An isotype-specific trans-acting factor is defective in a mutant B cell line that expresses HLA-DQ, but not -DR or -DP.

The B lymphoblastoid cell line clone 13 (a subclone of the mutant cell line P3JHR-1) has been found to express high levels of HLA-DQ; by contrast, HLA-DR and -DP antigens are not expressed and cannot be induced by interferon gamma. Northern blot analysis using gene-specific probes indicated that the lack of surface expression of the DR and DP antigens is due to a marked decrease in the levels of steady-state RNA for both the alpha and beta chains. Southern blots demonstrated that none of the transcriptionally repressed genes are grossly deleted. Preparations of interspecific transient heterokaryons between clone 13 and the class II antigen-positive murine B cell lymphoma, A20, resulted in reactivation of the DRA gene and surface expression of both the DR and DP molecules. The efficiency of the DRA promoter relative to the DQB promoter is markedly and specifically diminished in clone 13 (P3JHR-1) as compared with the parental cell line, Jijoye, as assayed both by transient expression of appropriate chloramphenicol acetyltransferase gene (CAT) constructs and by in vitro transcription analysis. These data clearly demonstrate the existence of an isotype-specific trans-acting factor, and provide direct evidence that the highly homologous class II genes have distinct regulatory mechanisms.

Soluble CD14 participates in the response of cells to lipopolysaccharide.

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

Structural relationship between the soluble and membrane-bound forms of human monocyte surface glycoprotein CD14.

The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD14, was determined by carboxypeptidase Y digestion and compared with the complete amino acid sequence of this protein as predicted from the structure of cloned cDNA [Goyert et al. Science 239, 497-500 (1988)]. The soluble antigen isolated from urine appears to lack eight C-terminal amino acid residues predicted for the full-size translation product, but possesses a major part of the C-terminal hydrophobic domain originally suggested as the membrane-spanning segment. The CD14 antigen can be removed from the monocyte surface by phosphatidylinositol-specific phospholipase C treatment, indicating that this glycoprotein is anchored in the membrane by a phospholipid and is not a transmembrane protein. The soluble form occurring in serum and in supernatants of cultured monocytes thus probably arises by phospholipase-mediated cleaving off the cell surface antigen. A sensitive sandwich ELISA was developed using a monoclonal anti-CD14 antibody, MEM-18, and polyclonal rabbit anti-CD14 antiserum for quantitation of the soluble antigen concns in sera and cell culture supernatants. Using this assay, the antigen present in the supernatant of phospholipase treated peripheral blood mononuclear cells could be estimated. The assay was also used for estimation of the concns of the soluble form of the CD14 antigen in human sera.

The CD14 molecule participates in regulation of IL-8 and IL-6 release by bronchial epithelial cells.

The soluble form of the leukocyte membrane antigen CD14 is known to increase the sensitivity of endothelial and epithelial cell lines to bacterial lipopolysaccharide (LPS). This molecule also directly induces cytokine production in monocytes. Here, the effect of sCD14 and LPS on the release of IL-6 and IL-8 by human bronchial epithelial cells (HBECs) was studied. Soluble CD14 induced cytokine production both in the presence and absence of LPS. In addition, neither sCD14 nor anti-CD14 monoclonal antibody which blocks the interaction of LPS with CD14 had any effect on the binding of LPS to HBECs. These data suggest that sCD14 may induce the release of IL-6 and IL-8 from HBECs. However, the binding of LPS to bronchial epithelium appears to be mediated by CD14-independent mechanisms.

Human monocyte activation induced by an anti-CD14 monoclonal antibody.

An anti-CD14 mAb RoMo-1 rapidly induces in human monocytes a transient oxidative burst activity as detected by chemiluminescence assay. Pretreatment of these cells with the mAb markedly suppresses the monocyte chemiluminescence response to opsonized zymosan. In addition, the antibody induces a significant increase of IL-1 production and secretion by mononuclear cells, comparable to a similar effect of rIFN-gamma or LPS. Electron microscopy demonstrates internalization of the CD14 molecules after interaction with the mAb in a characteristic receptor-like manner.

Monoclonal antibodies against human leucocyte antigens. IV. Antibodies against subunits of the LFA-1 (CD11a/CD18) leucocyte-adhesion glycoprotein.

Six monoclonal antibodies were prepared that recognize a broadly expressed antigen composed of noncovalently associated 95 kDa and 180 kDa subunits. Antibodies MEM-25, MEM-83 and MEM-95 are shown to react with a determinant(s) present on the isolated larger subunit identified as the CD11a glycoprotein, MEM-48 reacts with the isolated smaller subunit which is identical to the CD18 glycoprotein. Antibodies MEM-30 and MEM-94 recognize probably a determinant in the CD11a/CD18 complex (i.e., LFA-1 antigen) dependent on association of the constituent chains. Cytotoxic activity of NK cells in inhibited by antibodies MEM-25, MEM-95 and less strongly by MEM-30 and MEM-94.

Monoclonal antibodies against human leucocyte antigens. I. Antibodies against beta-2-microglobulin, immunoglobulin kappa light chains, HLA-DR-like antigens, T8 antigen, T1 antigen, a monocyte antigen, and a pan-leucocyte antigen.

Monoclonal antibodies against several human leucocyte cell surface antigens were prepared and characterized: B2M-02, a non-cytotoxic antibody against beta-2-microglobulin; MEM-09 and MEM-40, against immunoglobulin kappa-type light chains; MEM-12, MEM-24G and MEM-32B, all against a monomorphic determinant in MHC class II antigens, presumably HLA-DR, dependent on the association of alpha and beta chains; MEM-15 and MEM-18, against a monocyte antigen of 55 kDa; MEM-28, against a 200 kDa antigen expressed on all leucocytes; MEM-31, against the T8 antigen of the cytotoxic/suppressor T-lymphocyte subpopulation; MEM-32, against the T1 antigen of T lymphocytes.

Monoclonal antibodies against human leucocyte antigens. III. Antibodies against CD45R, CD6, CD44 and two newly described broadly expressed glycoproteins MEM-53 and MEM-102.

Monoclonal antibodies against several human leucocyte cell surface antigens were prepared and characterized: (1) MEM-56, MEM-93, MEM-66, and MEM-104 against the CD45R antigen (220 kDa and 205 kDa mol. wt. forms of the leucocyte common antigen CD45 expressed on B-lymphocytes, myeloid cells and a subpopulation of T-lymphocytes); (2) MEM-98 and MEM-100 against the T-lymphocyte antigen CD6 (mol. wt. 100 kDa); (3) MEM-85 against the broadly expressed antigen CD44 (mol. wt. 85 kDa) which was recently shown by us to be identical with the lymphocyte homing receptor; (4) MEM-53 against a newly described broadly expressed antigen of 35-40 kDa mol. wt. and (5) MEM-102 against another newly described glycoprotein of 40 kDa mol. wt. anchored in membrane through a phosphatidylinositol moiety.

Monoclonal antibodies against human leucocyte antigens. II. Antibodies against CD45 (T200), CD3 (T3), CD43, CD10 (CALLA), transferrin receptor (T9), a novel broadly expressed 18-kDa antigen (MEM-43) and a novel antigen of restricted expression (MEM-74).

The specificities of several monoclonal antibodies described by us earlier were determined more exactly: (1) antibodies MEM-31 and MEM-32 are directed against CD8 and CD5 T cell antigens, respectively: (2) MEM-15 and MEM-18 react with the CD14 monocyte antigen; (3) MEM-28 recognizes the CD45 pan-leucocyte antigen. Several new monoclonal antibodies are described: (4) MEM-55 and MEM-58, reactive with subsets of CD45-related molecules; (5) MEM-43, recognizing a broadly expressed 18-kDa glycoprotein; (6) MEM-57, reacting with the CD3 antigen complex: (7) MEM-59 recognizing the CD43 leucocyte antigen; (8) MEM-74, which reacts with an unidentified antigen strongly expressed on several cell lines and many leukaemic cells but absent from most resting leucocytes; (9) MEM-75, directed against transferrin receptor, and (10) MEM-78, recognizing CD10 (CALLA) antigen.

Physiological enzymatic cleavage of leukocyte membrane molecules.

Certain membrane molecules are enzymatically cleaved from the cell surface and then released into the extracellular medium in the form of soluble fragments. This process, commonly initiated by cell stimulation, may regulate the surface expression of such molecules, and may also be responsible for the production of their soluble forms in vivo. Here, Vladimír Bazil provides an overview of the molecules that are cleaved from cells, focusing particularly on leukocyte receptors. In addition, he discusses the mechanisms and putative enzymes involved in this process, as well as the potential physiological significance of such events.

Shedding of the CD44 adhesion molecule from leukocytes induced by anti-CD44 monoclonal antibody simulating the effect of a natural receptor ligand.

The CD44 adhesion molecule, playing an important role in leukocyte extravasation, was down-regulated by PMA and ionomycin on granulocytes and by an immobilized or soluble anti-CD44 mAb both on granulocytes and lymphocytes. Soluble labeled CD44 molecules of lower apparent molecular mass as compared to their membrane counterparts were isolated from culture supernatants of stimulated surface iodinated cells. Shedding rather than internalization is the mechanism found to be responsible for the loss of CD44 from the cell surface. The size of the soluble CD44 shed from the cells stimulated in vitro corresponds to soluble CD44 isolated from human serum. These data suggest that shedding, induced by anti-CD44 antibody simulating the effect of a natural CD44 ligand, is an important regulatory mechanism controlling surface CD44 expression on leukocytes in vivo.

Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes.

CD14, expressed on the surface of monocytes as a phospholipid-linked protein, is a receptor for serum LPS binding protein/LPS complex. It was specifically down-modulated after stimulation of monocytes by physiologic activating/differentiating agents such as bacterial LPS and IFN-gamma, by the pharmacologic agents PMA and calcium ionophore A23187, and by anti-CD14 antibodies. The down-modulation was almost totally blocked at 4 degrees C or at pH 4.5 and markedly inhibited by the protease inhibitors diisopropylfluorophosphate and PMSF. A soluble labeled CD14 was isolated from culture supernatant of surface iodinated monocytes after their activation, indicating that CD14 is shed from the cell surface rather than internalized. The size of the soluble CD14 shed from the monocytes in vitro was smaller than that of either the membrane-bound form or a soluble CD14 cleaved from the cell surface by phosphatidyl inositol-specific phospholipase C, but identical to the size of one of the two major soluble CD14 forms normally found in human serum. These data suggest that CD14 shedding induced by monocyte stimulation may play an important role in the regulation of surface CD14 expression.

Metalloprotease and serine protease are involved in cleavage of CD43, CD44, and CD16 from stimulated human granulocytes. Induction of cleavage of L-selectin via CD16.

CD43, CD44, CD16, and L-selectin have been previously shown to be enzymatically cleaved from stimulated leukocytes. However, little is known about the enzymes involved in these processes. Here, metalloprotease(s) inhibitable by 1,10-phenanthroline together with serine protease(s) inhibitable by N alpha-p-tosyl-L-lysine chloromethyl ketone and 3,4-dichloroisocoumarin are shown to be involved in the cleavage of CD43, CD44, and CD16 but not in the cleavage of L-selectin on granulocytes. In addition, mAbs that recognize these individual receptors and induce their specific cleavage did not initiate cleavage of the others. In one case only, L-selectin, cleavage was also triggered by mAbs interacting with CD16 (the low affinity Fc gamma R). Thus, this mechanism represents a novel pathway of L-selectin cleavage induction.

CD43, the major sialoglycoprotein of human leukocytes, is proteolytically cleaved from the surface of stimulated lymphocytes and granulocytes.

CD43, the major sialoglycoprotein of human leukocytes, whose expression is defective in patients with the Wiskott-Aldrich syndrome, was down-regulated by phorbol 12-myristate 13-acetate (PMA) on granulocytes but not on lymphocytes. However, CD43 expressed on both of these leukocyte subpopulations was down-regulated after crosslinking by anti-CD43 monoclonal antibodies, a stimulation that may simulate the effect of a natural CD43 ligand. Soluble, labeled CD43 molecules were isolated from culture supernatants of both surface-iodinated granulocytes activated by PMA and lymphocytes stimulated with anti-CD43 antibodies. Thus, in this case down-regulation represents release from the cell surface into the culture medium, rather than internalization. The apparent molecular masses of the released molecules and of soluble CD43 isolated from human serum were identical. Importantly, PMA-induced down-regulation of CD43 on granulocytes was markedly blocked both by the metalloprotease inhibitor 1,10-phenanthroline and by the serine protease inhibitors N alpha-(p-tosyl)-L-lysine chloromethyl ketone and Pefabloc SC, which inhibit two different classes of proteases, thus indicating that the release is proteolytic.

Apoptosis of human hematopoietic progenitor cells induced by crosslinking of surface CD43, the major sialoglycoprotein of leukocytes.

Interactions of hematopoietic progenitor cells (HPC) with bone marrow stroma, mediated by adhesion molecules, are assumed to be critically important to the regulation of hematopoiesis. However, the specific roles of individual adhesion molecules involved in these interactions are poorly understood. Here, a monoclonal antibody, MEM-59, recognizing CD43, an adhesion molecule highly expressed on HPC, is shown to induce apoptosis in this cell population. This process operates at the single-cell level, and its initiation requires crosslinking of surface CD43 and the presence of cytokines. In contrast to HPC, more differentiated cells originating from this primitive cell population, as well as peripheral lymphocytes, do not undergo apoptosis in response to the CD43-mediated stimulation. Thus, CD43 may function as a negative regulator of early hematopoietic events, delivering a signal for apoptosis of HPC.

Resistance of human hematopoietic stem cells to a monoclonal antibody recognizing CD43.

Hematopoietic progenitor cells (HPC) interact with bone marrow stroma by adhesion molecules which are thought to be critically important to the regulation of hematopoiesis. The specific roles of individual adhesion molecules involved in these interactions remain poorly understood. A monoclonal antibody (mAb) recognizing CD43, an adhesion molecule highly expressed by HPC, induces apoptosis in CD34hiLin- marrow cells. This process operates at a single-cell level, and the initiation of apoptosis requires crosslinking of surface CD43 and the presence of cytokines. In contrast to HPC, more differentiated hematopoietic cells do not undergo apoptosis in response to the CD43-mediated stimulation. Not all progenitor cells undergo apoptosis upon stimulation of CD43. Dividing progenitor cells are most affected, whereas more primitive, quiescent cells survive anti-CD43 mAb treatment. These surviving cells: A) are enriched for cobblestone area-forming cells; B) repopulate fragments of human fetal bone implanted into CX.B-17 severe combined immunodeficiency (SCID/hu) mice; C) have a potential to differentiate in vivo to myeloid and lymphoid cells, and D) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate tha cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis compared to HPC and that CD43 may function as a negative regulator of early events occurring during hematopoiesis.

Gene for human CD59 (likely Ly-6 homologue) is located on the short arm of chromosome 11.

The CD59 (MEM-43) antigen, which probably is a human homologue of mouse Ly-6 antigens, is a broadly expressed Mr 18,000-25,000 human leucocyte surface glycoprotein recognized by monoclonal antibody MEM-43. Ten mouse-human T-lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, were analyzed for expression of CD59 by indirect immunofluorescence and immunoblotting with MEM-43 antibody. Karyotypic analysis of the tested clones showed that the presence of human chromosome 11 correlated with the expression of CD59 in all clones tested. Three other human chromosome 11-encoded antigens, 4F2 (Trop-4), Leu 7 (HNK-1, CD57), and lymphocyte homing receptor, were expressed concordantly with CD59. A more exact localization of the gene for CD59 was obtained by the study of Chinese hamster-human cell hybrids containing short or long arm deletions of human chromosome 11. CD59 segregated with hybrids containing part of the short arm of human chromosome 11, but not with the hybrids containing the long arm. Based on these studies we assign the gene for CD59 to region p14-p13 of the short arm of chromosome 11.

Transcription of a subset of human class II major histocompatibility complex genes is regulated by a nucleoprotein complex that contains c-fos or an antigenically related protein.

Transcriptional regulation of the human major histocompatibility complex class II genes requires at least two upstream elements, the X and Y boxes, located in the -50- to -150-base-pair region of all class II promoters. The DRA and DPB promoters contain phorbol ester-responsive elements overlapping the 3' side of their X boxes. Mutation of this sequence down-regulates the efficiency of the DRA promoter, suggesting that a positive regulator(s) binds to this site. In this report, anti-sense c-fos RNA and an anti-c-fos antibody were used to show that the product of the protooncogene c-fos or an antigenically related protein is a component of a complex that binds to the X box and is required for maximal transcription from the DRA and DPB promoters. As c-fos (or its related proteins) cannot bind alone to DNA, these results suggest that it may dimerize with other members of the JUN/AP-1 family, such as hXBP1, to participate in the activation of a subset of class II major histocompatibility complex genes.

Recombinant soluble CD14 inhibits LPS-induced tumor necrosis factor-alpha production by cells in whole blood.

CD14 functions as a cell surface receptor for LPS in the activation of monocytes/macrophages and neutrophils by endotoxin. To assess the utility of soluble forms of the CD14 receptor as a possible therapeutic for endotoxin shock, we have produced recombinant human soluble CD14 using a baculovirus expression system. We find that the recombinant protein is not only expressed on the surface of the insect cells as a glycosyl phosphatidylinositol (GPI)-anchored protein, but is also released into the culture medium as a soluble form that lacks the GPI anchor. Functional analyses of recombinant human soluble CD14 show that it binds specifically to LPS and can inhibit the LPS-induced release of TNF-alpha by macrophages and mononuclear cells as well as by cells in whole human blood when used at concentrations of approximately 70 micrograms/ml. Thus, soluble CD14 may be useful as an adjunct in the treatment of endotoxin shock.

A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells.

CD43 (the major sialoglycoprotein of leukocytes) is an adhesion molecule broadly expressed on hematopoietic cells. A monoclonal antibody recognizing this molecule induces apoptosis of lineage marker-negative bone marrow hematopoietic progenitor cells (HPCs) that express CD34 at a high density (CD34hiLIN-). However, not all cells within this population undergo apoptosis on stimulation via CD43. Dividing progenitor cells are most highly affected, whereas more primitive quiescent cells survive anti-CD43 monoclonal antibody treatment. These surviving cells (1) are enriched for cobblestone area-forming cells, (2) repopulate fragments for human fetal bone implanted into C.B-17 scid/scid mice, (3) have a potential to differentiate in vivo to myeloid and lymphoid cells, and (4) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate that cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis as compared with HPCs. Thus, CD43 may be specifically involved in the regulation of HPC proliferation.