CD14lowCD16high: A cytokine-producing monocyte subset which expands during human immunodeficiency virus infection

Infection with the human immunodeficiency virus HIV-1 is associated with the expansion of a CD14^lowCD16^high monocyte subset in peripheral blood. This subset, which represents a minor subpopulation of monocytes in healthy individuals, increases during HIV infection and, in patients with AIDS, may represent up to 40% of the total circulating monocyte cell population. The CD14^lowCD16^high circulating monocytes co-express MAX.1, p150,95 and HLADR which are typical of tissue macrophage markers. These cells also express higher levels of intracellular interleukin (IL)-1α and tumor necrosis factor (TNF)-α than the CD14^highCD16^low monocyte population from the same patients. The CD14^lowCD16^high cells also express low levels of CD35, CD11a and CD4 in common with normal monocytes. When cultured in vitro, monocytes from HIV-seropositive individuals differentiated within a few hours into an elongated fibroblastoid shape characteristic of migratory cells. Our results suggest that the expansion of the CD14^lowCD16^high monocyte subset, which produce high amounts of TNF-α and IL-1α, may participate in the immune dysfunction observed during HIV infection.     

人外周血树突状细胞培养和地塞米松对其分化的影响作用

目的分离培养和鉴定人外周血树突状细胞（DC）,以及探讨地塞米松对其分化的影响作用。方法密度梯度离心法分离人外周血单个核细胞,贴壁后加入GM-CSF、IL-4和LPS培养,部分组另加入地塞米松,观察细胞形态学、流式标志和DC与T淋巴细胞共培养后的增殖变化。结果外周血单核细胞诱导培养后具有DC形态学特征,CD83表达上调,CD14表达下调,DC与T淋巴细胞共培养后呈增殖反应。培养液中加入地塞米松后CD83表达下调,CD14表达上调,DC与T淋巴细胞共培养后增殖反应减弱。结论外周血单核细胞经联合细胞因子可诱导为DC;地塞米松可使DC在功能上处于不成熟状态。     

The role of tumor necrosis factor (TNF)-α in the antitumor effect of intrapleural injection of Lactobacillus casei strain Shirota in mice

The involvement of several cytokines in the antitumor effect induced by intrapleural (i.pl.) injection of heat-killed cells of Lactobacillus casei strain Shirota (LC 9018) in mice was investigated. Injection of LC 9018 i.pl. into Meth A fibrosarcoma (Meth A)-bearing mice not only significantly prolonged the survival of the mice, but also effectively inhibited the accumulation of malignant pleural fluid in the thoracic cavity. In the thoracic cavity of tumor-bearing mice treated with LC 9018, we observed large amounts of several cytokines including interleukin (IL)-1β, interferon (IFN)-γ, IL-12 and tumor necrosis factor (TNF)-α. Both anti-IFN-γ and anti-IL-12 monoclonal antibody (mAb) treatments partially diminished the antitumor activity of LC 9018 in vivo, while the treatment of anti-IL-1β mAb did not influence the survival of the mice. However, anti-TNF-α mAb treatment completely abolished the antitumor effect of LC 9018 in vivo, suggesting that in this model LC 9018 has a survival-prolonging effect involving certain cytokines. Moreover, i.pl. injection of mouse recombinant TNF-α into Meth A-bearing mice pretreated with anti-TNF-α mAb partially restored the survival-enhancing effect of LC 9018. These results led us to conclude that TNF-α induced by i.pl. injection of LC 9018 plays an important role in the antitumor effect of LC 9018 in vivo. Received: 22 February 1999     

The involvement of CD14 in stimulation of cytokine production by uronic acid polymers

In this study the molecular mechanisms behind the stimulatory activities of the uronic acid polymers poly mannuronic acid (poly M), high M alginate and oxidized cellulose (C60XY) were investigated and compared with lipopolysaccharide (LPS). The cytokine-inducing abilities of the uronic acid polymers and LPS were examined on CD14-positive human monocytes and CD14-negative U373 astrocytoma cells. It was found that LPS induced monocytes and U373 cells to produce tumor necrosis factor (TNF) and interleukin(IL)-6, respectively, by different mechanisms. The poly uronic acids induced monocytes to produce TNF, but with 100-1000 times less potency compared to LPS. On U373 cells, LPS at concentrations ? 32 ng/ml resulted in a dose-related IL-6 production, whereas the poly uronic acids had negligible effects even at 1 mg/ml. The binding data demonstrate that only the CD14-positive monocytes in the peripheral blood mononuclear cells population bound poly M. Furthermore, poly M was found to bind to CD14 in the presence of serum. Antibodies against CD14 also inhibited the TNF-inducing activity of the three uronic acid polymers tested. In conclusion, these results demonstrate that uronic acid polymers induce TNF production through mechanisms which involve CD 14.     

系统性红斑狼疮患者骨髓间充质干细胞细胞因子分泌及其与病情活动的相关性研究

目的 探讨系统性红斑狼疮（SLE）患者骨髓间充质干细胞（MSCs）细胞因子分泌及其与疾病活动的相关性.方法 采用密度梯度离心和贴壁分离法对11例SLE患者和6例正常人骨髓MSCs进行分离培养,流式细胞术鉴定MSCs.取P2代细胞,半定量RT-PCR检测SLE患者骨髓MSCs白细胞介素-6（IL-6）,IL-7,IL-11,巨噬细胞集落刺激因子（M-CSF）和干细胞因子（SCF）的表达.结果 SLE患者MSCs均表达CD29、CD44和CD105,不表达CD14、CD34、CD45和HLA-DR.两组P2代MSCs均表达IL-6、IL-7、IL-11、M-CSF和SCF.SLE患者组MSCs IL-6、IL-7表达降低（P＜0.01）,IL-7的表达和SLE的活动评分（SLEDAI）呈负相关（r=-0.891,P＜0.05）.结论 SLE患者MSCs细胞因子分泌异常,可能与SLE血液系统损害及病情活动相关.     

Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture

The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme-linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in prmary culture. In PHA-driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)-2, IL-4, IL-5, IL-3, interferon (IFN)-γ, IL-10, IL-6, IL-12 and tumor necrosis factor (TNF)-α. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL-4, IL-5, IL-6 and IL-10, but little or no IFN-γ. IL-2, IL-3, TNF-α and IL-12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN-γ, IL-2 and TNF-α. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.     

Two distinct non-T helper type 2 interleukin-4 cell subsets in mice as revealed by single-cell cytokine analysis

We have previously defined four murine CD4⁺ peripheral T cell subsets, fractions (Fr.) I – IV, based on expression of the 6C10 and 3G11 determinants (Hayakawa, K. and Hardy, R. R., J. Exp. Med. 1988. 168: 1825). These subsets also show distinctive levels of other cell surface markers: the two minor subsets, Fr. III and Fr. IV, are both CD45RB^low/-, L-selectin (Mel-14)^? and CD44^hi, characteristic of secondary T cells. The patterns and levels of cytokine production by individual cells in each subset were determined by bioassay for interleukin (IL)-2/IL-4 or IL-4/interferon (IFN)-γ production after anti-CD3 stimulation. Our data revealed that these four phenotypically defined subsets largely coincide with clusters of cells showing uniform distinctive cytokine profiles, i.e. IL-2⁺/IFN-γ^?/IL-4^? (Fr. I and Fr. II, L-selectin⁺), IL-2⁺/IFN-γ⁺/IL-4⁺ (Fr. III, L-selectin^?), and IL-2^?/IFN-γ^low/-/IL-4⁺ (Fr. IV, L-selectin^?). Besides these subsets, an L-selectin-negative cell subfraction within Fr. II appears to represent a transitional population between the IL-2⁺/IFN-γ^?/IL-4^? stage and the IL-2⁺/IFN-γ⁺/IL-4⁺ stage. Taken together, these results demonstrate the presence of two IL-4⁺ secondary T cell subsets with distinct cytokine production patterns, and show that the majority of IL-4⁺ cells found in healthy adult laboratory mice co-produce IFN-γ, and thus are not typical T helper type 2 cells.     

Blockade of CD40-CD40 ligand pathway induces tolerance in murine contact hypersensitivity

Interactions between CD40 on antigen-presenting cells and its ligand (CD40L) on T cells has been implicated in T cell-mediated immune responses. Previously, we have shown that contact hypersensitivity (CHS), a cell-mediated cutaneous immune response in reaction to haptens, could be subclassified based on whether the hapten primed for Th1 or Th2 cytokines in cells isolated from draining lymph nodes. We also found that tolerance to a Th2-priming hapten could be induced only by simultane blockade of the CD40-CD40L and B7-CD28 at the time of sensitization. Here we demonstrate that blockade of CD40-CD40L signaling alone induces long-lasting unresponsiveness to the Th1 hapten 2,4-dinitrofluorobenzene (DNFB), and inhibits antigen-specific T cell proliferation in vitro. We find that CD40-CD40L signaling is required in the sensitization but not elicitation phase of DNFB-induced CHS, as treatment of mice with anti-CD40L monoclonal antibody (mAb) does not affect the response to hapten challenge in previously sensitized and untreated animals. Examination of cytokine production shows that anti-CD40L mAb decreases interferon-γ production by draining lymph node cells from DNFB-sensitized mice, and reciprocally increases interleukin (IL)-4 production. Consistent with this Th1 to Th2 immune deviation, anti-CD40L mAb prevents the induction of IL-12 mRNA in regional lymph nodes, an event which is normally seen within 12 h following hapten sensitization. In contrast, suppression of CHS by CTLA4Ig decreased the production of all cytokines by draining lymph node cells. Together, these data show that blockade of the CD40-CD40L pathway by itself is sufficient to induce tolerance to DNFB-induced CHS, and that this is associated with blockade of IL-12 induction and Th1 to Th2 immune deviation.     

Production of IL-12, IL-23 and IL-27p28 by bone marrow-derived conventional dendritic cells rather than macrophages after LPS/TLR4-dependent induction by Salmonella Enteritidis

Induction of the interleukin-12 (IL-12) cytokine family comprising IL-12, IL-23, IL-27, and IL-12p40 by intracellular pathogens is required for orchestration of cell-mediated immune responses. Macrophages (MΦ) have been shown to be a source of IL-12 following TLR4-dependent activation by Salmonella (S.). In this study another antigen-presenting cell type, the conventional dendritic cell (cDC), was analyzed and its cytokine responses compared with those of MΦ. We generated bone marrow-derived conventional dendritic cells (BMDC) and macrophages (BMMΦ) by incubating murine bone marrow cells with supernatants containing granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), respectively. Stimulation of BMDC and BMMΦ with S. enterica serovar Enteritidis (SE) or LPS resulted in the release of IL-12 and IL-23 by BMDC but not by BMMΦ. Furthermore, BMDC secreted approx. 20-fold more IL-12p40 and IL-27p28 than BMMΦ. However, BMDC and BMMΦ produced similar levels of IL-10. Using BMDC originating from wild-type (wt), TLR2^def and TLR4^def mice, we show that in BMDC the induction of IL-12, IL-23, and IL-27p28 by SE is dependent on TLR4, whereas low-level production of p40 is also mediated by pattern recognition receptors (PRR) other than TLR4. Interestingly, LPS- and SE-provoked responses of BMDC were remarkably similar indicating that LPS is the primary danger molecule of SE. Taken together, our results point to cDC rather than MΦ as the major producers of the IL-12 family members during in vitro infection with SE. The mechanisms of recognition of SE, however, appear to be the same for cDC and MΦ