Expression of a 33-kDa antigen Tm 1 on lymphocyte surface after modulation of cell surface antigens by IgM monoclonal antibody

The mAb Tm 1 was obtained from a fusion of SP2/O tumor cells with spleen cells from CF1 mouse immunized with T cells modulated by an IgM anti-CD3 mAb.mAb Tm 1 reacted with IgM anti-CD3 modulated T cells (66.6%) but not with unmodulated T cells (4.4%). Tm 1 was not expressed on T cells modulated with either IgG2a or IgG1 anti-CD3 mAb. Immunoprecipitation from 125I-labeled CD3-modulated T cells showed that Tm 1 Ag is a single polypeptide of 33 kDa under reducing and nonreducing conditions. Kinetic studies revealed that Tm 1 was detectable on T cells 10 min after incubation and maximally expressed after 4 h of incubation with IgM anti-CD3 mAb. CD3 expression was markedly modulated by this anti-CD3 mAb after the same period of incubation. Studies with cycloheximide revealed that Tm 1 expression on T cells does not require new protein synthesis. Tm 1 expression persisted long after CD3-reexpression 24 h later. Tm 1 was present on a small fraction of circulating T cells, B cells, and monocytes and absent from granulocytes, platelets, E, and thymocytes. Tm 1 was not expressed on T cells after various activation stimuli but was expressed on B cells upon activation. Additional studies indicate that IgM mAb against other T cell differentiation Ag and IgM mAb against B cell Ag also lead to the expression of Tm 1 on these cells. Thus, modulation of surface Ag by IgM mAb externalizes this cytoplasmic Ag. However, one exception has been noted. Purified mAb Tm 1 was not mitogenic and was unable to block either the T cell proliferation induced by 12-O-tetradecanoyl phorbol-13-acetate plus anti-CD3 mAb and other T cell stimuli, or the B cell proliferation induced by B cell mitogens. The role of Tm 1 on lymphocyte function remains to be determined.     

Human blood dendritic cell-like B cells isolated by the 5G9 monoclonal antibody reactive with a novel 220-kDa antigen.

We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40-60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either kappa- or lambda-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9- PBMC, 5G9- B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.     

Activated T cells and monocytes have characteristic patterns of class II antigen expression

The expression of human histocompatibility class II Ag was measured on activated T cells and monocytes by quantitative mAb binding in direct two-color immunofluorescence. Monocytes activated by IFN-gamma bound an average of 2 x 10(6) DR-specific mAb, 3 x 10(5) DQ-specific mAb, and 7 x 10(5) DP-specific mAb per cell. For T cells activated by anti-CD3, a subpopulation bound 1 x 10(5) DR-specific mAb, 5 x 10(4) DQ-specific mAb and 5 x 10(4) DP-specific mAb per cell. These measurements were obtained after establishing a base line of class II Ag expression on resting B cells and monocytes. Resting B cells and those monocytes that were positive for class II Ag bound identical amounts of mAb; 3 x 10(4) DR-specific mAb, 3 x 10(3) DQ-specific mAb and 2 x 10(4) DP-specific mAb. However, most resting monocytes (75%) expressed only DR Ag. In the process of studying the expression of class II Ag on T cells, it was necessary to define and analyze the activated T cell state. Cell cultures activated with 0.3 ng/ml anti-CD3 had the highest expression of class II Ag on T cells, whereas those activated with 3.0 ng/ml anti-CD3 had the highest expression of IL-2R on T cells. Addition of IL-2 had no further effect on DR Ag expression on T cells but did up-regulate IL-2R expression. Reducing the initial monocyte concentration before activating T cells increased class II Ag expression on T cells without affecting IL-2R expression. The results obtained on T cell activation suggest that perhaps a lymphokine may be made by CD3-activated T cells which induces class II Ag expression on T cells.     

Bovine afferent lymph veiled cells differ from blood monocytes in phenotype and accessory function.

Veiled cells have been prepared from bovine afferent lymph and their surface phenotype has been compared with that of blood monocytes using a panel of mAb that define bovine leukocyte Ag. It has been observed that the expression of CD1c and a putative bovine CD45R Ag is restricted within these populations to afferent lymph veiled cells (ALVC), whereas CD11b is expressed only on monocytes. The CD11a and CD11c specificities and an additional Ag defined by mAb 1LA24 (p110/75) are expressed by both cell types, although CD11a and p110/75 are found only on subpopulations of ALVC. In a comparison of accessory function, we have found that ALVC are considerably superior to blood monocytes both as stimulators in mixed leukocyte cultures and as presenters of soluble Ag to primed T cells. This superiority appears to be associated with an increased capacity to form clusters with responding lymphocytes. In addition, we have established that the capacity of ALVC to present soluble Ag to Ag-specific T cell clones resides almost exclusively in a subpopulation defined by the phenotype p110/75+ CD11a-.     

The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.     

Enhancement of human neutrophil functions by a monoclonal antibody directed against a 19-kDa antigen.

A mouse IgG mAb termed P1C3 was raised against A23187-treated human peripheral blood neutrophils and has been shown to recognize an Ag with an apparent molecular mass of 19 kDa, herein named p19. This p19 Ag was weakly expressed at the cell surface of resting human peripheral blood neutrophils and monocytes, but its cell surface expression was dramatically increased upon activation of these cell types with different secretagogues, including FMLP, PMA, and the calcium ionophores A23187 and ionomycin. A large latent pool of p19 molecules became accessible by immunofluorescence flow cytometry after cell permeabilization of resting neutrophils. A practically total translocation of the intracellular pool of this p19 molecule to the plasma membrane was achieved under appropriate cell stimulation, which induced an almost total degranulation of neutrophil secretory granules. The p19 Ag was absent from platelets, PBL, as well as from the human promyelocytic cell line HL-60, the human promonocytic cell line U937, and the human lymphoid cell lines Daudi and Jurkat. The p19 Ag was also expressed by circulating and/or interstitial neutrophils and monocytes in distinct tissues examined. The mAb P1C3 was found to enhance several neutrophil responses, such as chemotaxis, cell adhesion, phagocytosis, and respiratory burst. These data indicate that the mAb P1C3 recognizes an intracellular Ag in human resting mature neutrophils and monocytes, which upon cell activation is translocated to the cell surface and is able to affect cell functionality.     

Identification of a human endothelial cell antigen with monoclonal antibody 44G4 produced against a pre-B leukemic cell line

Staining of a variety of human tissue sections (lymph node, tonsil, spleen, thymus, kidney, lung, and liver) by the indirect immunoperoxidase method indicated that mAb 44G4, produced against a human pre-B leukemic cell line, was strongly reactive with vascular endothelium. All other cell types observed in these tissues were unreactive. Immunofluorescence staining of endothelial cells isolated from umbilical cord vein and grown in culture confirmed that mAb 44G4 recognized a surface membrane component of vascular endothelium. Granulocytes, monocytes, B and T lymphocytes, and T lymphocytes cultured in the presence of PHA for 72 h did not express the 44G4 Ag. mAb 44G4 reacted weakly with leukemic cells from 28 of 41 patients with non-T cell acute lymphocytic leukemia and 4 of 7 patients with acute myelocytic leukemia, whereas 8 of 10 cases of T cell acute lymphocytic leukemia were negative. Moderate reactivity with leukemic cell lines of pre-B and myelomonocytic origin was also observed. The level of 44G4 Ag on umbilical endothelial cells was three to five times that of leukemic cell lines and 25 times the average levels observed on leukemic cells isolated from patients. Immunoprecipitation of lysates prepared from surface-iodinated endothelial cells and the immunizing pre-B leukemic cell line revealed that the 44G4 Ag from both cell types was composed of two subunits of apparent m.w. 95,000 linked by disulfide bond(s). Comparison of the cellular localization and subunit structure of 44G4 to that of known Ag suggests that it represents a previously undescribed marker of endothelial cells.     

Identification and characterization of a hamster monoclonal antibody anti-2.28 directed against a 70-kilodalton activation antigen on human monocytes

Hamster mAb against activated human monocytes were examined for their reactivities against monocyte activation Ag. One mAb, anti-2.28, stained only monocytes activated with LPS plus IFN-gamma, but not unactivated peripheral blood monocytes, polymorphonuclear leukocytes, lymphocytes, RBC, and platelets. However, it stained peripheral blood T cells activated with PMA plus anti-CD3 and peripheral blood and tonsillar B cells activated with PMA plus anti-mu. Of the 35 cell lines of diverse origin examined for immunofluorescence staining by anti-2.28, only EBV-transformed cell lines showed strong staining by this mAb. One pre-B cell line, Nalm-12, could be induced by PMA to exhibit intermediate staining. Immunoprecipitation studies identified the 2.28 Ag as a 70- to 85-kDa monomer. Immunofluorescence staining, immunoprecipitation, and peptide mapping studies indicated that 2.28 was different from a number of monocyte and lymphocyte surface Ag including Mo3e, B-4 (CD19), B-5, CD39, and the G28-8 Ag Bgp 95. These studies suggest that 2.28 may be a novel hemopoietic non-lineage-specific activation Ag.     

Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18

We established two monoclonal antibodies (mAbs) which specifically recognize human 'functionally inactive' recombinant IL-18, and IL-18 protein polymorphism was examined using human monocytes and macrophages (M phi). In 6 day GM-CSF-treated M phi, an 'inactive' IL-18-recognizing mAb 21 detected the IL-18 proform (24 kDa) and a 48-kDa protein, which were gradually increased concomitant with maturation stage. Majority of the 24- and 48-kDa forms were barely detectable with other mAbs recognizing 'active' IL-18. No reagents including Toll stimulators up-regulated these IL-18 populations in M phi. The 21-recognizable IL-18 species were separated using an anion-exchanger column and their IFN gamma-inducing activity was assessed with human lymphocytes plus IL-12. Virtually no as yet known activity was detected with these IL-18 species. After processed with M phi proteases, an 18-kDa form was generated to express the IFN gamma-inducing activity, although the activity was far weaker than that of control 'active' IL-18. These observations suggested that large amounts of various IL-18 species are produced with monocyte-M phi differentiation and most of these IL-18 species are functionally 'inactive' in terms of the reported IL-18 function even after proteolytic 18-kDa conversion.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both 125I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20% of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of 3H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both ¹²⁵I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20 % of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of ³H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton chain and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.

C33 Ag was originally identified by mAb inhibitory to syncytium formation induced by human T cell leukemia virus type 1. The Ag was shown to be a highly heterogeneous glycoprotein consisting of a 28-kDa protein and N-linked oligosaccharides ranging from 10 to 50 kDa. In the present study, cDNA clones were isolated from a human T cell cDNA expression library in Escherichia coli by using mAb C33. The identity of cDNA was verified by immunostaining and immunoprecipitation of transfected NIH3T3 cells with mAb. The cDNA contained an open reading frame of a 267-amino acid sequence which was a type III integral membrane protein of 29.6 kDa with four putative transmembrane domains and three putative N-glycosylation sites. The C33 gene was found to belong to a newly defined family of genes for membrane proteins, such as CD9, CD37, CD53, CD63, and TAPA-1, and was identical to R2, a cDNA recently isolated because of its strong up-regulation after T cell activation. Availability of mAb for C33 Ag enabled us to define its distribution in human leukocytes. C33 Ag was expressed in CD4+ T cells, CD19+ B cells, CD14+ monocytes, and CD16+ granulocytes. Its expression was low in CD8+ T cells and mostly negative in CD16+ NK cells. PHA stimulation enhanced the expression of C33 Ag in CD4+ T cells by about 5-fold and in CD8+ T cells by about 20-fold. PHA stimulation also induced the dramatic size changes in the N-linked sugars previously shown to accompany human T cell leukemia virus type 1-induced transformation of CD4+ T cells.     

Conformational structure of a monoclonal anti-idiotypic antibody to the monoclonal anti-adenocarcinoma-associated carbohydrate antibody YH206.

The mAb AI206 (IgG1) is an anti-Id antibody of mAb YH206 (IgM) to adenocarcinoma-associated carbohydrate Ag and inhibits the reaction of mAb YH206 to YH206 Ag at low concentrations. By Western blot analysis, mAb AI206 only reacted with unreduced mAb YH206, whereas it did not react with reduced mAb YH206. Furthermore, mAb AI206 reacted with IgM subunit (180 kDa), F(ab')2 (110 kDa), and F(ab) (50 kDa) of pepsin-treated unreduced mAb YH206. Thus, mAb AI206 recognized the structure of F(ab) of mAb YH206. The mAb YH206 reacted with unreduced mAb AI206, F(ab')2 (110 kDa), and F(ab) (50 kDa) of pepsin-treated unreduced mAb AI206. It is presumed that mAb YH206 and mAb AI206 recognize each other in an unreduced condition but not a reduced condition. The recognition of such a conformational Id on F(ab) is important. Because mAb YH206 recognized the carbohydrate on YH206 Ag as well as the peptide on mAb AI206, the conformation on F(ab) of mAb AI206 may mimic the carbohydrate structure on YH206 Ag. In fact, YH206 antibody activity was induced in syngeneic mouse serum immunized with mAb AI206. These observations suggest that the internal image of YH206 carbohydrate Ag is preserved within the conformational Id on F(ab) of mAb AI206.     

A monoclonal antibody against a novel 20-kDa protein induces cell adhesion and cytoskeleton-dependent morphologic changes.

A new murine IgA mAb (JKT.M1), developed against Jurkat T cells chronically infected with HIV IIIB induces in vitro homotypic aggregation in several hemopoietic cell lines. The JKT.M1 Ag is expressed on a wide variety of cell types including human lymphocytes, monocytes, platelets, RBC, human umbilical vein endothelial cells, many T cell lines, myelomonocytic cell lines, and a primate kidney cell line. The JKT.M1 Ag shows differential expression on myelomonocytic cells; it is present on K562 and HL60 cell lines, which represent precursors of E and monocytes, respectively, but is not expressed on the surface of U937 and THP-1 cell lines, which appear to represent intermediate cell types of the monocytic cell lineage. However, the JKT.M1 Ag is expressed on mature peripheral blood monocytes and the MonoMac cell line. Immunoprecipitation from cell lysates (Jurkat, SupT1, PBMC, MonoMac) with the JKT.M1 mAb yields a 20-kDa Ag with few if any carbohydrate residues as determined by N-glycanase and neuraminidase treatments. The pI appears acidic by two-dimensional gel analysis, and the nonreduced form migrates more slowly than the reduced form when analyzed by SDS-PAGE suggesting the presence of intramolecular disulfide bridge(s). JKT.M1 mAb-induced cell adhesion is shown to be divalent cation- and temperature-dependent. The adhesion induced by JKT.M1 mAb is inhibited by 20 microM cytochalasin B and also by 2 mM 2-deoxyglucose plus 10 mM sodium azide suggesting that cytoskeletal changes and metabolic energy are required. Aggregation induced by JKT.M1 appears to be independent of CD43, CD44, and VLA4 (CD29/CD49d), mAb against which have also been shown to induce homotypic cell adhesion. Anti-CD18 mAb have been shown to inhibit homotypic aggregation in other studies but failed to do so in the present study. Thus JKT.M1-induced adhesion also appears to be independent of CD18, the beta-chain of leukocyte integrins. However, like mAb against LFA-1, immobilized JKT.M1 stimulates a T cell line to undergo dramatic morphologic changes which could be enhanced by the addition of phorbol ester. These data suggest that the novel 20-kDa molecule recognized by the JKT.M1 mAb may trigger cell adhesion through a previously undescribed mechanism.     

Identification of a human natural killer cell target cell antigen with monoclonal antibodies

mAb have been derived against NK cell-sensitive target cells in an effort to identify the target cell structure involved in Ag recognition by NK cells. Several mAb were selected for further study based on their preliminary target cell binding characteristics. Flow cytometry demonstrated that each of these mAb bound to a series of NK-sensitive target cells of various origins (e.g., K562 and Molt-4) while having little or no reactivity with several NK-resistant target cell lines (e.g., SB and Daudi). Functional studies revealed that two of the mAb were able to inhibit the lysis of NK-sensitive K562 target cells by freshly isolated, endogenous NK cells in a dose-dependent fashion. Further, these mAb also could inhibit the killing of K562 target cells by both activated NK cells and cultured lymphokine-activated killer cells, as well as the cytolysis of other NK-sensitive target cells by each of these effector cell populations. Control experiments with another mAb which bound to the target cells but did not inhibit lysis implied that the effects of these mAb on NK cell function was not the result of steric hindrance. Single cell conjugate assays demonstrated that the mAb inhibited NK cell lysis via the inhibition of binding (recognition). Biochemical analysis of this target cell structure revealed that it was a molecule of approximately 42 kDa which may exist as a homodimer in its native state. Thus, it appears that the mAbs identify an unique Ag on the surface of NK cell-sensitive target cells which is involved in NK cell Ag recognition.     

A monoclonal antibody that detects a specific human neutrophil antigen involved in phorbol myristate acetate- and formyl-methionyl-leucyl-phenylalanine-triggered respiratory burst.

A mouse IgM mAb termed P1E3 was raised against resting human peripheral blood neutrophils and has been shown to recognize a cell-surface Ag with an apparent molecular mass of 155 kDa, as assessed by immunoprecipitation analysis. In addition to the main 155-kDa protein, an additional band of about 210 kDa was also recognized by P1E3 in Western blot analysis. Sequential immunoprecipitation assays showed that the Ag recognized by P1E3 differed from the CD29 and CD45 Ag. However, sequential immunoprecipitation assays carried out with two distinct anti-CD15 mAb and P1E3 showed that P1E3 reacted with CD15 or with a CD15-like Ag. P1E3 stained strongly resting human peripheral blood neutrophils, hardly reacted with peripheral blood monocytes and did not react with PBL and platelets, as assessed by immunofluorescence flow cytometry. P1E3 inhibited the respiratory burst induced by PMA or FMLP, but not the oxidative response induced by Con A or the calcium ionophores A23187 or ionomycin. Furthermore, P1E3 inhibited the activation of the Na+/H+ antiporter in response to PMA or FMLP and the phosphorylation of a protein of about 50 kDa in response to PMA. However, preincubation of neutrophils with P1E3 did not affect the increase in cytosolic free calcium concentration induced by FMLP. These data suggest that the Ag recognized by P1E3 may play a role in modulating the activation of the respiratory burst induced by PMA or FMLP, and that P1E3 seems to affect protein kinase C-mediated signal transduction mechanisms coupled to the induction of the respiratory burst.     

A monoclonal antibody recognizing 68- to 75-kilodalton protein(s) associated with the human IL-1 receptor

We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R.     

IFN-gamma plus glucocorticoids stimulate the expression of a newly identified human mononuclear phagocyte-specific antigen

Glucocorticoid hormones, although able to exert profound immunosuppressive effects, do not suppress mononuclear phagocyte activation by IFN-gamma and may even enhance it. For example, expression and functional activity of the high affinity FcR for IgG on human mononuclear phagocytes (FcR gamma I) is increased by IFN-gamma and is maximal after co-treatment with IFN-gamma plus the glucocorticoid dexamethasone (DEX). To determine whether there are other mononuclear phagocyte surface Ag that are regulated in this manner, hybridomas were prepared using IFN-gamma-plus-DEX-treated human monocytes as immunogen. Five IgG1 mAb (Mac 2-8, 2-38, 2-48, 2-49, and 2-158) were developed that recognize a trypsin-sensitive mononuclear phagocyte-specific surface Ag of Mr 155,000. There was no detectable reactivity of these mAb to lymphocytes or granulocytes or to several cell lines, including U-937 and HL-60. The p155 Ag was detected on monocytes and increased significantly with time of culture or after treatment with DEX. Expression was maximal after co-treatment with rIFN-gamma plus DEX, but was inhibited or unaffected by treatment with IFN-gamma alone. For freshly isolated cells, expression of the p155 Ag was highest on peritoneal macrophages. Our results indicate that the p155 Ag is a newly identified Ag of the human mononuclear phagocyte lineage and may represent, in the least, a phenotypic marker of monocyte differentiation or maturation.     

Regulation of nuclear antigen expression on the cell surface of human monocytes.

The expression of cell surface nuclear Ag was studied by examining the binding of anti-histone mAb to viable human peripheral blood cells. Freshly isolated cells showed no binding of these mAb. However, in vitro culture in the presence of LPS induced a dose-dependent expression of cell surface nuclear Ag on monocytes (M3+ cells). The addition of IL-1 beta to cultures also induced expression of cell surface nuclear Ag, whereas IFN-gamma was without effect. Release of nuclear material into the supernatants over time was demonstrated by using a chromatin-specific sandwich ELISA. Analysis of the DNA in the released nuclear material demonstrated banding at multiples of 190 bp, suggesting the release of polynucleosomes. Although LPS was required for cell surface nuclear Ag expression, it did not affect the release of nuclear material into the supernatants. The ability of monocytes to bind exogenous chromatin was studied by adding biotinylated-chromatin to PBL and detection with FITC-avidin. Freshly isolated PBL bound no chromatin, but when PBL were cultured in the presence of LPS, monocytes bound chromatin in a saturable manner. The LPS induction of the capacity to bind exogenous chromatin was blocked by cycloheximide. These data suggest that monocyte activation is associated with the expression of a chromatin (?nucleosome)-binding receptor and that this receptor is capable of binding nuclear material released into the cellular milieu. Monocytes may thus provide an important mechanism for the removal of extracellular nuclear material at sites of cell death and/or inflammation. The binding of nuclear Ag to cell surfaces and potential abnormalities of this binding may play a role in the induction of antinuclear antibodies and/or tissue damage in diseases such as SLE.