Impaired accessory cell function in a human dendritic cell line after human immunodeficiency virus infection

We generated human dendritic cell (DC) hybridoma cell lines by fusing HGPRT-deficient promonocytic U937 cells with immature DCs obtained by culturing peripheral blood monocytes with interleukin-4 (IL-4; 1,000 U/ml) and granulocyte-macrophage colony-stimulating factor (100 U/ml) for 7 days and mature DCs by treatment with tumor necrosis factor alpha (12.5 microg/ml) for 3 days. Only one fusion with immature DCs was successful and yielded four cell lines--HB-1, HB-2, HB-3, and HB-9--with an overall fusion efficiency of 0.0015%. The cell lines were stable in long-term culture, displayed morphological features typical of DCs, and expressed distinct class I and class II molecules not present on U937 (A*031012, B*51011, Cw*0701, DRB3*01011 52, and DR5*01011). A representative cell line, HB-2, that expressed DC markers including CD83, CD80 and CD86 could be induced to produce IL-12 through CD40 stimulation. After human immunodeficiency virus (HIV) infection, there was impairment of antigen-presenting cell (APC) function, which was manifested by an inability to stimulate allogeneic T-cell responses. There was no change in expression of major histocompatibility complex class I and class II antigens, CD83, CD40, CD4, CD11c, CD80, CD86, CD54, and CD58, or IL-12 production in the HIV-infected HB-2 cells. The HIV-infected HB-2 cells induced T-cell apoptosis in the cocultures. T-cell proliferation could be partially restored by using ddI, indinivir, and blocking anti-gp120 and anti-IL-10 antibodies. Our data suggest that there are multiple mechanisms that DCs use to inhibit T-cell responses in HIV-infected patients. The HB-2 cell line could be a useful model system to study APC function in HIV-infected DCs.     

Induction of selective biological responses to chemoattractants in a human monocyte-like cell line

The availability of monocyte cell lines that can be induced to differentiate in a predictable fashion can provide important tools for the study of the biochemical mechanisms of specific cellular responses. The U937 human monocyte cell line was previously shown to differentiate into chemotactically responsive cells when incubated with supernatants of lectin-stimulated lymphocytes (conditioned medium). Considering the heterogeneous nature of stimulated lymphocyte supernatants, attempts were made to identify well-defined agents that could reproducibly induce U937 cell differentiation. Both dimethyl sulfoxide and dibutyryl cAMP induced expression of receptors for the N-formylated oligopeptide chemoattractants in U937 cells. Unstimulated U937 cells contained no detectable receptors. After cells were exposed to 1 mM dibutyryl cAMP, 1.3% dimethyl sulfoxide, or 5% conditioned medium for 72 h, the average number of oligopeptide chemoattractant receptors per U937 cell was 33,000, 4,000, and 3,400, respectively. Specific binding proteins for the chemoattractants were identified by covalent affinity labeling on the differentiated U937 cells as well as on normal human monocytes. Cells exposed to conditioned medium responded chemotactically, secreted lysosomal enzymes, and formed superoxide anion when incubated with the chemoattractant. Treatment of U937 cells with dibutyryl cAMP resulted in the most reproducible and rapid increase in the number of chemoattractant receptors as well as in chemotactic responsiveness. The receptors on dibutyryl cAMP-treated cells and on dimethyl sulfoxide-treated cells initiated chemotaxis and lysosomal enzyme secretion in response to chemoattractants, but not the formation of superoxide anion. These findings demonstrate that development of the chemotactic and respiratory burst functions during the differentiation of a monocyte-like cell line can occur independently.     

Antibody-mediated enhancement of BK virus infection in human monocytes and a human macrophage-like cell line

We have monitored BK virus (BKV) antigen expression and multiplication in human monocytes and in a human macrophage (M luminal diameter)-like cell line (U937) in the presence or absence of dilution series of human or rabbit anti-BKV antisera. After infection with BKV alone, restricted expression (structural antigens and T-antigen) and multiplication was recorded in monocytes from some donors, while in U937 cells and monocytes from other donors, no signs of viral activity were detected. Monocyte cultures established from the same donor at different times demonstrated antigen expression/multiplication on two occasions but not on the third. A pronounced enhancement of BKV expression/multiplication in human monocytes and multiplication in U937 cells was seen with some dilutions of all antisera (human and rabbit) used. The pattern of enhancement and the dilution resulting in maximum viral activity was constant and seemed to be determined by the serum, but the exact level of enhancement for a given serum differed considerably in monocytes from different donors and seemed to be determined by the cells. In the latter respect, monocytes taken from the same donor some weeks apart showed variations at the same level, as did cells from different donors. PMA (phorbol-12-myristate-13-acetate) stimulation of monocytes and U937 cells resulted in stronger antibody enhancement in terms of infectivity, without affecting the number of monocytes showing antigen expression. No expression/multiplication of BKV was detected in the murine M luminal diameter-like cell line P338 DI.     

A neomycin-resistant cell line for improved production of monoclonal antibodies to cell surface antigens

Overgrowth of hybridomas by proliferating spleen T cells often hinders the production of monoclonal antibodies to certain antigens. To overcome this problem we derived neomycin-resistant fusion partner SP2 neoR.1 by transfection of SP2/0-Ag14 cells with plasmid pSVTK neo beta. Hybridomas obtained with SP2 neoR.1 grew optimally in the presence of the neomycin analog G418 at concentrations which blocked the proliferative response of T cells to mitogenic stimuli. The advantage of using SP2 neoR.1 and G418 under conditions where spleen cell proliferation occurs after fusion was demonstrated with hybridomas derived from a rat immunized with mouse helper T cell lines.     

Association of some cell surface antigens of lymphoid cells and cell surface differentiation antigens with early rat pregnancy

Monoclonal antibodies and the immunoperoxidase technique were used to localize some cell surface antigens of rat lymphoid cells and cell surface differentiation antigens on cryostat sections of early rat pregnancies. The W3/13 leucocyte sialoglycoprotein was detected almost constantly on trophoblast. The immunoglobulins were more associated with mother's rather than with embryo-derived tissues. We were unable to detect considerable amounts of class I and class II major histocompatibility complex-derived antigens on trophoblast and adjacent decidual cells. The Ia+ cells of the lymphocyte type were occasionally detected in the sites exhibiting presence of immunoglobulins. The Thy-1 cell surface differentiation antigen was detected on the cells producing Thy-1+ material among decidual cells. Depletion of Thy-1 was followed by the regression of decidualized tissue. The OX-2 antigen, known as minor glycoprotein of rat thymocytes, was detected on trophoblast cells and endothelia of decidual vessels, the latter exhibiting also class I major histocompatibility complex-derived antigens. The non-pregnant uterine tissues, as well as the oviduct epithelium were also investigated. The possible role of some of these antigens in the maintenance of the 'immunologically privileged' stage of trophoblast, and in the control of the rearrangement of maternal tissues surrounding the embryo, is discussed.     

Quantitative measurement of Fc receptor activity on human peripheral blood monocytes and the monocyte-like cell line, U937, by laser flow cytometry

The expression of high affinity Fc receptors for IgG (FcRI) on the cell line U937 has been measured by flow cytometry, using fluorescein isothiocyanate-conjugated human IgG (FITC-IgG) calibrated spectrofluorimetrically against lasergrade fluorescein (Fl). A standard curve is presented, relating the effective fluorescence in terms of fluorescein equivalents per IgG molecule, to the degree of conjugation of FITC-IgG. The flow cytometer was calibrated with commercially available fluorescein-coupled latex beads. The quantitation of FcRI, in terms of sites per cell and affinity constants, was compared with a radioligand assay performed concurrently on the same cell population. Good agreement between the two assays was observed. The Fc receptors on peripheral blood monocytes were measured in unpurified lysed blood by gating on forward/side scatter. Monomer IgG binding to monocyte FcRII or FcRIII cannot be measured in direct IgG radioligand analyses because of the low affinity of these receptors and their low numbers per cell. However, flow cytometry may be employed for measuring both high and low affinity ligand-FcR interactions, using monomer FITC-IgG.     

HIV infection of monocytes inhibits the T-lymphocyte proliferative response to recall antigens, via production of eicosanoids.

Human monocytes infected in vitro with human immunodeficiency virus (HIV) soon after adherence to plastic substrate demonstrated a significantly decreased ability to restimulate autologous immune T-lymphocyte proliferation after exposure to soluble (tetanus toxoid) and particulate [herpes simplex virus (HSV)] antigen. Incubation with the cyclo-oxygenase inhibitor, indomethacin (2-5 microM), prevented inhibition of antigen-stimulated lymphocyte proliferation. The inhibitory activity was identified in ultrafiltrates containing the low molecular weight fraction (less than 3000 MW) of supernatants from HIV-infected monocyte cultures. This activity was significantly and markedly reduced in similar ultrafiltrates prepared from indomethacin-treated cultures. Increased concentrations of prostaglandin E2 (PGE2) were detected in ultrafiltrates from HIV-infected monocyte cultures compared with uninfected cultures and cultures preincubated with indomethacin. Ultrafiltrates were inhibitory when added during the presentation of antigen to T lymphocytes but not when removed from monocyte cultures prior to the addition of lymphocytes. In addition, ultrafiltrates inhibited antigen-stimulated lymphocyte proliferation and PHA-induced lymphocyte proliferation to the same extent. These data indicate that cyclo-oxygenase products of arachidonic acid, including PGE2, are produced in excess by HIV-infected monocytes and that PGE2 and perhaps other cyclo-oxygenase products are implicated in the inhibition of antigen-stimulated lymphocyte proliferation via a direct effect on T lymphocytes.     

T-cell subsets in multiple sclerosis: a comparative study between cell surface antigens and function

Pokeweed mitogen (PWM)-driven immunoglobulin synthesis (IgG, IgA, IgM) and concanavalin A (Con A)-stimulated suppression of allogeneic mixed leukocyte reaction (MLR) were studied and compared to T-cell subsets defined by monoclonal antibodies OKT4 and OKT8 in patients with multiple sclerosis (MS). The group of patients with active progressive MS showed diminished suppressor activity as measured by T-cell functional tests and also an elevated OKT4/OKT8 ratio. The group of MS patients in remission did not show these abnormalities. However, this correlation between functional tests and T-cell phenotypes was not found when separate individuals were considered within the subgroups of MS. Since neither OKT4 nor OKT8-reactive cells represent homogeneous functional subsets of T cells, the OKT4/OKT8 ratio does not account for the functional immunological status of separate individuals but rather provides a global evaluation of T-cell subset disturbances in different groups of diseases.     

The kinetic study on cell surface antigens of a hybridoma between human B cells and a mouse B-cell line

Human B cells obtained from tonsils were fused with a mutant clone of a murine B-cell line in the presence of polyethylene glycol and dimethyl sulfoxide. THT12.58, a subclone of the human-mouse B-cell hybridoma, was shown to express human B-cell surface antigens on the cell membrane, namely B1, B2, I2, human IgM and IgD derived from human B cells by analysis of flow microfluorometry (FMF), as well as murine B-cell markers; the amount of these human B-cell markers on THT12.58 did not change for more than 1 year after establishment. Interestingly, the expression of the human B-cell antigens significantly decreased after treatment of the cells with B-cell stimulatory factors (BSF) obtained from the supernatant of the culture of PHA-P activated T cells; this was followed by significant enlargement in cell size compared with the control. A marked decrease in the amount of each antigen on the hybrid was observed when treated with Staphylococcus aureus Cowan I strain (SAC) in addition to BSF. In contrast, the expression of these markers on the cells increased after exposure of the cells to recombinant human interferon-gamma (rINF-gamma). Additionally, the effect of BSF on the generation of IgM-secreting cells by human B cells markedly decreased after absorption of BSF with the hybrid cells. These results suggest that THT12.58 may possess a receptor for BSF on the cell surface, and be capable of differentiating into much more mature stage of B-cell lineage after exposure to BSF. Thus, this kind of a human-mouse B-cell hybridoma with human B-cell differentiation antigens can be a good model to investigate the kinetics of cell surface antigens and characterization of a receptor for BSF on human B cells.     

Impaired in vitro survival of monocytes from patients with HIV infection.

In vitro survival of monocytes (MO) was studied in 59 patients with HIV infection of different clinical stages. MO from 61 donors and 12 healthy seronegative homosexual men were also examined. Compared with the number of MO seeded, the percentage of adherent monocyte-derived macrophages (MDM) present after 10 days was significantly lower in patients with HIV infection than in the controls. However, the number of viable, non-adherent MO/MDM was similar in patients and controls. Our data indicate markedly decreased in vitro survival of MO from patients with HIV infection. After 10 days, the MDM population in the patient cultures was significantly less differentiated than the control cells, assessed by immunocytochemical staining with monoclonal antibodies against differentiation antigens. Reduced in vitro survival of MO/MDM was associated with low numbers of CD4+ and CD8+ lymphocytes in blood, reduced lymphocyte mitogen responses, presence of HIV p24 antigen in serum and advanced clinical stage. Decreased in vitro survival of MO/MDM may be associated with HIV replication in the cells. Although the level of HIV replication in the cultures was low as assessed by measurement of HIV p24 antigen in culture supernatants and staining of MO/MDM for HIV antigens, cytopathogenic effects of HIV or HIV products cannot be ruled out.     

生殖细胞核因子及Oct-4在P 19细胞系诱导分化中的表达

目的 体外培养未成熟畸胎瘤细胞（P19细胞）并诱导分化,检测生殖细胞核因子（GCNF）及Oct-4在P 19细胞诱导分化前后的表达。
方法 用全反式维甲酸（ATRA）诱导P 19细胞分化,通过间接免疫荧光染色和RT-PCR方法检测GCNF及Oct-4在P 19细胞诱导分化前后的表达。
结果 Oct-4在P 19细胞诱导前呈强阳性表达,诱导2 d后表达下降;而GCNF在P 19细胞诱导前呈阴性表达,诱导2 d后呈阳性表达。
结论 GCNF参与了恶性生殖细胞肿瘤的体外分化。GCNF可能是通过下调Oct-4基因的表达参与了恶性生殖细胞肿瘤的分化。     

Biological significance of the antibody response to HIV antigens expressed on the cell surface

Summary Human antibodies to HIV antigens expressed on the surface of infected cells may inhibit cell fusion with uninfected CD 4-positive cells and mediate killing of the infected cells by effector cells bearing the Fc receptor.Sequential sera from ten HIV-antibody seroconverted men, of which five progressed to ARC or AIDS (CDC stage IV) during the follow-up period of two years, were tested for the ability to inhibit CD 4-dependent cell fusion, (CFI) and to mediate antibody-dependent cellular cytotoxicity (ADCC).Nine patients developed HIV-specific ADCC and seven CFI-antibodies using the HIV strain HTLV-IIIB as target antigen. These antibodies appeared approximately at the same time 2–12 months after primary infection, defined as antibody seroconversion or antigenaemia. ADCC antibodies were detectable at higher titers as compared to CFI-antibodies. All sera of asymptomatic individuals (CDC stage II and III) were CFI antibody positive and had a higher mean ADCC titer as compared to sera from patients progressing to AIDS or ARC.ADCC and CFI antibodies coincided in some cases in the complete absence of core antibodies. Because the relationship between ADCC and CFI was not exclusive it is concluded that distinct domains of the HIV envelope induce natural antibodies mediating ADCC and CFI.     

Longitudinal study of leukocyte functions in homosexual men seroconverted for HIV: rapid and persistent loss of B cell function after HIV infection

The early effects of infection with human immunodeficiency virus (HIV) were investigated in homosexual men who had seroconverted for anti-HIV antibodies. Leukocyte functional activities were determined in longitudinally collected peripheral blood mononuclear cell samples. During the first 10 months following seroconversion, anti-CD3 monoclonal antibody-induced T cell proliferation, monocyte accessory function and T helper activity on B cell differentiation in a pokeweed mitogen-driven system were not affected. In contrast, from the moment of seroconversion on, B cells of seroconverted men failed to produce immunoglobulin in the pokeweed mitogen-driven system. This defect was not restored by addition of normal CD4+ T cells. Immunoglobulin synthesis induced by Staphylococcus aureus and interleukin 2 decreased gradually, until it was completely lost 10 months after seroconversion. In addition, proliferation in response to anti-IgM or Staphylococcus aureus by B cells from HIV seroconverted men was decreased. The lack of inducible in vitro B cell activity was not accompanied by elevated spontaneous Ig synthesis by B cells of the seroconverted men. In the second group of men studied during the 2nd year following seroconversion, T helper activity on normal B cell differentiation significantly decreased, whereas anti-CD3-induced T cell proliferation and monocyte accessory function were not significantly affected. Our results demonstrate that in almost all HIV-infected individuals B cell functional defects are the first leukocyte abnormalities observed preceding defects in T helper activity.     

gp63 homologues in Trypanosoma cruzi: surface antigens with metalloprotease activity and a possible role in host cell infection

gp63 is a highly abundant glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed predominantly in the promastigote but also in the amastigote stage of Leishmania species. In Leishmania spp., gp63 has been implicated in a number of steps in establishment of infection. Here we demonstrate that Trypanosoma cruzi, the etiological agent of Chagas' disease, has a family of gp63 genes composed of multiple groups. Two of these groups, Tcgp63-I and -II, are present as high-copy-number genes. The genomic organization and mRNA expression pattern were specific for each group. Tcgp63-I was widely expressed, while the Tcgp63-II group was scarcely detected in Northern blots, even though it is well represented in the T. cruzi genome. Western blots using sera directed against a synthetic peptide indicated that the Tcgp63-I group produced proteins of approximately 78 kDa, differentially expressed during the life cycle. Immunofluorescence staining and phosphatidylinositol-specific phospholipase C digestion confirmed that Tcgp63-I group members are surface proteins bound to the membrane by a GPI anchor. We also demonstrate the presence of metalloprotease activity which is attributable, at least in part, to Tcgp63-I group. Since antibodies against Tcgp63-I partially blocked infection of Vero cells by trypomastigotes, a possible role for this group in infection is suggested.     

Release from a human monocyte-like cell line of two different soluble forms of the lipopolysaccharide receptor,CD14

Lipopolysaccharide (LPS) stimulates mononuclear phagocytes to synthesize and secrete immunoregulatory and inflammatory molecules such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). LPS forms complexes with either the serum protein termed LPS-binding protein or a serum factor, septin. These complexes are more stimulatory than LPS alone. The myeloid differentiation antigen CD14 is known to be the receptor for such complexes. In the present study, by using a monocytic cell line, we demonstrate the release of two different soluble forms of CD14 (sCD14) which are secreted by different mechanisms. We show that the two sCD14 forms differ in their electrophoretic mobility, two-dimensional gel electrophoretic patterns, sensitivity to endoglycosidases and peptide maps. One of the sCD14 molecules, apparent molecular mass 48 kDa, was found in supernatants of both surface iodinated and [³⁵S] methionine biosynthetically labeled cells. The other sCD14 molecule (56 kDa) was found labeled only in supernatants of [³⁵S] methionine-labeled cells. Furthermore, purified 48 kDa sCD14 enhanced the LPS-induced TNF-a and IL-6 release by the monocytic cells suggesting that a cell-surface signal transducer molecule may be involved in signaling. The data suggest a possible novel role for sCD14 in the monocyte response to LPS.     

Hepatic vascular endothelial cells heterogenously express surface antigens associated with monocytes,macrophages and T lymphocytes

Summary During studies of the antigenic and functional properties of hepatic sinusoidal lining cells in situ, we found that only the sinusoidal endothelial cells share antigens with a peripheral blood macrophage subset capable of presenting soluble antigens and triggering autologous mixed lymphocyte reactions. They were HLA-DR+, OKM1–, OKM5+. Vascular endothelial cells in the portal areas and central veins were HLA-DR+, OKM1– and OKM5–. The sinusoidal endothelial cells also expressed an antigen found on helper/inducer (OKT4 and Leu3 a) T lymphocytes. Thus, the present study suggests that endothelial cells in different anatomic compartments in the liver heterogenously express surface antigens associated with monocytes, macrophages and T lymphocytes and possess distinct immunological functions.     

The human monocyte-like cell line THP-1 expresses Fc gamma RI and Fc gamma RII.

THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized Fc gamma R expression on this cell line by flow cytometry, radiolabeled IgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-Fc gamma RI, II, and III mAb, and a rabbit anti-Fc gamma RIII F(ab')2 demonstrated that only Fc gamma RI and Fc gamma RII are expressed by these cells. A panel of anti-Fc gamma RIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (Fc gamma RI) and of 42 to 53 kDa (Fc gamma RII). Fc gamma R expression was determined by binding of radioiodinated human IgG1 (to detect Fc gamma RI), mAb IV.3 (to detect Fc gamma RII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 x 10(-9) M) for IgG1 were found on THP-1 cells. Interferon-gamma (IFN gamma) upregulated Fc gamma RI expression by THP-1 cells 2.8-fold, whereas Fc gamma RI on U937 cells was increased six- to eight-fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor-alpha (TNF alpha), and vitamin D3 had no effect on IgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFN gamma treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D3 treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.3 identified 50,000 sites per cell. Treatment of THP-1 cells with IFN gamma, TNF alpha, PMA, or vitamin D3 had no effect on Fc gamma RII expression. That Fc gamma RI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human IgG1 as well as mouse IgG2a mAb to Fc gamma RII inhibited immune complex binding by 76 to 84%, whereas mouse IgG1 mAb to Fc gamma RII had minimal effect on immune complex binding. Fc gamma R expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D3 was able to induce the expression of CD14, a marker of mature monocytic phenotype.     

Persistent infection with human cytomegalovirus in a lymphoblastoid cell line.

Cells from a line of human lymphocytes originating from a leukemic patient were persistently infected with human cytomegalovirus (HCMV). The infected culture has persistently yielded HCMV with titers ranging from 2 × 10⁴ to 3 × 10⁵ PFU/ml over a period of 1 year. Infectious center and fluorescent antigen assays and electron microscopic examination indicated that 1–10% of the cells were infected. It appears that persistent infection is due to a balance between release of virus and the growth of uninfected cells rather than to a defective or temperature-sensitive mutant of HCMV. The treatment of persistently infected cultures with anti-HCMV serum resulted in curing the virus infection. Cured cells in culture grew at the same rate as normal uninfected cells and became resistant to HCMV infection and relatively resistant to HSV infection.