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Rheumatoid factors (RF) and the disease-specific anti-citrullinated protein autoantibodies (ACPA) coexist in the joints of rheumatoid arthritis (RA) patients where they probably contribute to synovitis. We investigated the influence of IgM and IgA RF on the FcR- and complement-dependent effects of ACPA immune complexes (ACPA-IC). When stimulated by ACPA-IC formed in the presence of IgM RF or IgA RF fractions purified from RA serum pools, M-CSF-generated macrophages skewed their cytokine response toward inflammation, with increases in the TNF-α/IL-10 ratio and in IL-6 and IL-8 secretion, and decreases in the IL-1Ra/IL-1β ratio. In the IgM RF-mediated amplification of the inflammatory response of macrophages, the participation of an IgM receptor was excluded, notably by showing that they did not express any established receptor for IgM. Rather, this amplification depended on the IgM RF-mediated recruitment of more IgG into the ACPA-IC. However, the macrophages expressed FcαRI and blocking its interaction with IgA inhibited the IgA RF-mediated amplification of TNF-α secretion induced by ACPA-IC, showing its major implication in the effects of RF of the IgA class. LPS further amplified the TNF-α response of macrophages to RF-containing ACPA-IC. Lastly, the presence of IgM or IgA RF increased the capacity of ACPA-IC to activate the complement cascade. Therefore, specifically using autoantibodies from RA patients, the strong FcR-mediated or complement-dependent pathogenic potential of IC including both ACPA and IgM or IgA RF was established. Simultaneous FcR triggering by these RF-containing ACPA-IC and TLR4 ligation possibly makes a major contribution to RA synovitis., (Copyright © 2015 by The American Association of Immunologists, Inc.)

A single chain variable fragment (scFv) of CB515, a complement-independent bactericidal monoclonal IgM against a relapsing fever Borrelia, was constructed to investigate the region wherein the unique bactericidal function resides. Monomeric CB515 scFv (26 kDa) was capable of binding its Ag on whole organisms and by immunoblot. This binding was shown to be species and serotype-specific to the 19 kDa variable small protein, recognized by its parent monoclonal IgM. A dose-dependent bactericidal effect of the CB515 scFv was detected by direct enumeration of spirochetes. Spirochetes incubated with the CB515 scFv before inoculation into mice grew into escape mutants, whereas spirochetes incubated with an irrelevant scFv developed as the original infecting serotype. This bactericidal effect, as seen at the ultrastructural level, was due to disruption of the outer membrane and to severe membrane blebbing eventually progressing to lysis. These results indicate that the variable region of CB515 is responsible for this bactericidal activity and that the constant region of the Ab is dispensable.

The physiological relevance of naturally occurring IgM-ALA remains to be elucidated. These autoantibodies are present from birth and increase in diverse inflammatory states that are both infectious and noninfectious. Clinical observations showing significantly less acute allograft rejections in recipients having high IgM-ALA levels, led us to investigate whether IgM-ALA could have a functional role in attenuating T cell mediated inflammatory responses. In pursuit of this hypothesis, we did studies using IgM purified from the serum of normal individuals, patients with end stage renal disease, and HIV-1 infection. All preparations of IgM immunoprecipitated certain receptors e.g., CD3, CD4, CCR5, and CXCR4 from whole cell lysates but failed to immunoprecipitate IL-2R and HLA Ags. In physiological doses IgM down-regulated CD4, CD2 and CD86 but not CD8 and CD28, inhibited T cell proliferation, decreased production of certain proinflammatory cytokines e.g., TNF-alpha, IL-13 and IL-2, but not IFN- gamma, IL-1beta, GM-CSF, IL-6 and IL-8 and inhibited leukocyte chemotaxis. These inhibitory effects were more pronounced when using IgM from patients with high levels of IgM-ALA and these inhibitory effects were significantly reduced after using IgM preabsorbed with leukocytes. IgM-ALA binding to leukocytes was found to be highly specific, as <10% of IgM secreting B cell clones had IgM-ALA specificity with some clones having specificity for either T cells or monocytes. These findings support the concept that IgM-ALA provides an innate mechanism to regulate T cell mediated inflammatory responses.

In prior studies, we show that naturally occurring IgM anti-leukocyte autoantibodies (IgM-ALA) bind to CD3, CD4, CCR5, and CXCR4 receptors. These observations prompted us to determine whether IgM-ALA have a role in inhibiting HIV-1 infectivity by inhibiting viral entry into cells. We show that purified IgM, but not IgG, from individual sera of both normal and HIV-1 infected individuals is highly inhibitory (>95%) to HIV-1 viral infectivity both in vitro using PHA plus IL-2 activated PBL and in vivo using the human PBL-SCID mouse. Inhibition was observed with physiological doses of purified serum IgM and even after IgM was added 3 days postinfection in the in vitro assays. Absorbing purified serum IgM either with leukocytes or immobilized recombinant CD4 significantly decreased (>80%) the inhibitory effect on HIV-1 infectivity. IgM inhibited by >90% syncytia formation with the X4-IIIB infected SupT-1 cells indicating therefore that IgM inhibits viral attachment to core-receptors. IgM mediated anti-HIV-1 activity was highly specific as only certain IgM-ALA, obtained from human B cell clones inhibited HIV-1. IgM from certain HIV-1 infected individuals were not inhibitory to some R5-HIV-1 viral strains indicating that certain HIV-IgM may lack Abs reactive to strain specific coreceptor epitopes. These data indicate that an innate immune mechanism which is present from birth i.e., IgM-ALA, has a role in inhibiting HIV-1 viral entry into cells. Validation of this data with other in vivo models will be needed to determine whether in vivo administration or enhancement of IgM-ALA, e.g., through a vaccine, could prolong the asymptomatic state in HIV-1 infected individuals.

Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4+CD25+Foxp3+ Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25(high) cells, but not Foxp3+ cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25-Foxp3+ or CD25(low)Foxp3+ cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25(high)Foxp3+ cells results from the re-expression of CD25 on peripheral populations of CD25-Foxp3+ but not from the conversion of peripheral Foxp3-) cells. CD25(high)Foxp3+ repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25+Foxp3+ cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25+Foxp3+ cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4+CD25-Foxp3+ cells and/or conversion of CD4+CD25-Foxp3- cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3+ cells after anti-CD25 treatment and observe that TGF-beta is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3+ cell numbers and a normal ratio of Treg:non-Treg cells.

Cross-linking cell surface molecules with IgM Abs is a specific approach for activating cells in vitro or in vivo. Dendritic cells (DC) activated with a human B7-DC (PD-L2)-specific IgM Ab can induce strong antitumor responses and block inflammatory airway disease in experimental models, yet the Ab-mediated molecular events promoting these responses remain unclear. Analysis of human or mouse DC treated with the B7-DC cross-linking Ab revealed PI3K-dependent phosphorylation of AKT accompanied by mobilization of NF-kappaB. Ab-activated DC up-regulated expression of cytokine and chemokine genes in an NF-kappaB-dependent manner. Importantly, PI3K-->AKT-->NF-kappaB activation was found to be indispensable for B7-DC cross-linking Ab-mediated protection of DC from cell death caused by cytokine withdrawal. Although other DC activators similarly protect DC from cell death, a synergy between cross-linking B7-DC and ligating RANK was observed. The parallel signaling events induced in human and mouse DC demonstrate that activation of cells using IgM Ab results in a response governed by a common mechanism and support the hypothesis that B7-DC cross-linking using this Ab may provide beneficial therapeutic immune modulation in human patients similar to those seen in animal models.

Splenic marginal zone (MZ) and follicular mantle (FO) B cells differ in their responses to stimuli in vitro and in vivo. We have previously shown that MZ cells exhibit greater calcium responses after ligation of membrane IgM (mIgM). We have now investigated the molecular mechanism underlying the difference in calcium responses following ligation of mIgM and studied the response to total B cell receptor ligation in these two subsets. We compared key cellular proteins involved in calcium signaling in MZ and FO cells. Tyrosine phosphorylation and activity of phospholipase C-gamma 2 and Syk protein tyrosine kinase were significantly higher in MZ cells than in FO cells after mIgM engagement, providing a likely explanation for our previous findings. Tyrosine phosphorylation of CD22 and expression of Src homology 2-containing inositol phosphatase and Src homology 2-containing protein tyrosine phosphatase-1 were also higher in the MZ cells. Expression and tyrosine phosphorylation of Btk, BLNK, Vav, or phosphatidylinositol 3-kinase were equivalent. In contrast, stimulation with anti-kappa induced equivalent increases in calcium and activation of Syk in the two subsets. These signals were also equivalent in cells from IgM transgenic, J(H) knockout mice, which have equivalent levels of IgM in both subsets. With total spleen B cells, Btk was maximally phosphorylated at a lower concentration of anti-kappa than Syk. Thus, calcium signaling in the subsets of mature B cells reflects the amount of Ig ligated more than the isotype or the subset and this correlates with the relative tyrosine phosphorylation of Syk.

The IgM mAbs 12A1 and 13F1 are protective and nonprotective, respectively, against lethal Cryptococcus neoformans infection in mice. To better understand the variables that contribute to IgM efficacy against C. neoformans, we studied the effects of inoculum size, route of infection, and Ab dose for each of these mAbs. mAb 13F1 did not prolong survival under any condition studied. mAb 12A1 prolonged survival after the administration of certain Ab doses after i.p. infection with defined inocula and promoted phagocytosis, agglutination, and the formation of inflammatory cell rings around yeast cells in vivo. Large Ab doses of mAb 12A1 resulted in either no protection or enhanced infection, consistent with a prozone-like effect. Investigation of this phenomenon revealed that the fungal cell was protected against microbicidal nitrogen-derived oxidants when large amounts of Ab were bound to the C. neoformans capsule. mAb 12A1 was opsonic in vitro for peritoneal, but not splenic or alveolar macrophages. In summary, our results indicate that IgM efficacy against C. neoformans is a function of the route of infection, inoculum, and Ab dose and is associated with its ability to promote opsonization, agglutination, and phagocytic ring formation in vivo. The occurrence of the prozone-like phenomenon implies that high Ab titers are not necessarily beneficial in assuring protection against certain pathogens and that caution should be exercised in using high Ab titer as a measure for vaccine efficacy.

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.

We and others previously observed that IgM and CD40 stimulation in murine B cells resulted in activation of extracellular signal-regulated kinase (ERK), a subfamily of mitogen-activated protein kinase. The present study demonstrated that ERK was rapidly phosphorylated and translocated to the nucleus in murine B cells upon stimulation with CD40, whereas it was preferentially localized within the cytosol after stimulation with IgM, suggesting that signaling through CD40 and IgM differentially regulates ERK subcellular localization. Costimulation with CD40 and IgM (CD40/IgM) resulted in subcellular localization of ERK within the cytosol, supporting the notion that stimulation with IgM delivers the signal responsible for inhibition of ERK nuclear transport. Consistent with these observations, IgM and CD40/IgM stimulation resulted in activation of ribosomal S6 kinase, which is a cytoplasmic substrate for ERK, whereas CD40 stimulation had little effect on its activity. Disruption of the microtubule by colchicine in WEHI231 cells resulted in reduction of ERK activity in IgM signaling, but not in CD40 signaling, compatible with the notion that the microtubule network may hold cytoplasmic ERK activity mediated by IgM stimulation. These results support the notion that ERK could mediate different effector functions in B cells upon stimulation with IgM and CD40.

Fas ligand (FasL) and Fas are mediators of apoptosis, which are implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and cytotoxicity mediated by CD8+ T cells. Fas is also believed to be involved in several central nervous system diseases, but until now, the effector cells expressing FasL in the brain have not been identified. We investigated the expression levels of Fas and FasL with the stimulation of cytokines and the possible effector cells targeting Fas-bearing cells. Our data demonstrated that: 1) FasL is expressed constitutively on astrocytes taken from a fetus or an adult and that its expression increases when these cells are treated with IL-1, IL-6, or TNF-alpha in which the pretreatment of IFN-gamma triggers astrocytes to express more FasL; 2) astrocytes induce apoptosis in MOLT-4 cells through FasL; 3) Fas is also expressed constitutively and is up-regulated by IL-1, IL-6, or TNF-alpha in which the pretreatment of IFN-gamma triggers astrocytes to express more Fas; 4) apoptosis occurs when fetal astrocytes are treated with agonistic anti-Fas IgM Ab after culture with IFN-gamma and TNF-alpha; and 5) TNF-related apoptosis inducing ligand is up-regulated in fetal astrocytes with stimuli of IL-1 or TNF-alpha. These findings suggest a possible role of astrocytes in the induction of apoptosis in central nervous system diseases.

HIV-infected cells aberrantly express a high level of antigenic glycosidic structures such as GM2 and Gg4. Some normal sera containing natural IgM Abs to GM2 and/or Gg4 cause C-mediated cytolysis of HIV-infected cells. In the present study we demonstrated that a human IgM anti-GM2 mAb (L55 Ab) can induce cytolysis of HIV-infected cells. Increased GM2 expression by HIV-1 infection of a human T cell line (MOLT4), a human monocyte cell line (U937), and human lymphoblastoid cells was confirmed by immunofluorescence staining with L55 Ab. These infected cells were readily lysed by L55 Ab in the presence of fresh human serum as a C source that alone did not cause cytolysis. L55 Ab also had the ability to destroy HIV-1 particles via C-mediated lysis. By adding L55 Ab together with human C to mixed culture of HIV-infected cells and naive cells, HIV-1 replication was significantly suppressed, and this effect was synergistic when L55 Ab was combined with a reverse transcriptase inhibitor and a proteinase inhibitor. Therefore, a human IgM anti-GM2 mAb may be effective in treating HIV-infected patients, especially when used together with chemotherapeutic agents.

We and others have demonstrated previously the occurrence of cC1qR/CaR, a receptor for the collagen-like stalks of complement component C1q, on endothelial cells. In the present study we investigated whether binding of C1q to endothelial cells resulted in enhancement of cytokine or chemokine production. HUVEC produced 82 +/- 91 pg/ml of IL-8, 79 +/- 113 pg/ml of IL-6, and 503 +/- 221 pg/ml of monocyte chemoattractant peptide-1 (MCP-1) under basal conditions. Incubation with C1q resulted in a time- and dose-dependent up-regulation of IL-8 (1012 +/- 43 pg/ml), IL-6 (392 +/- 20 pg/ml), and MCP-1 (2450 +/- 101 pg/ml). This production is dependent on de novo protein synthesis, as demonstrated by the detection of specific mRNA after C1q stimulation, and inhibition of peptide production in the presence of cycloheximide. The production of all factors was inhibited (69 +/- 7%) by the collagenous fragments of C1q, while the C1q globular heads only induced 13 +/- 11% inhibition. When HUVEC were incubated with C1q in the presence of aggregated IgM, enhanced production of IL-8 (2500 +/- 422 pg/ml), IL-6 (997 +/- 21 pg/ml), and MCP-1 (5343 +/- 302 pg/ml) was found. Furthermore, F(ab')2 anti-calreticulin partially inhibited the production of IL-8, confirming at least the involvement of cC1qR/CaR. These experiments suggest that in an inflammatory response C1q not only is able to activate the complement pathway, but when presented in a proper fashion also might induce the production of factors that contribute to acute phase responses and recruitment of inflammatory cells.

CD19 enhances membrane IgM (mIgM) signaling and is required for B lymphocyte responses to T-dependent Ags. CD19 is tyrosine phosphorylated when mIgM is ligated and binds SH2 domain-containing signaling proteins. We suggest that the basis for phosphorylation is the association of CD19 with Syk and other components of the mIgM complex. IgM, CD22, Ig-alpha, Ig-beta, and Syk were coimmunoprecipitated with CD19 from detergent lysates of B lymphocytes. The association was maintained with a chimeric form of CD19 containing only the transmembrane domain and the membrane proximal 17 amino acids of the cytoplasmic domain encoded by exon 6. This sequence is sufficient to mediate the association, as a synthetic peptide of the exon 6-encoded region adsorbs IgM and Syk. Deletion of the juxtamembrane 17 amino acids of the cytoplasmic domain encoded by CD19 exon 6 abolishes association of CD19 with the mIgM complex. Deletion of these amino acids, which contain no tyrosines, also reduces mIgM-induced tyrosine phosphorylation of the remainder of the CD19 cytoplasmic domain. Coligating this mutant CD19 to mIgM restores phosphorylation. Thus, a discrete region of the cytoplasmic domain regulates the tyrosine phosphorylation of CD19 in the activation of B cells by mIgM.

The effects of IL-10 on murine B cell proliferation in vitro were investigated. IL-10 inhibited LPS-induced B cell proliferation with an EC50 of approximately 500 pg/ml. IL-10-mediated inhibitory activity was not overtly associated with cytotoxicity or induction of apoptosis. The presence or the absence of T cells and mononuclear phagocytes did not affect the inhibitory activity of IL-10 on LPS-induced proliferation. LPS-stimulated, IL-10-exposed B cells progressed from G0 or from M to G1A of the cell cycle, but were inhibited from entry into subsequent phases. IL-10 had no discernible effect on B cell proliferation elicited with goat anti-mouse IgM plus IL-4. Moreover, cross-linking, but not mere ligation, of surface Ag receptors restored LPS-induced B cell proliferation in the presence of IL-10. The proliferative response to ligation of CD40 with anti-CD40 Abs was also not inhibited by IL-10, and as observed with goat anti-mouse IgM, the presence of such Abs in IL-10-containing B cell cultures allowed for the proliferative response to LPS. A variety of other Abs reactive with murine B cell surface markers were ineffective at modulating the response to IL-10. IL-1, IL-2, IL-4, IL-5, IL-6, IFN-gamma, and TGF-beta were also ineffective in this regard. These observations suggest that IL-10 has a role in the suppression of inappropriate B cell proliferation, i.e., proliferation by B cells that have not effectively interacted with relevant Ag or CD40 ligand.

Pentameric IgM and dimeric IgA both contain disulfide bonds between cysteines located in the secretory tailpieces of the heavy chains. To compare the influences of the mu and alpha tailpieces on the polymeric structure, we have replaced amino acids in the tailpiece of the human mu-chain with amino acids found in the corresponding positions in the human alpha tailpiece. We show that an IgM with an alpha tailpiece (IgM L561H, Y562V, L566V, S569A, D570E, T571V, and A572D) as well as IgM L561H, Y562V, and IgM A572D have a size distribution similar to that of wild-type IgM. However, one IgM mutant with a mu/alpha hybrid tailpiece (IgM L566V, S569A, D570E, T571V, and A572D) is secreted as a mixture of mainly hexamers, pentamers, tetramers, and dimers. The tetramers and dimers are specifically formed and secreted at the expense of pentamers and hexamers; no alterations in polymerization or secretion rates were observed. We have also incorporated the mu, alpha, and hybrid mu/alpha tailpieces to a human IgG3 or IgGL309C mutant. The IgG-tailpiece mutants are poorly secreted, but the secreted fractions contain multimeric molecules. Each of the mutants that contain both the L309C mutation and a secretory tailpiece forms mainly hexamers; however, small differences in polymer distribution exist for the different tailpieces. Comparison of the influence of different tailpieces on IgM and IgG polymeric structures suggests that the function of a specific tailpiece is dependent on other parts of the heavy chain, which can vary for different isotypes.

Intravenous Ig, composed principally of IgG, prevents complement attack by inhibiting C3 and C4 uptake onto target cells and tissues. Using two different models, Ab-sensitized SRBC and BSA-anti BSA solid phase immune complexes, we have examined the complement inhibitory capacity of three Ig classes (IgG, IgM, IgA) focussing on inhibition of C4 uptake. It was found that on both a weight and molar basis, monomeric serum IgA and IgM were far more active than IgG (weight efficiency ratios were 1.0, 20.8, and 236.3, and molar efficiency ratios 1.0, 24.0, and 1382.9 for polyclonal IgG, IgA1, and IgM, respectively). Monoclonal IgM were less active than polyclonal IgM (50% inhibition was achieved in SRBC model by 0.022, 0.30, 1.6, and 1.6 mg/ml of polyclonal IgM and monoclonal IgM from patients Lew, Will, and Pri). Secretory IgA was less active than serum IgA1 and similar in inhibitory activity to IgG (weight and molar efficiency ratios 1.5 and 0.6 compared with IgG). All tested preparations were less active in the solid phase immune complex model than in the sensitized cell model. A mixture of Igs of different isotypes was somewhat more active than any isotype alone. These results suggest that polyclonal serum IgA and IgM can also be considered for active therapy in diseases accompanied by the activation of classical complement pathway.

Given the range of mIg-binding affinities expressed by Ag-specific B cells, the ligand:receptor affinity threshold for achieving full B cell activation via the mIgM-mediated signaling pathway is quite high. Several recombinant, or semi-purified, cytokines were found to reduce the very high mIgM:ligand affinity threshold for induction of human B cell S phase entry by bivalent, affinity-diverse anti-IgM mAbs without notably affecting the lower affinity threshold for G1-related RNA synthesis. Two-stage culture experiments suggested that one major means by which IL-4, IL-2, and low m.w. B cell growth factor lower the affinity threshold for S phase entry is an indirect one, i.e., rescue of B cells whose mIg engagements with Ag are of sufficient affinity for achieving G1 entry, but of insufficient affinity for initiating the late-phase mIgM-mediated signals needed for the G1-->S phase transition. IL-4 had additional effects in early G1. In contrast to the above cytokines, IFN-gamma, did not function as an independent cell cycle progression factor, but rather required the concomitant presence of mIgM-cross-linking ligand for enhancement. A greater potential of multivalent anti-IgM-dextran conjugates to trigger S phase entry in the absence of cytokines was found to reflect a greater potential for initiating mIgM signals during the late phase in B cell activation. The results indicate that progression of mIgM receptor-activated B cells past a G1-->S phase restriction point is dependent upon continued signal transduction via either the mIgM receptor and/or a cytokine receptor signaling pathway. When mIgM-engaging ligands are ineffective at initiating late-phase signals, due to limited size and binding site valency and/or affinity, ancillary signal transduction through cytokine receptors becomes most relevant.

Cell-cell adhesive interactions involve numerous receptor/ligand interactions that play a crucial role in the development of immune function. Engagement of multiple cell-surface molecules on B lymphocytes generates intracellular signals through a tyrosine kinase-dependent pathway that activates cell-surface adhesion receptors and thereby induces homotypic cell-cell adhesion. Homotypic adhesion is mediated in part through LFA-1/ICAM-1 and other heretofore unknown adhesion receptors. In this study, evidence of a regulatory role for CD45 in the induction of homotypic adhesion is suggested. A new mAb (HAB-1) was developed that inhibits homotypic adhesion in B cell lines induced through MHC class I and class II, CD19, CD20, CD21, CD40, and Leu-13 cell-surface molecules. Although binding of this mAb strongly inhibited cell-surface Ag-induced homotypic adhesion at mAb concentrations as low as 0.1 microgram/ml, it exhibited no effect on homotypic adhesion induced by phorbol esters. Binding of the HAB-1 mAb to lymphocytes altered the pattern of cellular protein tyrosine phosphorylation, but did not have a global inhibitory effect on cell activation because it did not have major effects on the growth of mitogen-activated lymphocytes. Immunoprecipitation studies revealed that the HAB-1 mAb identified an epitope present on all isoforms of CD45. The HAB-1 mAb may identify a unique epitope of CD45 because this mAb had a unique staining pattern when assessed by indirect immunofluorescence staining. The HAB-1 mAb was similar to some other CD45 mAb that have the capacity to amplify CD2-induced proliferation of blood lymphocytes. However, only 1 of 12 other anti-CD45 mAb tested had a similar inhibitory effect on adhesion. Homotypic adhesion of lymphocytes may therefore be governed by a regulatory system of cell-surface molecules that generate positive and negative signals that either trigger adhesion or, like CD45, directly down-regulate adhesion. This highlights the significance of adhesive events that result from surface molecules being engaged by their natural ligands during lymphocyte activation.

Opsonization of the C3b receptor (CR1) on phagocytic cells with C3b enhances both attachment of targets to the cells and subsequent IgG-dependent ingestion of these targets. To explore mechanisms involved in this increased phagocytosis, we adhered cultured human monocytes to surfaces pre-coated with CR1 ligand or control proteins and quantitated ingestion of sheep E opsonized with IgG alone. Three ligands for CR1 resulted in markedly enhanced phagocytosis of targets when compared individually to a panel of non-ligands, as determined by both the proportion of monocytes ingesting targets (percent phagocytosis) and by the number of targets ingested per 100 monocytes (phagocytic index). The ligands included purified C3b, iC3, and Fab fragments of 1B4, a monoclonal anti-CR1, which resulted in a percent phagocytosis of 56.3 (p less than 0.01), 59.0 (p less than 0.01), and 54.4 (p less than 0.02) and a phagocytic index of 281.2 (p less than 0.01), 281.1 (p less than 0.01), and 247.1 (p less than 0.02), respectively. Control proteins including human serum albumin, hemoglobin, Fab fragments of anti-fibronectin, anti-beta 2 microglobulin, and MOPC 21, and Fc fragments of 1B4 and MOPC 21 produced no significant stimulation of phagocytosis, nor did F(ab')2 fragments of monoclonal anti-CR3, M1/70. CR1-specific augmentation of target ingestion was apparent with monocytes cultured in serum-free medium for 1 to 7 days, but was not seen with freshly elutriated cells. Phagocytosis of unopsonized or IgM-coated targets was minimal. These results suggest that the adherent monocytes are primed by CR1 cross-linking for enhanced FcR-mediated phagocytosis even when the CR1 ligand is not present on the targets. This contrasts with the behavior of CR3, and demonstrated functional divergence between these C3 fragment receptors in the phagocytic process.

Pretreatment of MTV-induced BALB/cfC3H mammary tumor cells with autologous serum results in increased spleen cell cytotoxic activity and the recruitment of previously inactive spleen cells to cytotoxic activity against the target cells. These recruiting antibodies are tumor-specific for individual tumors; pretreatment with such serum of target cells of an MTV-induced mammary tumor obtained from a different BALB/cfC3H female results in blocking of spleen cell activity. The autologous recruiting factors are active at dilutions of 1000 or more of whole serum are found in the 19S fraction after gel filtration.