N-terminal and central regions of the human CD44 extracellular domain participate in cell surface hyaluronan binding

CD44 molecules are cell surface receptors for hyaluronan (HA). To define regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bind to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3F12, BU-75, and HP2/9) of 21 studied were identified that at least partially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected Jurkat cells. Analysis of reactivity of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of reactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demonstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived from the T-ALL cell line, CEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-type CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at position 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to histidine mutation at aa position 154. Taken together, our studies demonstrated that there are three epitopes to which HA-blocking mAbs bind in the extracellular domain of CD44, and that the CD44 N-terminal proximal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to cell surface CD44.     

CD44 variants but not CD44s cooperate with beta1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis

The expression of osteopontin (OPN), CD44 variants, and integrins has been correlated with tumorigenesis and metastasis. Here we show that these proteins cooperate to enhance cell motility. First, we demonstrate that several different CD44 variants bind to OPN in an arginine-glycineaspartic acid-independent manner, but that the standard form of CD44 does not. These CD44 variants bind to both the amino- and COOH-terminal portions of OPN independently of the arginine-glycine-aspartic acid sequence, suggesting that multiple domains on OPN can be bound by the CD44 variants. Antibodies directed against the integrin beta1 subunit are able to inhibit this binding. The binding of CD44 variants to OPN is significantly augmented by both anti-CD44s and anti-CD44v antibodies. This augmentation by anti-CD44 antibodies is OPN specific and, again, can be blocked by anti-beta1 antibodies. Finally, we show that OPN binding by CD44 variants/beta1-containing integrins promotes cell spreading, motility, and chemotactic behavior.     

Fine epitope specificity of antibodies to region II of the Plasmodium vivax circumsporozoite protein correlates with ability to bind recombinant protein and sporozoites

Recent work has suggested that important B- and T-cell epitopes on the circumsporozoite protein (CSP) of Plasmodium vivax lie external to the major repeat regions of the protein. We have studied two naturally exposed human populations (Caucasian and Papua New Guineans) and determined the antibody response to yeast-derived recombinant CSPs, overlapping synthetic peptides spanning amino acids 76 348 of the Belem P. vivax CSP and overlapping peptides representing the variant repeats of the VK247 strain of P. vivax. We have demonstrated that the P. vivax CSP-specific antibody response is directed towards areas within the repeat region as well as areas external to this; but the dominant epitopes recognized by the two populations studied, were distinct. One epitope, lying external to the repeats and recognized by both populations, partially overlaps an area of the protein referred to as region II-plus. Sera from malaria-exposed Papua New Guineans and Thais contained antibodies to this epitope (V22, single letter amino acid sequence TCGVGVRVRRRVNAANKKPE) which were capable of recognizing sporozoites, as determined by quantitative inhibition IFA. Seventeen percent of PNG sera had antibodies to this peptide compared with 33% who had antibodies to the central repeats of the protein. Immunization of mice with recombinant CSP did not induce antibodies to V22. However, immunization with overlapping peptide epitopes representing this region (V21 or V22) induced specific antibodies but only two sera recognized both V21 and V22 and, by inference, the overlapping peptide sequence (TCGVGVRVRR). Antibodies in these two sera could bind recombinant CSP in ELISA; however, in contrast, nine sera which recognized either V21 or V22 alone did not bind CSP. Only one of two sera containing antibodies recognizing CSP stained P. vivax sporozoites. This serum also recognized an epitope dependent upon two amino acids aminoterminal to V22. These data suggest that the fine specificity of antibodies is a critical determinant for binding to both recCSP and sporozoites.     

Neutralising antibodies to the granulocyte colony-stimulating factor receptor recognise both the immunoglobulin-like domain and the cytokine receptor homologous domain

To define regions of the granulocyte colony-stimulating factor (G-CSF) receptor that are important for ligand binding, neutralising monoclonal antibodies to the human receptor have been produced. Eleven antibodies recognised six different receptor epitopes. Antibodies from three of the epitope groups were able to detect the receptor by western blotting but did not inhibit G-CSF binding. The other three antibody groups inhibited G-CSF binding either completely (groups 1 and 2) or partially (group 3). All the antibodies inhibited proliferation of BA/F3 cells expressing the G-CSF receptor to varying extents. By using human-marine chimeric receptors, the binding sites of the antibodies were mapped to the immunoglobulin-like domain (groups 1 and 3), the cytokine receptor homologous domain (group 2) or the fibronectin type III domains (groups 4 to 6). These results show that the immunoglobulin-like and cytokine receptor homologous domains of the receptor are important for ligand binding and subsequent signalling.     

Calcium- and calmodulin-dependent PMA-activation of the CD44 adhesion molecule

The ability of the CD44 adhesion molecule to interact with its ligand hyaluronic acid (HA) is tightly regulated. CD44-positive mouse LB lymphoma cells are unable to bind HA unless activated by the tumor promoter phorbol 12-myristate 13-acetate (PMA). PMA causes a dose-dependent increase in both CD44 expression level and HA-binding capacity, with the binding of HA observed only above a threshold amount of CD44 molecules. This induction of HA-binding as well as the increase in CD44 expression are prevented by cycloheximide, suggesting a requirement for new additional CD44 molecules on the cell surface and/or cooperating proteins. In the present study, we have investigated which of the signal transduction pathways activated by PMA leads to the increased CD44 expression with subsequent acquisition of HA-binding capacity. By comparing the influence of each inhibitory agent on PMA-activated LB lymphoma cells versus that on a constitutive HA-binder cell line derived from LB cells (designated HA9 cells), we could distinguish between an effect on the PMA-activation phase and a one on the HA-binding phase. Our data show that the PMA-induced HA-binding could not be blocked by agents inhibiting protein kinase C (PKC) (staurosporine, sphingosine, polymyxin B, quercetin) or genestein, an inhibitor of tyrosine protein kinases. However, this PMA response was strongly inhibited by calmodulin antagonists (chlorpromazine, trifluoperazine, W-7) and the calcium blocker verapamil. The calmodulin antagonists inhibited the PMA-induced increase in CD44 expression on LB cells, but had no influence on the ability of the constitutive HA-binder HA9 cell line to interact with HA, indicating an effect on the PMA induction phase rather than on the binding itself. Verapamil also blocked the PMA-induced increase in CD44 expression on LB cells, but in addition it slightly reduced the ability of the HA9 cells to bind HA without affecting their CD44 expression level. In conclusion, our data suggest that CD44 activation by PMA is calcium and calmodulin dependent, rather than mediated by protein kinase C.     

Active immunity against the CD4 receptor by using an antibody antigenized with residues 41-55 of the first extracellular domain

Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.     

Peptide-specific antibodies as probes of the topography of the Ca2+-binging motifs in alphaIIbbeta3

Antibodies against synthetic peptides corresponding to four Ca2+-binding motifs of the alphaIIb subunit have been obtained and used as molecular probes to analyze the topography of the alphaIIbeta3 complex. The specificity of the antibodies has been characterized by ELISA and Western immunoblotting in terms of binding capacity and affinity to the isolated alphaIIbbeta3 and its alphaIIb subunit. Our data suggest that: (a) all four Ca2+-binding motifs of the alphaIIb are partially exposed on the surface of the intact molecule and accessible to antipeptide antibodies. However, they are not in close vicinity to the ligand recognition domain since the antibodies do not produce complete inhibition of platelet aggregation. (b) The conformation of amino acid stretches which form the second Ca2+-binding motif of alphaIIb is particularly dependent upon the presence of cation, and this region undergoes significant conformational alterations upon Ca2+ expulsion.     

Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding

CD44 is a cell surface receptor for the glycosaminoglycan hyaluronan (HA). Not all CD44-positive cells bind HA, and binding ability is strictly regulated. Three different HA binding states have been defined: inactive, inducible (by certain CD44-specific monoclonal antibodies), and constitutively active. The observation that sets of genetically related cell lines representing different HA binding states showed correlated differences in N-glycosylation of CD44, and that inhibition of N-glycosylation enhanced HA binding (Lesley et al., J. Exp. Med., 182: 431-437, 1995) led us to examine directly whether specific N-glycosylation site modifications were involved in regulating the HA binding function. CD44-negative, -active, and inducible cell lines were stably transfected with mutant constructs in which each of the five N-glycosylation sites of murine CD44 had been separately inactivated. Ability to bind soluble HA was examined over a range of CD44 expression levels. For the active cell line, AKR1, transfectants for all N-glycosylation mutants bound HA as well as did transfectants for wild type CD44. No inhibitory effects of inactivating specific N-glycosylation sites were observed. HA binding was activated when two of the mutant constructs were transfected into a novel CD44-negative inducible cell line. Inactivation of N-glycosylation sites at residues 25 or 120 converted the inducible cell line to constitutively active, whereas inactivation of other sites had little or no effect. Fusion proteins secreted from inactive, inducible, or active cell lines were purified, bound to beads, and assayed for HA binding activity by flow cytometric analysis. Fusion proteins derived from inactive, inducible, and constitutively active cells exhibited three distinguishable "threshold" densities required for HA binding ability. The results imply that the CD44 molecules produced in cells in these three activation states have intrinsic differences in HA binding function. Treatment of the fusion proteins with neuraminidase altered the HA binding state, and glycosylation mutations that affected the phenotype of the inducible cell line lowered the threshold required for HA binding of CD44-immunoglobulin fusion proteins derived from the inducible cell line. Thus, alterations of glycosylation of CD44 itself can affect HA binding ability as manifested by a change in HA binding state.     

A cysteine residue located in the transmembrane domain of CD44 is important in binding of CD44 to hyaluronic acid

In the transmembrane domain and cytoplasmic domain of human CD44 protein there are two cysteine residues. These two cysteines are conserved in all known mammalian CD44 proteins. The functions of these cysteine residues are not known. Site-specific mutagenesis was used to create CD44 mutant proteins lacking either one or both of these cysteine residues. Wild-type CD44 and mutant CD44 genes were transfected into CD44- Jurkat cells to establish stable transfectants. These transfectants were used to study whether these two cysteine residues are important in the binding of CD44(H) to fluorescein-conjugated hyaluronic acid (F-HA). Jurkat transfectant bearing wild-type CD44 did not bind F-HA, unless they were stimulated in vitro with immobilized anti-CD3 monoclonal antibody. Anti-CD3 antibody also stimulated the binding of F-HA in Jurkat CD44.C295A transfectant in which the cytoplasmic cysteine residue has been replaced with alanine. In contrast, anti-CD3 antibody failed to stimulate the binding of F-HA in Jurkat transfectant (CD44.C286A), in which the transmembrane domain cysteine 286 has been replaced with an alanine, and in Jurkat transfectant CD44.2C2A, in which both of the cysteine residues have been altered. Binding can also be induced with a monoclonal anti-CD44 antibody (F-44-10-2) in Jurkat wild-type CD44 and Jurkat CD44.C295A transfectants but not in CD44. C286A transfectant. These results provide evidence that the transmembrane domain of CD44, more specifically the cysteine residue in the transmembrane domain, is important for both activation-induced and anti-CD44 antibody-induced binding of soluble HA.     

Magnetic resonance tomography in diagnosis and therapy follow-up of patients with congenital hip dysplasia and hip dislocation

PROBLEM: Human chrionic gonadotropin (hCG) is a placental glycoprotein hormone, a heterodimeric molecule, consisting of alpha and beta chains. It induces the synthesis of progesterone, which is essential for the maintenance of the fertilized egg. Antibodies directed against hCG can, therefore, prevent pregnancy and serve as a vaccine. hCG belongs to the glycoprotein hormone family and shares the alpha chain with the other members. The beta chain is a hormone-specific subunit that is unique to hCG, but still possesses 85% amino acid homology with the beta chain of luteinizing hormone (LH), which means that prolonged immunization with hCG produces antibodies that cross-react with LH. METHOD OF STUDY: We have taken an approach involving the mutation of beta hCG to eliminate cross-reactive epitopes without affecting the natural folding of the polypeptide chain and thus the unique beta hCG-specific epitopes. RESULTS: Several mutants have been constructed that have maintained the binding to hCG-specific monoclonal antibodies (mAbs) but have lost the ability to bind to a panel of LH cross-reactive mAbs. To investigate the immunogenicity of selected mutants, mice were immunized with expression plasmid DNA, containing the gene for wild-type beta hCG and two mutants: mutant 3, with four amino acid substitutions (68 Arg-->Glu; 74 Arg-->Ser; 75 Gly-->His; 79 Val-->His), and mutant 7, with a single amino acid substitution (68 Arg-->Glu). CONCLUSIONS: Although both mutants were able to elicit antibody responses in at least some animals, the levels were less than those seen with the wild-type beta hCG DNA, and there seems still to be a residual cross-reactivity with LH. Attempts to improve the immunogenicity of the mutants and to further modify the sequence to remove the cross-reactivity are currently underway.     

Building synthetic antibodies as adhesive ligands for integrins

An antibody engineering strategy was employed to build high affinity ligands and antagonists of integrins alpha v beta 3 and alpha IIb beta 3. Previously, we inserted the integrin recognition motif, RGD, into the antigen binding site of a human antibody and selected the optimal flanking sequences from a phage-display library (Barbas, C. F., Languino, L. R., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10003-10007). The resulting antibody, Fab-9, blocked the function of integrin alpha v beta 3 but also bound to the ligand binding site of platelet integrin alpha IIb beta 3. In this report, the antibody engineering effort has been extended by 1) redesigning Fab-9 to achieve specificity for platelet integrin alpha IIb beta 3, 2) building non-RGD-containing antibodies that bind the ligand binding site of both beta 3-integrins, and 3) testing the hypothesis that peptides derived from complementarity determining regions (CDR) can be used to emulate the activity of the parent synthetic antibody. These goals were accomplished by subjecting the original antibody, Fab-9, to a "motif optimization" (MTF). A phage library was constructed in which the residues flanking the RGD motif in Fab-9 were maintained, but the RGDX sequence was randomized. This library was panned on purified alpha IIb beta 3 to identify high affinity binders. Four function-blocking antibodies lacking RGD, but with specificity for alpha IIb beta 3, were characterized. The antibody with the highest preference for alpha IIb beta 3, MTF-10, had an adhesion sequence of KGDN. This sequence is similar in primary structure to the active sequence within the disintegrin barbourin, which also antagonizes alpha IIb beta 3 (Scarborough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried, V. A., Campbell, A. M., Nannizzi, L., and Charo, I. F. (1991) J. Biol. Chem. 266, 9359-9362). MTF-10 had a 70-fold higher affinity for alpha IIb beta 3 than alpha v beta 3. Through our selection strategy, we also identified several antibodies that lack RGD but still blocked ligand binding to both integrins with high affinity. Therefore, the RGD sequence is not necessary for a high affinity interaction with the ligand binding site of beta 3-integrins. Further investigation showed that the activity of inhibitory antibodies could be emulated by synthetic peptides derived from the protein sequences of the antibody's HCDR3. CDR-derived peptides blocked ligand binding to integrins and maintained essentially the same specificity as the parent antibody.     

Hyaluronan induces monocyte chemoattractant protein-1 expression in renal tubular epithelial cells

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that accumulates in the renal interstitium in immune-mediated kidney diseases. The functional significance of such HA deposition in the kidney has not been elucidated. Several studies have suggested that HA may exhibit proinflammatory effects. Since chemokines such as monocyte chemoattractant protein-1 (MCP-1) play an important role in the recruitment of leukocytes in renal injury, this study tested whether HA and its fragments could promote MCP-1 production by renal parenchymal cells. Mouse cortical tubular cells were stimulated with fragmented HA or with high molecular weight HA (Healon) in vitro and were examined for MCP-1 expression. Fragmented HA, but not Healon, increased MCP-1 mRNA within 30 min with a peak after 2 h. In addition, a 10-fold increase of MCP-1 protein in the supernatant was found after a 6-h stimulation with fragmented HA. The enhanced MCP-1 mRNA and protein expression in response to HA was dose-dependent between 1 and 100 microg/ml. Upregulation of MCP-1 protein production could be blocked by preincubation with actinomycin D or cycloheximide, suggesting that MCP-1 mRNA and protein expression in response to HA are based on de novo synthesis. The HA-stimulated MCP-1 production was also inhibited with anti-CD44 antibodies, suggesting that MCP-1 is upregulated at least in part by signaling through CD44. In summary, fragmented HA markedly stimulates renal tubular MCP-1 production by mechanisms that involve binding to the HA receptor CD44. It is hypothesized that the accumulation of HA in immune renal injury could participate in the recruitment and activation of inflammatory cells in vivo through production of MCP-1.     

Proglumide as a morphine adjunct in cancer pain management

PURPOSE: To determine the expression of CD44 isoforms in cultured human trabecular meshwork (HTM) and to discuss their possible relationship with outflow facility. METHODS: CD44 isoform expression in cultured HTM was qualitatively examined using immunohistochemistry and RT-PCR analysis. RESULTS: Immunohistochemistry of cultured HTM showed intense staining with a CD44s antibody, and with antibodies against CD44 exon 7, exon 11-12 and exon 14. By RT-PCR, at least three isoforms of CD44 were expressed in HTM: CD44s, CD44v-III and CD44v-I. CONCLUSIONS: At least three isoforms of CD44 are expressed in the HTM. CD44 may play a role in binding and turnover of hyaluronic acid in the trabecular meshwork, thereby regulating outflow facility.     

Antigenic structure of the central conserved region of protein G of bovine respiratory syncytial virus

Epitopes were resolved at the amino acid level for nine monoclonal antibodies (MAbs) directed against the central conserved region of protein G of bovine respiratory syncytial virus (BRSV-G). Peptide binding studies showed which amino acids in the epitope contributed to antibody binding. The details of the epitopes were compared with the high-resolution structure of a synthetic peptide corresponding to the central conserved region of BRSV-G, and this indicated which face of the central conserved region is the antigenic structure. The major linear epitope of the central conserved region of BRSV-G is located at the tip of the loop, overlapping a relatively flat surface formed by a double disulfide-bonded cystine noose. At least one, but possibly two sulfur atoms of a disulfide bridge that line the conserved pocket at the center of the flat surface, is a major contributor to antibody binding. Some of the residue positions in the epitope have mutated during the evolution of RSV-G, which suggests that the virus escaped antibody recognition with these mutations. Mutations that occur at positions 177 and 180 may have only a local effect on the antigenic surface, without influencing the structure of the backbone, whereas mutations at positions 183 and 184 will probably have major structural consequences. The study explains the antigenic, structural, and functional importance of each residue in the cystine noose which provides information for peptide vaccine design. Additionally, analysis of the epitopes demonstrated that two point mutations at positions 180 and 205 define the preliminary classification of BRSV subgroups.     

Generation of neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa exotoxin A

Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.     

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.     

Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.     

Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated beta2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P approximately 110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P approximately 110. The inhibition of P approximately 110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin-transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin-IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin-IgG-triggered PMN/PMN aggregation as well as P approximately 110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered beta2-integrin-dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the beta2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P approximately 110.     

The role of charged residues mediating low affinity protein-protein recognition at the cell surface by CD2

Insights into the structural basis of protein-protein recognition have come principally from the analysis of proteins such as antibodies, hormone receptors, and proteases that bind their ligands with relatively high affinity (Ka approximately 10(9) M-1). In contrast, few studies have been done on the very low affinity interactions mediating cell adhesion and cell-cell recognition. As a site of protein-protein recognition, the ligand binding face of the T lymphocyte cell-cell recognition molecule, CD2, which binds its ligands 10(4)- to 10(5)-fold more weakly than do antibodies and proteases, is unusual in being both very flat and highly charged. An analysis of the effect of mutations and ionic strength on CD2 binding to its ligand, CD48, indicates that these charged residues contribute little, if any, binding energy to this interaction. However, the loss of these charged residues is shown to markedly reduce ligand-binding specificity. Thus, the charged residues increase the specificity of CD2 binding without increasing the affinity. This phenomenon is likely to result from a requirement for electrostatic complementarity between charged binding surfaces to compensate for the removal, upon binding, of water interacting with the charged residues. It is proposed that this mode of recognition is highly suited to biological interactions requiring a low affinity because it uncouples increases in specificity from increases in affinity.     

Cross reacting antibodies against keyhole limpet haemocyanin may interfere with the diagnostics of acute schistosomiasis

The number of individuals catching schistosomiasis has increased with the popularity of 'primitive tourism' in Africa. Highly immunogenic material originating from the intestine of intravascular adult schistosomes gives rise to an antibody response making possible early identification of infected individuals using serology. Antibodies against gut associated antigens (anti-GAA), detected by indirect immunofluorescence microscopy employing sections of adult worms as antigen may occur before the onset of egg production. In the present study we show that this well known schistosomiasis-specific anti-GAA staining reaction can be confused with a similar staining reaction with ducts of both male and female worms. Antibodies with duct reactivity were seen in sera both from schistosomiasis-patients and patients with some other invasive worm infections. Cross reactive anti-duct antibodies appear to have different specificity. One cross reactive antibody reacted with antigenic epitopes present in keyhole limpet haemocyanin (KLH). Anti-duct reactivity could be inhibited by absorption with KLH. This was most obvious in the trichinellosis patient sera.