Apoptotic human lymphocytes have diminished CD4 and CD8 receptor expression.

We used quantitative multiparameter flow cytometric assays to simultaneously detect viable, apoptotic, and necrotic human peripheral blood mononuclear cells (PBMC) and immunophenotyped lymphocyte subsets within the PBMC. Apoptosis was induced by a spectrum of treatments, including camptothecin, cisplatin, dexamethasone, hyperthermia, staurosporine, and etoposide in anti-CD3 mAb-stimulated cells and by cyclohexamide in both quiescent and stimulated cells; apoptosis in the latter was augmented by anti-fas mAb. We found that CD4(+) and CD8(+) cells were significantly underrepresented in the apoptotic PBMC and that the percentage of CD4(+) and CD8(+) PBMC each markedly decreased as apoptosis increased. This suggested that surface expression of these receptors was lessened on apoptotic CD4(+) and CD8(+) cells. This was directly confirmed by observation of sorted CD4(+) PBMC. This analysis of a wide variety of apoptotic stimuli demonstrates that diminished CD4 and CD8 surface receptor expression is a common feature of human T lymphocyte apoptosis.

Werner syndrome lymphoblastoid cells are sensitive to camptothecin-induced apoptosis in S-phase.

Werner Syndrome (WRN) is an autosomal recessive disorder showing an endogenous mutator phenotype in combination with an elevated risk of predominantly mesenchymal cancer. The gene mutated in WRN patients codes for 3'-->5' DNA helicase and 3'-->5' exonuclease activities. We have found similar S-phase arrest in both WRN and control cells after treatment with the DNA-topoisomerase-I-trapping drug camptothecin; this may be responsible for the drug-exposure-related growth inhibition seen in both cell types. A clearer phenotypic difference between WRN and control immortalized B-cell lines (LCLs) is obtained by examining cell death. The mechanism of camptothecin-induced cell death in WRN-deficient LCLs appears to be through apoptosis, a phenotype that strongly differentiates WRN-deficient from wild-type LCLs. We hypothesize that, in cells deficient for WRN function, a topoisomerase-I-DNA intermediate persists. Conflict with DNA replication may lead to apoptosis, increased mutation rates, and cancer in WRN.

An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants.

Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to this genotoxic agent, which are intermediate to those of wild-type and homozygous mutants. 4NQO is shown to induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the question of an enhanced sensitivity of the relatively prevalent heterozygous carriers to certain environmental genotoxic agents.

The human leukocyte integrin CD11a promoter directs expression in leukocytes of transgenic mice.

The human CD11a molecule is expressed specifically on lymphocytes, monocyte/macrophages, and neutrophils, in which it mediates important adhesion-related functions. We used 1.7 kb of regulatory sequences upstream from the human CD11a gene transcription start site to drive expression of a modified human CD4 reporter gene in transgenic mice. The transgene was expressed in a tissue-specific fashion on all leukocytes and paralleled endogenous mouse CD11a expression. All five founder mice expressed the transgene, providing evidence for integration site-independent expression. However, expression was not proportional to transgene copy number. These studies indicate that (1) the mutated human CD4 serves as an excellent reporter for analysis of leukocyte-specific promoters; (2) the CD11a regulatory unit used here represents a novel reagent for targeting gene expression to leukocytes; and (3) additional regulatory regions will be required for copy-number-dependent activity of CD11a regulatory sequences.

Transfected leukocyte integrin CD11b/CD18 (Mac-1) mediates phorbol ester-activated, homotypic cell:cell adherence in the K562 cell line.

The CD11b/CD18 leukocyte integrin molecule mediates diverse neutrophil adherence-related functions, including cell:cell and cell:extracellular matrix attachments. To study the individual role of this leukocyte integrin in cell adherence in hematopoietic cells, we expressed the CD11b/CD18 complex on the surface of K562 cells, a cell line derived from an individual with chronic myelogenous leukemia in blast crisis. We used an amphotrophic retroviral vector designated LCD18SN, harboring the complete coding sequence for the CD18 subunit, to transfer the CD18 cDNA into K562 cells and select stable cell lines. The CD11b subunit in the expression plasmid pREP4 was transfected into these K562/CD18 cells by electroporation and stable cell clones were selected. These K562 cells possessed RNA and intracellular protein for each subunit, and they expressed the CD11b/CD18 heterodimer on the cell surface. When CD11b/CD18 expressing K562 cells were stimulated with phorbol myristate acetate (50 ng/mL) for 24 to 48 hours, these K562 cells formed dense cell:cell aggregates. This homotypic aggregation required both activation of the CD11b/CD18 complex and the induction of the counter-receptor for CD11b/CD18 on the conjugate cell. This cell line will (1) enable the structure-function relationships between cell activation and homotypic adherence to be assessed, (2) provide the opportunity to identify accessory molecules required for activation of the CD11b/CD18 complex, and (3) facilitate the identification of novel ligands for the CD11b/CD18 complex.

Description of the leukocyte function-associated antigen 1 (LFA-1 or CD11a) promoter.

The CD11a/CD18 (leukocyte function-associated antigen 1 or LFA-1) leukocyte integrin is expressed at high levels on the cell surface of T lymphocytes and macrophages, where it mediates homotypic and heterotypic adherence between leukocytes and other cell types by binding to intracellular adhesion molecules 1 and 2 on the conjugate cell. To initiate studies of the molecular regulation of expression of the CD11a molecule, we isolated genomic clones corresponding to the 5'-flanking region of CD11a, identified the transcriptional start sites for CD11a, and characterized the CD11a promoter sequence in transient expression assays. The CD11a promoter (1.7 kb) directed functional activity of a heterologous reporter gene in the T-lymphocyte cell line Jurkat and the myeloid cell line HL-60 but did not direct functional activity in three different nonleukocyte cell lines. Deletional analysis of the CD11a promoter sequence indicated the presence of distinct, cell-type-specific regulatory sequences with the region from -40 to -17 relative to the transcription start sites responsible for most of the in vitro activity of the CD11a promoter in the Jurkat T-cell line, and the promoter sequence located within the first 17 bp relative to the transcription start sites responsible for CD11a promoter activity in the HL-60 cell line. Identification of the CD11a promoter provides the opportunity to identify unique cis-acting elements and trans-acting factors responsible for the cell-type-specific expression of CD11a in human leukocytes. Further, the CD11a promoter may be useful in transgenic constructs and in retroviral vectors to direct expression of heterologous genes selectively in leukocytes.

Identification of the promoter of the myelomonocytic leukocyte integrin CD11b.

The CD11b (or macrophage-1 antigen; MAC-1) subunit of the leukocyte integrin family forms a noncovalently associated heterodimeric structure with the CD18 (beta) subunit on the surface of human granulocytes and monocyte/macrophages, where it enables these myeloid cells to participate in a variety of adherence-related activities. Expression of the CD11b subunit is restricted to cells of the myelomonocytic lineage and depends upon the stage of differentiation with the most mature myeloid cells expressing the highest levels of CD11b. To study the regulation of CD11b expression, a genomic clone corresponding to the 5' region of the CD11b gene was isolated from a human chromosome 16 library. Primer extension and RNase protection assays identified two major transcriptional start sites, located 90 base pairs and 54 base pairs upstream from the initiation methionine. DNA sequence analysis of 1.7 kilobases of the 5' flanking sequence of the CD11b gene indicated the absence of a "CAAT" or "TATA" box; however, potential binding sites for the transcription activators Sp1, PU.1, ets, and AP-2 are present, as well as retinoic acid response elements. The 1.7-kilobase CD11b promoter sequence displayed functional activity in transient transfection assays in the monocytic cell line THP-1 and the myeloid cell line HL-60. In contrast, this 1.7-kilobase promoter sequence did not display functional activity in the Jurkat T-lymphoid cell line. Detailed characterization of the CD11b promoter sequence should provide insight into the molecular events regulating the tissue-specific and developmental stage-specific expression of the CD11b molecule in myelomonocytic cells.

Regulation of expression of the leukocyte integrin CD11a (LFA-1) molecule during differentiation of HL-60 cells along the monocyte/macrophage pathway.

The CD11a/CD18 (LFA-1) leukocyte integrin receptor mediates homotypic and heterotypic leukocyte adhesion by binding to one of two defined ligands, ICAM-1 or 2, on the conjugate cell. In this study we investigated the molecular regulation of expression of the CD11a subunit during myeloid differentiation of HL-60 cells. Induction of monocyte/macrophage differentiation of HL-60 promyelocytic leukemia cells with PMA results in an increase in CD11a surface Ag expression and the acquisition of CD11a/CD18-mediated homotypic adherence. These changes are accompanied by a 40-fold increase in CD11a mRNA levels. Nuclear run-on transcription assays indicate that the increase in CD11a mRNA in PMA-induced HL-60 cells is not caused by an increase in CD11a RNA transcription. We assessed the posttranscriptional regulation of CD11a using two methods. By using actinomycin D to block RNA transcription, we demonstrate that the CD11a mRNA half-life in HL-60 cells is prolonged after PMA treatment. Inhibition of protein synthesis with cycloheximide also results in enhanced expression of CD11a mRNA in HL-60 cells without increasing CD11a transcription. These findings indicate that, in HL-60 cells induced with PMA to differentiate along the monocyte/macrophage pathway, CD11a expression is regulated primarily at the posttranscriptional level by a labile protein. Identification of the specific CD11a RNA sequences, and the proteins that bind to these sequences may provide insight into lineage commitment during human monocyte/macrophage differentiation.

Ig lambda-producing B cells do not show feedback inhibition of gene rearrangement.

In order to study the regulation of expression of Ig lambda genes we have analyzed lambda-producing hybridomas derived from transgenic mice which harbor a functionally rearranged kappa transgene. We also analyzed lambda-producing hybridomas from nontransgenic mice. Surprisingly, all but one of the transgenic lambda-hybridomas co-produce kappa L chains. Also, in contrast to transgenic kappa-hybridomas, most lambda-hybridomas have rearranged endogenous kappa genes despite the presence of transgenic kappa-chains and endogenous H chains. Analysis of spleen cells and hybridomas from nontransgenic mice shows that about 20% of lambda-producing B cells in the spleen co-produce kappa, and a similar proportion of lambda-hybridomas from normal spleens produce both kappa- and lambda-chains. The data argue strongly against the strictly sequential expression of kappa and lambda genes. We present a new model for the regulation of kappa and lambda gene expression, whose key feature is the distinction between a kappa cell lineage in which Ig gene rearrangement is susceptible to feedback by a complete antibody molecule at the pre-B cell stage, and a kappa lambda B cell lineage which does not show feedback inhibition during B cell development.

High expression of cloned immunoglobulin kappa gene in transgenic mice is restricted to B lymphocytes.

Immunoglobulin genes are normally expressed only in cells of the B lymphocyte lineage after a variable (V) and constant (C) gene rearrangement has occurred. To study the control of immunoglobulin gene expression in a defined situation, we have produced transgenic mice by microinjecting a rearranged mouse immunoglobulin kappa gene (designated pB1-14) into fertilized mouse eggs. We present here the analysis of six different kappa-transgenic mouse lines. All the transgenic mice express the microinjected kappa gene in a completely tissue-specific fashion. Transcripts from pB1-14 are found at a high level in the spleen, but are undetectable in nonlymphoid tissues of testis, liver, kidney, heart, muscle, brain and thyroid gland. In lymphoid cell subpopulations, the level of pB1-14 transcripts is correlated with the relative number of B cells; there is no correlation with the proportion of T lymphocytes. We concluded, therefore, that the microinjected kappa gene contains target sequences for B lymphocyte-specific gene activation signals that override the influence of the integration site.

IgA hybridomas: a method for generation in high numbers.

An immunization regimen has been developed which yields a high frequency of hybridomas producing IgA isotype, antigen-specific antibody when spleen cells from immunized mice are fused with non-immunoglobulin secreting murine myeloma cells. Germfree BALB/c mice were carrier-primed with sheep erythrocytes (SRBC) by gastric intubation (GI) for 2 consecutive days followed 1 week later by GI with trinitrophenyl (TNP)-haptenated SRBC. After 7 days, spleen cells were fused with non-immunoglobulin secreting myeloma cells (X63-Ag8.653), and 2-3 weeks later, culture wells were scored for hybrid clones. Of 240 culture wells plated, 157 wells (65.4%) exhibited clones producing anti-TNP antibodies as determined by enzyme-linked immunosorbent assay. A total of 50 specific cell lines were established, of which 27 clones (54%) produced IgA isotype anti-TNP antibodies, while the anti-TNP antibodies produced by the remaining 23 clones were approximately equally distributed between the IgM and IgG isotypes. The IgA and IgM monoclonal antibodies were more effective in hemagglutinating TNP-SRBC than were IgG isotype antibodies. This study describes a method for production of a high number of antigen-specific IgA hybridomas which will allow production of IgA monoclonal antibodies to important antigens on mucosally-associated pathogens, and thus allow elucidation of functions of IgA antibody at mucosal surfaces.

Immunoglobulin bound in vivo to Fc receptor-positive cells in human central nervous system tumors.

Fifteen human central nervous system tumors of various histopathologic types were assessed qualitatively and quantitatively by indirect immunofluorescence for the presence of in vivo bound IgG, IgA, and IgM. The tumors were selected to reflect varying degrees of infiltration with Fc receptor-positive macrophages. The major purpose of the study was to determine the relative contribution of immunoglobulin (Ig) bound to tumor cells as compared to Ig bound to the Fc receptor-positive host macrophages. Of the 15 tumors, 1 tumor contained no detectable IgG, IgA, or IgM, 2 tumors contained only IgG and IgA bound in a smooth, homogeneous pattern to the surface of tumor cells, and 8 contained only IgG, IgA, and IgM attached to Fc receptor-positive cells. Four tumors contained significant numbers of tumor cells with cytoplasmic Ig, and two of those tumors also were infiltrated with Fc receptor-positive cells with membrane-associated Ig. Ig was removed from Fc receptor-positive cells but not from tumor cells by prolonged washing of sections of tumor at 37 degrees C, which suggested that Ig was associated with Fc receptors. That possibility was strengthened by the observation that the IgG subclass distribution of the Fc receptor-associated Ig was predominantly IgG1 and IgG3, whereas no predominant subclass existed for IgG bound to tumor cells. Furthermore, the Fc receptor-associated Ig appeared to be in the form of antigen-antibody complexes because it had a granular quality and because IgA and sometimes even IgM were involved in the Fc receptor-bound complexes.

The failure of microglia in normal brain to exhibit mononuclear phagocyte markers.

The origin of brain macrophages or "reactive microglia" has been the subject of considerable controversy. The fundamental question is whether or not there is a morphologically and functionally distinct population of cells, called microglia, which are resident in normal brain and differentiate into macrophages in response to inflammatory stimuli. The present study was performed to determine if any cells in the normal brain have the common markers of mononuclear phagocytes; phagocytosis, IgGFc receptors or macrophage specific antigens. In studies of the newborn and the adult murine brain and adult human brain no cells were detected which had any of those markers, although the highly sensitive marker methods were capable of detecting mononuclear phagocytes in all other tissues where they are known to occur. The results suggest that microglia, if they exist as a distinct cell type, are unrelated to mononuclear phagocytes. Furthermore, they suggest, but do not prove, that all inflammatory macrophages are derived from hematogenous precursors.

T-lymphocytes and macrophages in primary murine fibrosarcomas at different stages in their progression.

The relative contribution of lymphocytes, macrophages, and granulocytes to the cell content of primary 3-methyl-cholanthrene-induced murine fibrosarcomas was determined at different stages in their progression by differential cell analysis on enzyme-derived single-cell suspensions. Furthermore, immunohistological analyses were performed on the tumors to detect, quantitate, and determine the distribution of T-lymphocytes and macrophages. The T-lymphocyte content of small tumors was very high, and the T-cells were distributed throughout the tumor mass. As the tumor increased in size, there was a marked decrease in the relative T-cell content; most were located at the tumor periphery. Macrophages were present in significant numers in all tumors and appeared to increase in number as the tumors increased in size. Macrophages were distributed throughout the tumor mass, but generally they were more densely distributed near the tumor periphery. Granulocytes were present in low numbers in all tumors. Yeast phagocytosis was used to assess the functional capacity of the macrophage population. The phagocytic capacity of the macrophages was low in the small tumors, increased significantly as the tumors progressed, but dropped to relatively low levels in large tumors. The results represent a preliminary attempt to characterize the dynamics of host cell infiltration of primary immunogenic tumors.

Macrophages in giant cell tumours of bone.

Five giant cell tumours of bone were studied to determine the degree of macrophage infiltration and whether the giant cells expressed the characteristics commonly associated with macrophages, i.e., IgGFc and C3 receptors, phagocytosis and non-specific esterase activity. Macrophages were assessed in trypsin-derived tumour cell suspensions by IgGEAC rosette formation and in frozen sections of tumour by EA adsorption. The percentage of macrophages in cell suspensions from four of the tumours ranged from 11 to 40 per cent. Strong EA adsorption occurred over 35 to 95 per cent. of the tumours' surface and significant non-specific esterase positivity was observed in the tumour sections. The giant cells were receptor negative and non-phagocytic, but a low percentage of them expressed esterase activity. The results strongly suggest that despite the fact that large numbers of macrophages were present in the tumours, the giant cells were derived from cells other than macrophages.