesion molecules: their possible roles in normal human physiology and disease.

Human erythrocytes express a relatively large number of known adhesion receptors, despite the fact that red blood cells (RBCs) are generally considered to be nonadhesive for endothelial cell surfaces. Some of these adhesion receptors are expressed by many other tissues, while others have more limited tissue distribution. Some adhesion receptors, including CD36 and VLA-4, are only expressed by immature erythroid cells, while others are present on mature erythrocytes. The structure and function of these proteins is reviewed here. LW, CD36, CD58, and CD147 have been shown in other tissues to mediate cell-cell interaction. Other receptors, such as CD44, VLA-4, and B-CAM/LU, can mediate adhesion to components of extracellular matrix. In addition, their roles in normal erythropolesis, as well as in the pathophysiology of human disease, are summarized. The most convincing evidence for a pathophysiologic role for any of these receptors on erythrocytes comes from studies of cells from patients homozygous for hemoglobin S, as RBC adhesion is thought to contribute to vaso-occlusion. Thus, receptors such as B-CAM/LU may become targets for future therapy aimed at preventing or ameliorating this thrombotic process.

Large-scale use of red blood cell units containing alloantibodies.

Many transfusion services are reluctant to accept red blood cell (RBC) units containing antibodies. We evaluated the impact of accepting routine shipments of our region's inventory of alloantibody- positive RBC units over a 4-month period. All patients' samples received up to 30 days after transfusion of such units were evaluated for the presence of passively acquired antibody, and labor and reagent costs were determined. During the study period, we received 259 alloantibody-containing RBC units, and 253 of these were transfused to 187 patients. Follow-up samples were received on 99 of these 187 patients, and 10 of these patients had detectable passive antibody in posttransfusion antibody screening tests. Two patients had anti-C and -D and eight patients had anti-D. Due to our negotiation of a small discount for antibody-containing units and the use of 20 units based on labeled phenotype rather than antigen typing in our laboratory, we experienced a net savings of $3814 over the 4-month period. This savings was achieved despite some additional costs incurred, including costs of data entry and additional testing on patients' samples. We concluded that large-scale use of RBC units from donors with alloantibodies is safe and likely to have a minimal impact on a busy transfusion service's workload and costs. Furthermore, nationwide use of such units would help alleviate projected blood shortages.

Critical factors in basal cell adhesion molecule/lutheran-mediated adhesion to laminin.

Basal cell adhesion molecule (B-CAM) and Lutheran (LU) are two spliceoforms of a single immunoglobulin superfamily protein containing five Ig domains and comprise the sickle (SS) red cell receptor for laminin. We have now analyzed laminin binding to murine erythroleukemia cells transfected with various human B-CAM/LU constructs. B-CAM and LU bound equally well to laminin, indicating that the longer cytoplasmic tail of LU is not required for binding. However, binding of soluble laminin did require the presence of the membrane-proximal fifth immunoglobulin superfamily (IgSF) domain of LU, while deletion of IgSF domains 1, 2, 3, or 4 individually or together did not abrogate laminin binding. Under flow conditions, MEL cells expressing B-CAM, LU, and LU lacking domains 1, 2, 3, or 4 adhered to immobilized laminin with critical shear stresses over 10 dynes/cm2. However, MEL cells expressing LU lacking domain 5 bound to laminin poorly (critical shear stress = 2.3 dynes/cm2). Moreover, expression of only IgSF domain 5 of LU was sufficient to mediate MEL cell adhesion to immobilized laminin (critical shear stress >10 dynes/cm2). Finally, Scatchard analysis showed that SS red cells had an average of 67% more B-CAM/LU than normal red cells, and low density red cells from sickle cell disease patients expressed 40-55% more B-CAM/LU than high density SS red cells. B-CAM/LU copy number thus may also play a role in the abnormal adhesion of SS red cells to laminin.

Basal cell adhesion molecule/lutheran protein. The receptor critical for sickle cell adhesion to laminin.

Sickle red cells bind significant amounts of soluble laminin, whereas normal red cells do not. Solid phase assays demonstrate that B-CAM/LU binds laminin on intact sickle red cells and that red cell B-CAM/LU binds immobilized laminin, whereas another putative laminin binding protein, CD44, does not. Ligand blots also identify B-CAM/LU as the only erythrocyte membrane protein(s) that binds laminin. Finally, transfection of murine erythroleukemia cells with human B-CAM cDNA induces binding of both soluble and immobilized laminin. Thus, B-CAM/LU appears to be the major laminin-binding protein of sickle red cells. Previously reported overexpression of B-CAM/LU by epithelial cancer cells suggests that this protein may also serve as a laminin receptor in malignant tumors.

Leukocyte phenotypic changes in an in vitro model of ABO hemolytic transfusion reaction.

BACKGROUND: ABO antigen-antibody interaction in the presence of peripheral blood leukocytes (white cells) results in the production of a variety of proinflammatory cytokines. However, although tumor necrosis factor alpha has been shown to be derived at least primarily from monocytes, the range of cells activated by this process has not previously been reported. Therefore, changes in mononuclear cell surface antigen expression were studied, to determine which subsets of white cells appeared to be activated in the setting of ABO incompatibility. STUDY DESIGN AND METHODS: Group O peripheral blood mononuclear cells (PBMCs) were incubated in autologous plasma with group A or O red cells (RBCs) for up to 24 hours. White cell expression of activation and adhesion markers was measured at 2 and 24 hours by flow cytometry, using direct or indirect fluorescein or phycoerythrin labeling. RESULTS: Expression of lymphocyte activation markers CD25, CDw108, and CD109 was equivalent when PBMCs incubated with group A and O RBCs were compared. However, after 2 hours, mean fluorescence of CD14 on PBMCs incubated with group A RBCs was 65 percent of that on PBMCs incubated with group O RBCs and remained similarly decreased at 24 hours. CD44 expression was upregulated on PBMCs exposed to both group A and O RBCs, but it was increased significantly more on monocytes exposed to group A RBCs. The ability to bind hyaluronic acid was induced in approximately 42 percent of CD14+ monocytes exposed to group A RBCs but in no cells exposed to group O RBCs. CONCLUSION: Downregulation of CD14 and increased binding of hyaluronic acid reflects monocyte activation in this model. No evidence of lymphocyte activation was found, supporting the hypothesis that ABO transfusion reactions primarily activate monocytes.

Expression of cell adhesion molecule CD44 in primary tumors of the liver: an immunohistochemical study.

CD44, a widely distributed integral membrane protein, has been implicated in tumor invasion and metastatic spread in some human carcinomas and lymphomas. In this study, 35 cases of hepatocellular carcinoma from 32 patients (11 cholangiocarcinomas, 9 hepatic adenomas, and 5 cases of focal nodular hyperplasia, a non-neoplastic lesion) were examined by immunohistochemical methods for expression of CD44. The mouse monoclonal antibody A3D8 was used on formalin-fixed, paraffin-embedded tissue; this antibody does not distinguish between standard CD44 and splice variants. Positive membrane staining was seen in 13 of 35 cases of hepatocellular carcinoma (12 of 32 patients), 8 of 11 cases of cholangiocarcinoma, and 1 of 9 cases of hepatic adenoma. The strongest staining for CD44 was seen in two cases of fibrolamellar carcinoma, but CD44 expression was otherwise not related to degree of tumor differentiation. All five cases of focal nodular hyperplasia were negative for CD44. In non-neoplastic liver, hepatocytes were negative; sinusoidal lining cells and portal lymphocytes were positive; bile ducts and proliferating bile ductules were focally positive in some cases. Anatomic stage at time of presentation was similar in both groups of patients, with most patients presenting with stage III or IV disease. A trend towards slightly longer survival in patients whose hepatocellular carcinomas were CD44 negative was noted. These results show that aberrant CD44 expression is present in a subset of hepatocellular carcinomas and in most cholangio-carcinomas. The relationship between CD44 expression and tumor spread is unclear in this group of tumors, but is unlikely to be a simple association between CD44 expression and metastatic potential.

Biologic functions of blood group antigens.

In the past few years, we have learned a great deal about the biologic function of structures bearing blood group antigens. Some blood group antigen-bearing proteins function as major transport channels within the erythrocyte membrane; these include the anion transporter (band 3: Diego and Wright antigens), the water channel (aquaporin: Colton antigens), and the urea transporter (Kidd antigens). At least two erythrocyte blood group antigen proteins have complement regulatory functions: the complement receptor type 1 (CR1, CD35: Knops antigens) and decay accelerating factor (DAF, CD55: Cromer antigens). Some blood group antigens reside on proteins with known receptor functions, such as the chemokine receptor (Duffy) and the hyaluronan receptor (Indian). The Cartwright antigens reside on an enzyme, acetylcholinesterase, and the Kell antigens reside on a protein that belongs to the CALLA-related family of neutral metalloproteinases. Finally, some blood group antigens reside on proteins that serve crucial structural functions necessary to normal erythrocyte lifespan and morphology. These proteins include band 3, glycophorins C/D (bearing the Gerbich antigens), and the Rh proteins. Both oligosaccharide and protein blood group antigens may act as receptors for bacterial, viral, and parasitic infectious agents.

A blood group-related polymorphism of CD44 abolishes a hyaluronan-binding consensus sequence without preventing hyaluronan binding.

CD44 is a widely expressed integral membrane protein that acts as a receptor for hyaluronan (HA) and is proposed to be important to cell-extracellular matrix interaction. The Indian (In) blood group antigens reside on CD44, and most individuals express the Inb antigen. Homozygosity for the Ina allele occurs as a rare event and is associated with production of alloantibody to the common Inb antigen after transfusion or pregnancy. The present study demonstrates that a single point mutation (G252 --> C) causes an Arg46 --> Pro substitution, which is responsible for the Inb/Ina polymorphism. Additional mutations were found in In(a+b-) cDNA but were not necessary to the antigenic phenotype as determined in site-directed mutagenesis studies. In studies using CD44 chimeric constructs, Arg46 has previously been shown to be crucial for maintenance of HA-binding ability to a CD44 peptide. However, the present study demonstrates that the Arg46 --> Pro substitution does not reduce HA binding to the intact CD44 protein, which contains two proposed extracellular HA-binding motifs. Down-regulation of HA binding to In(a+b-) CD44 by anti-CD44 monoclonal antibody (mAb) ligands, however, was weakened, although all mAbs tested bound In(a+b-) and In(a-b+) CD44 equally well. Competitive inhibition studies using human anti-Inb also showed that some mAbs that inhibit HA binding to CD44 may do so by interacting with a domain separate from, but affecting the structure of, the Inb epitope.