Monocyte-depleted Peripheral Blood Lymphocytes Research Articles

Cytotoxic T lymphocytes promote cytarabine-induced acute myeloid leukemia cell apoptosis via inhibiting Bcl-2 expression.

Acute myeloid leukemia (AML) remains difficult to cure due to its drug tolerance and refractoriness. Immunotherapy is a growing area of cancer research, which has been applied for the treatment of numerous types of cancer, including leukemia. The present study generated AML cell-specific cytotoxic T lymphocytes (CTLs) in vitro and investigated the effect of combining CTL treatment with one of the most commonly used drugs for the treatment of hematological malignancies, cytarabine, on AML cell apoptosis. Firstly, it was observed that monocyte-depleted peripheral blood lymphocytes from healthy donors could be used to generate large numbers of CD3+CD8+ CTLs through immune stimulation. These CD3+CD8+ CTLs could effectively recognize and induce the apoptosis of human Kasumi-3 AML cells. In addition, cytarabine-induced AML cell apoptosis was enhanced by CTL treatment. Western blotting revealed that Bcl-2 expression was downregulated in AML cells following cytarabine and CTL treatment, indicating that the synergistic effect of this treatment on AML cell apoptosis is due to the downregulation of Bcl-2. These results highlight the potential application of CTL immunotherapy for the treatment of AML. Further studies optimizing the specificity and potency of CTLs, and identifying favorable combinations with other chemotherapeutic drug are required.

Antibody penetration of viable human cells. I. Increased penetration of human lymphocytes by anti-RNP IgG

Antibody penetration of viable cells and interaction with intracellular antigens may have major consequences for immunopathological processes in connective tissue diseases. We have reported previously that antibody can penetrate viable human lymphocytes. To assess further the role of antinuclear antibodies in this process, peripheral blood lymphocytes (PBMC) were incubated with FITC-conjugated IgG fractions from sera containing anti-RNP (anti-RNP IgG), Ro(SS-A), La(SS-B) and dsDNA antibodies and control sera for 24 h. Using crystal violet to quench cell surface staining, intracellular fluorescence of viable lymphocytes was quantified on the flow cytometer. It was noted that anti-RNP IgG entered 46.4 +/- 7.2% of lymphocytes which was significantly higher than anti-Ro(SS-A) (29.9 +/- 4.1%, P less than 0.05), La(SS-B) (22.0 +/- 7.5%, P less than 0.01) IgG and control IgG (28.8 +/- 2.1%, P less than 0.05) and not statistically different from anti-dsDNA IgG (32.6 +/- 14.3%). Inhibition experiments showed that the increased number of cells penetrated by anti-RNP IgG was a specific process. Time-course studies showed that anti-RNP IgG entry into cells was different from pooled control IgG. With anti-RNP IgG, positive-staining lymphocytes gradually increased in number from 12 to 24 h incubation, whilst with pooled control IgG, the peak was reached within 5 min. Dual staining experiments suggested that whereas both anti-RNP IgG and pooled control IgG entered B and NK cells, anti-RNP IgG also entered T cells. Using IgG F(ab')2 and Fc fragments from either anti-RNP IgG or pooled control IgG to compete with their FITC-conjugated counterparts indicated that the entry of anti-RNP IgG into-viable cells appeared to involve both F(ab')2 and Fc fragments, and pooled control IgG depended exclusively on the Fc portion of IgG. Further investigation by incubating anti-RNP IgG with 35S-methionine-labelled monocyte-depleted PBMC (MD-PBMC) suggested that anti-RNP IgG might react with the corresponding antigens either on the cell surface or within the cytoplasm.

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and function on cultured human glomerular epithelial cells.

Glomerular epithelial cells are involved in extracapillary inflammation (crescents) but the mechanisms of this extracapillary accumulation of macrophages, epithelial cells and occasional lymphocytes are unknown. Human glomerular parietal epithelial cells express ICAM-1 and VCAM-1 on immunohistological stains of renal biopsies. We studied the expression of these cell adhesion molecules on cultured human glomerular epithelial cells (HGEC), their regulation by pro-inflammatory cytokines, and their role in mediating the adhesion of concanavalin A (Con A)-activated peripheral blood mononuclear cells. Human glomerular epithelial cells in culture constitutively express ICAM-1 and VCAM-1. The expression of ICAM-1 was not significantly altered by tumour necrosis factor-alpha (TNF-alpha) (P = 0.32), IL-1 beta (P = 0.24), interferon-gamma (IFN-gamma) (P = 0.66) or IL-4 (P = 0.85). VCAM-1 expression was increased by all four cytokines, but only significantly so by IL-4 (P = 0.0001). Con A-stimulated, monocyte-depleted peripheral blood lymphocytes bound to human glomerular epithelial cells, median 28.9% (range 14.5-37.9%). This adherence was significantly inhibited by anti-ICAM-1 (P = 0.03) and anti-LFA-1 (P = 0.02), but not by anti-VCAM-1 (P = 0.13) or by antibody to von Willebrand factor (P = NS). The interaction between ICAM-1 on HGEC and LFA-1 on mononuclear cells may be important in the pathogenesis of extracapillary inflammation in glomerulonephritis.

Plant polyphenols induce differential regulation of CD11b expression and function on monocytes and γδ T cells (97.11)

Abstract Leukocyte adhesion and migration is partially mediated by CD11b/CD18 (MAC-1, CR3). Earlier studies have demonstrated a role for green tea polyphenols in down-regulating CD11b on monocytes and CD8+ T cells. We have recently shown a stimulatory effect of unripe apple polyphenols (APP) on γδ T cells. Thus, we compared the effect of APP on γδ T cell and monocyte CD11b expression. Purified bovine monocytes and monocyte-depleted peripheral blood lymphocytes were cultured with APP for 24 hours. CD11b levels decreased on monocytes in response to APP. In contrast, a subset of γδ T cells responded to APP by up-regulating CD11b. Consistant with the changes in surface CD11b expression, APP treated γδ T cells showed increased adherence to plastic, whereas monocyte adhesion was reduced. The affects of APP on monocytes could be inhibited by anti-CD11b mAb, whereas the mAb had no effect on the APP response in γδ T cells, suggesting the two cell types respond to APP via different mechanisms. Polyphenols are typically thought of as anti-inflammatory agents, however, these data, as well as other ongoing studies, indicate they have a pro-inflammatory effect on γδ T cells. In vivo, plant polyphenols may enhance γδ T cell migration to sites of inflammation, where they can enact rapid immunoregulatory and innate-like responses. Funded by: NIH –NIAID DAIT-BAA-03 -41

Mechanisms of lymphocyte adhesion to cultured human corneal epithelial cells

PURPOSE. The authors studied the adhesion mechanisms between peripheral blood lymphocytes (PBL) and cultured human corneal epithelial (HCE) cells to investigate the lymphocyte interaction with corneal epithelial cells in the corneal immune response. METHODS. First, the authors examined the expression of inter-cellular adhesion molecule (ICAM)-1 or lymphocyte function-associated antigen (LFA)-3 on the normal human corneal epithelium and cultured HCE cells by an immunostaining technique and flow cytometry. Effects of inflammatory cytokines such as interferon (IFN)-? and tumor necrosis factor (TNF)-a, on ICAM-1 or LFA-3 expression on cultured HCE cells were also examined. Second, the authors performed an adhesion assay with 51 Cr-labeled monocyte-depleted PBL from normal, healthy volunteers and cultured HCE cells, with and without treatment of IFN-? or TNF-a in 96-well-plates for 1 hour at 37°C in 5% CO 2. After unbound PBL were removed, the radioactivity of the sample in each well was counted with a scintillation counter. In addition, the authors evaluated the blocking effects of monoclonal antibodies (mAbs) on the adhesion of PBL to the cultured HCE cells. RESULTS. ICAM-1 expression was not detected in the normal human corneal epithelium. However, the expression of ICAM-1 was detected on the cultured HCE cells with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. In addition, both IFN-? and TNF-a increased ICAM-1 expression on the cultured HCE cells dramatically. LFA-3 expression was detected in all cell layers of the normal human corneal epithelia. Neither IFN-? nor TNF-a had any effect on LFA-3 expression on the cultured HCE cells. The PBL adhesion to the HCE cells with and without treatment of IFN-? or TNF-a was blocked dominantly by anti-ICAM-1 or anti-LFA-1a mAb. Anti-LFA-3 mAb also blocked the PBL adhesion but had less blocking effect than anti-ICAM-1 or anti-LFA-1a mAb. Anti-very late activation antigen b, or anti-human leukocyte antigen (HLA)-class I or HLA-class II mAb had no effect on the PBL adhesion to the HCE cells. The adhesion percentile of the PBL applied to the HCE cells pretreated with IFN-? or TNF-a showed a dose-response curve dependent on the concentration of these cytokines. CONCLUSIONS. The results in the present study demonstrate that (i) adhesion of lymphocytes to HCE cells could be mediated by the LFA-1-ICAM-1 pathway and/or the CD2-LFA-3 pathway; (ii) the LFA-1-ICAM-1 pathway could be crucial in lymphocyte adhesion to HCE cells; (iii) IFN-? or TNF-a exerts an enhancing effect not only on the ICAM-1 expression on HCE cells but also on the adhesion of lymphocytes to HCE cells.

Diminished cell-cell binding by lymphocytes from healthy, elderly humans: evidence for altered activation of LFA-1 function with age.

We studied the kinetics of cell-cell adhesion by monocyte-depleted peripheral blood lymphocytes with a stirred-cuvette aggregometer using samples from healthy young (mean age = 25 years) and healthy elderly (mean age = 75 years) human donors. This was a very reproducible assay, as there was virtually no intraindividual variability and very little interindividual variability among donors of the same age group. In all cases, aggregation of cells began immediately upon addition of the protein kinase C activator phorbol myristate acetate (PMA) or the calcium ionophore ionomycin, and continued until reaching an asymptotic limit by 20 minutes or less. Unstimulated cells did not aggregate during this time. The rate of aggregate formation and total amount of aggregation (using young donors' cells) varied between PMA- and ionomycin-stimulated cells, suggesting different mechanisms for initiating cellular aggregation, or possibly, different peripheral blood lymphocyte subsets affected by these activating agents. However, for both stimuli, the rate of aggregation and the total amount of cellular aggregation were significantly lower with the elderly donors' cells. Further, aggregation was Ca2+/Mg2+ dependent, and the reaction required metabolically active cells, as the reaction was inhibited by the addition of sodium azide and 2-deoxy-D-glucose in both donor groups. Pretreating cells with the actin polymerization inhibitor cytochalasin B resulted in 35-40% inhibition of aggregation among young donors' cells, although, interestingly, there was no apparent effect upon the cells capable of forming aggregates in the old donor group. In both donor groups, aggregation by activated cells could be partially blocked by pretreating cells with monoclonal antibodies directed against the alpha and beta chains of the integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18). These results show that there is diminished cell-cell binding among lymphocytes from healthy, elderly humans, and this is partly due to altered activation of LFA-1 function with age.

In VitroInhibitory Effect of Docosahexaenoic and Eicosapentaenoic Acids on Human Endothelial Cell Production of Interleukin-6

The interaction between lymphocytes, cytokines, and endothelial cells (EC) is a key step in the inflammatory process. Interleukin-6 (IL-6), a pleiotropic cytokine in its effects, seems to be an early indicator of acute systemic inflammation. We examined the effects of polyunsaturated fatty acids (PUFA) on the production of IL-6 by human unstirnulated EC and by EC stimulated with TNF-α (100U/ml), IL-4 (100U/ml), LPS (1 μg/ml), or allogeneic peripheral blood lymphocytes (PBL). Immunoreactive IL-6 of twenty-four hour culture Supernatants was measured by sandwich ELISA. We demonstrated that the production of IL-6 was potentiated when EC were stimulated with TNF-α, IL-4, LPS, or monocyte-depleted PBL in comparison to unstimulated EC. The addition of n-3 PUFA to the culture medium (100 pglml DHA or EPA) significantly reduced the production of IL-6 by unstimulated EC and by EC stimulated with TNF-α, IL4, LPS or depleted PBL, whereas the n-6 PUFA (Arachidonic acid), even used at a similar concentration, was ineff...

Humoral modulation of lymphokine‐activated killer (LAK)‐cell induction in humans: IgG‐related and non‐IgG inhibitors in sera from cancer patients

Soluble inhibitors of lymphokine-activated killer (LAK) cell induction were characterized and purified from serous fluids from healthy donors or from patients with advanced cancer. Inhibitory activity in sera from cancer patients was partially absorbed with protein A agarose or anti-human IgG agarose. Following absorption, residual inhibition varied with individual sera, suggesting the presence of IgG-related and non-IgG inhibitory factors, and the proportions varied in the patient population. IgG, purified by affinity chromatography from ascitic fluids, from plasma of cancer patients, and from plasma of healthy donors, significantly inhibited IL-2 induction of LAK cells in a dose-dependent manner. Irrespective of the sources, inhibitory activity resided in the high-molecular-weight IgG fraction composed of IgG aggregates and immune complexes, but not monomeric IgG. Commercially prepared human IgG in aggregated form, but not in monomeric form, inhibited LAK cell induction in a dose-dependent manner. In contrast, neither bovine IgG nor human albumin affected LAK cell induction, even at higher concentrations. Aggregated human IgG inhibited LAK cell induction in unfractionated peripheral blood mononuclear cells (PBMC) but not in monocyte-depleted peripheral blood lymphocytes (PBL). Despite extensive (greater than 99%) depletion of IgG by protein-G affinity chromatography, the serous fluid from cancer patients displayed significant inhibitory activity. Fractionation of the IgG-depleted inhibitory materials by Sephacryl S-300, high-pressure ion-exchange column (HPIEC) or gel-permeation chromatography (HPGPC) demonstrated a 65-kDa inhibitor, distinct from IgG. Affigel-blue affinity chromatography of the 65-kDa fraction depleted albumin but did not remove the inhibitory activity, suggesting that the 65-kDa inhibitor is not serum albumin nor an albumin-bound component. These results suggest that serous fluids from patients with advanced-stage cancer contain 2 distinct regulators for LAK cell induction: (I) aggregated IgG and (2) a 65-kDa inhibitor, distinct from albumin.

Monocyte-dependent, serum-borne suppressor of induction of lymphokine-activated killer cells in lymphocytes from melanoma patients.

Both phytohemagglutinin-induced cytotoxicity and recombinant-interleukin-2 (rIL-2)-induced lymphokine-activated killer (LAK) activity against noncultured melanoma cells were significantly reduced when peripheral blood mononuclear cells (PBMC) from patients with metastatic melanoma were incubated in RPMI medium 1640 and 10% autologous human serum instead of 10% fetal calf serum, while serum from either healthy donors or patients with primary melanoma did not affect the level of cytotoxicity. The serum-mediated suppression was not restricted by major histocompatibility complex and was time-dependent. Addition of 10% human serum from the patients with metastatic melanoma [HS-Pt(m)] to the culture of PBMC with rIL-2 at the same time or 1 day after incubation significantly inhibited LAK activity. However, addition of 10% HS-Pt(m) 2 or 3 days after incubation did not inhibit LAK activity. Incubation of PBMC for 2 h with a high dose (10(4) U/ml) of rIL-2 in the presence of 10% HS-Pt(m), followed by incubation in the absence of either rIL-2 or HS-Pt(m), did not affect LAK cell activity. These results suggest that HS-Pt(m) inhibits the early stage of LAK cell differentiation, rather than the binding of rIL-2 to PBMC or a later stage in the differentiation. In contrast to PBMC, monocyte-depleted peripheral blood lymphocytes exhibited comparable levels of LAK activity when cultured with rIL-2 either in 10% fetal calf serum, 10% human serum from healthy donors or 10% HS-Pt(m). Addition of purified autologous monocytes to the culture of monocyte-depleted peripheral blood lymphocytes with rIL-2 suppressed LAK cell induction when 10% HS-Pt(m) was present. Thus serum-mediated suppression of LAK cell induction is largely dependent on the presence of monocytes, which may produce a secondary inhibitor that acts on lymphocytes. Addition of indomethacin to the culture did not reverse this monocyte-dependent serum-mediated suppression in a majority of cases, suggesting that prostaglandin E2 does not have a major role in the suppression.

HLA-linked nonresponsiveness to Cryptomeria japonica pollen antigen. I. Nonresponsiveness is mediated by antigen-specific suppressor T cell.

The objective of this study was to elucidate the cellular mechanism of IgE nonresponse to the Cryptomeria japonica (Japanese cedar) pollen antigen (CPAg), which was shown in our previous study to be HLA-linked (1). We established an assay system for the measurement of small amounts of anti-CPAg IgE antibody, both in an antigen-specific and isotype-specific manner, and a culture system to induce antigen-driven IgE antibody synthesis in vitro. By using these methods, we clarified that the function of the HLA-DR molecule in the CPAg-driven IgE response is similar to that of I-A or I-E molecule in mice, namely the product of immune response genes (Ir-genes), because anti-HLA-DR monoclonal antibody blocked the response, and the interaction between monocyte and monocyte-depleted peripheral blood lymphocytes (PBL) to respond to CPAg was restricted by HLA-DR. Furthermore, PBL from nonresponders revealed a specific IgE response to CPAg when the Leu-2+3- T cell fraction was depleted, thereby suggesting that even nonresponders have Leu-2-3+ T cell and B cell clones specific for CPAg, and they apparently show no response due to the presence of CPAg-specific Leu-2+3- suppressor T cells. This suppressor T cell fraction abolished the IgE response of the autologous B + monocyte + Leu-2-3+ T cell in a CPAg-specific manner. The current cellular analysis together with our previous genetic analysis strongly suggest that the HLA-linked IgE nonresponse to CPAg is mediated by CPAg-specific suppressor T cells. The HLA-linked gene controlling the nonresponsiveness to CPAg is thus designated as the immune suppression gene for CPAg (Is-CPAg). Mapping of Is-CPAg within HLA-DQ subregion is discussed.

Modulation of natural killer cell function by human alveolar macrophages

Lymphocyte natural killer (NK) activity appears to be important in resistance against viral infection and malignancy. Since pulmonary alveolar macrophages (PAM) derived from nonsmokers suppress NK activity in vitro, we asked if PAM derived from smokers modulated NK activity in a similar fashion. Bronchoalveolar lavage cells (BLC) were obtained from healthy nonsmokers and healthy smokers by bronchoalveolar lavage. Increasing numbers of BLC were cocultured with a fixed ratio of monocyte-depleted peripheral blood lymphocytes (PBL) and Cr 51-labeled K562 tumor target cells (PBL:K562, ratio 25:1; BLC:PBL ratios ranging from 0.02:1 to 1:1). Increasing numbers of BLC progressively suppressed the ability of either autologous or homologous PBL to lyse tumor targets. Smoker and nonsmoker BLC inhibited NK activity of homologous lymphocytes with similar potency. Suppression of NK activity by BLC was not reversed by the addition of indomethacin or catalase to cell cultures. Furthermore, suppression could not be reproduced by performing cytotoxicity assays in the presence of BLC culture supernatants. Lymphocytes boosted to high levels of NK activity by preincubation with α-interferon or interleukin-2 were still susceptible to suppression by BLC. Thus, PAM derived from smokers efficiently suppress lymphocyte NK activity, and the degree of suppression increases as the ratio of PAM to lymphocytes increases. Since smoking causes a bronchoalveolar accumulation of PAM, these inhibitory cells may result in profound local suppression of NK activity in smoker's lungs.

Modulation of the adherence of human lymphocytes to measles-infected cells by corticosteroids

Corticosteroids have been shown to have an inhibitory effect on many aspects of the human inflammatory and immunologic response. It is largely unknown by what mechanism corticosteroids alter the biologic function of lymphoid cells. One possible mechansim by which steroids could alter lymphocyte function is by modulation of surface receptors. The present study was designed to examine this possibility by the study of the effect of hydrocortisone (HC) and other steroids on the lymphocyte surface binding site for measles virus antigens. These receptors were detected by an assay involving the rosetting of lymphocytes to measles-infected Hela cells (Hela-K11). We showed that HC and several other steroids inhibit the adherence of monocyte-depleted peripheral blood lymphocytes to Hela-K11 cells (HC inhibition, 34 ± 12.1%, mean ± 1 SD). Inhibition could only be observed after the removal of glass-adherent cells. Modulation of lymphocyte functional surface determinants may be important in explaining some of the pharmacologic effects of corticosteroids on the immune system.