Dissociation between Allogeneic T Cell Stimulation and Interleukin-l or Tumor Necrosis Factor Production by Human Lung Dendritic Cells Laurent Pierre Nicod, Beatrice Galve-de Rochemonteix, and Jean-Michel Dayer Pulmonary Division and Division of Immunology and Allergy, University Hospital, Geneva, Switzerland
A small portion of human lung mononuclear cells are very potent stimulators of allogeneic resting T cells. Although several-fold more effective than phagocytic alveolar macrophages (AM) and blood monocytes (Mo), they do not produce more ofthe lymphocyte co-stimulators interleukin-I-alpha (IL-la), interleukinI-beta (IL-li3), or tumor necrosis factor-alpha (TNF-a) than did Mo. Blocking antibodies against IL-la, IL-li3, TNF-a, and IL-6 did not reduce T cell proliferation. These potent antigen-presenting cells (APC) are loosely adherent and do not have phagocytic inclusions. Most of them have the marker RFD, of dendritic cells (DC) rarely present on Mo or AM and have a strong tendency to form clusters with T cells like murine DC. Thus, we demonstrate an example in the human system of a dissociation between T cell activation and IL-l or TNF-a production by DC or Mo, implying a major role for other "co-stimulating signals" by lung APC with dendritic features. The presence of different APC with various co-stimulating signals may be of importance for T cell subsets modulation.
The majority of previous studies on lung immunity have focused on antigen presentation by alveolar macrophages (AM). However, several investigators demonstrated that human AM from bronchoalveolar lavage are usually poor accessory cells in in vitro lymphocyte proliferation assays (1-4) despite the presence of a high density of HLA-DR and HLADQ on more than 80% of AM (1). A decreased ability to produce interleukin-l (IL-l) (5, 6), an enhanced PG& production (7), a decreased density of LFA1 (8), or different carbohydrate composition of HLA-DR molecules on AM compared to peripheral blood monocytes (Mo) (9) are some of the reasons invoked to explain why AM are poorer than Mo in stimulating resting T cells. The presence of a potent accessory cell population with features of dendritic cells (DC) isolated from minced and enzyme-digested human lung has been described (10). More recently, after an adherence step, a high purity of DC was obtained when DC and a poorly functional accessory cell population were separated, based on the absence or presence, respectively, of autofluorescent phagocytic inclusions (11). The heterogeneity of lung accessory cells from bronchoalveolar lavage has previously been noted by other invesKey Words: blood monocytes, autofluorescent macrophages, dendritic cells, antigen-presenting cells (Received in original form October 30, 1989 and in revised form December 29, 1989)
Addresscorrespondence to: L. P. Nicod, Pulmonary Division, H6pital cantonal universitaire, 1211 Geneva 4, Switzerland. Abbreviations: autofluorescent macrophages, AM; antigen-presenting cells, APC; dendritic cells, DC; interleukin-1, IL-1; loosely adherent mononuclear cells, LAM; mononuclear cell factor, MCF; mixed leukocyte reaction, MLR; blood monocytes, Mo; pulmonary mononuclear cells, PMC; tumor necrosis factor, TNF. Am. J. Respir, Cell Mol. BioI. Vol. 2. pp. 515-522, 1990
tigators using cells from bronchoalveolar lavage (12, 13). The lack of specific markers has not allowed a good assessment of the location of the different antigen-presenting cells (APC) in the various compartments of the lung. T cell activation requires the interaction of the T cell receptor and the MHC complex (14). In addition, a "second signal;' or a co-stimulant, is necessary. IL-l is thought to be such a co-stimulant (15). However, additional signals may be provided by co-mitogenic factors such as IL-6 (16, 17), tumor necrosis factor-alpha (TNF-a) (18), or cell-cell interactions through membrane proteins (19). The variety of second signals from the different accessory cells may be of importance for the modulation of T cell subsets. Indeed, in a mouse model, it has been shown that T helper cells of type 1 are less dependent than T helper cells oftype 2 on IL-I expression by the APC (20). To study the mechanism of T cell activation by the small subset of pulmonary APC with dendritic features, we compared them with peripheral blood monocytes, precursors of most lung parenchymal macrophages (21). The majority of the potent lung APC are shown to have the marker RFD, of DC (22), not present in most blood monocytes and phagocytic macrophages. More importantly, the APC function of these dendritic-like cells appears unrelated to an enhanced capacity to produce IL-la, IL-l~, or TNF-a. This dissociation between T cell stimulation and IL-l or TNF-a secretion is a promising model to compare T cell modulation by dendritic cells and by monocyte-derived lung mononuclear cells.
Materials and Methods Reagents and Media Hanks' balanced salt solution with Ca2+ and Mg2+ (HBSS);
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
fetal calf serum (FCS); and RPMI 1640 with 2.5 mM Hepes containing penicillin (l00 Vlml), streptomycin (100 Vim!), and glutamine (l %) were reagents all obtained from GIBCO (Paisley, Scotland). Preparation of Cells from Lung Normal pulmonary tissue was obtained from surgical specimens. Tissue distant from a limited primary lung carcinoma was collected by a pathologist. All surgical specimens were smokers, but no patient had clinical or histologic evidence of active pulmonary infection at the time of collection of the specimens. Pulmonary mononuclear cells were obtained from whole lung by mincing and enzyme digestion, as previously described (10). Briefly, lung specimens were rinsed with HBSS to remove residual blood. The minced tissue was digested with type I collagenase (150 Vlml) and type II elastase (10 Uzml), both from Sigma Chemical Co. (St. Louis, MO). The enzyme-digested fragments were taped through a stainless steel screen and separated on a Ficoll-Hypaque density gradient of 1.079 g/dl (Ficoll-Paque; Pharmacia, Piscataway, NJ) to obtain between 150 X 106 to 450 X 106 pulmonary mononuclear cells (PMC), from 20 to 30 g of lung tissue. Viability as assessed by trypan blue was> 90%. Differential counts were performed on May-Grunwald Giemsa-stained smears; 70 to 80% were monocytes or macrophage-like cells, 20 to 30 % were typical lymphocytes, and 2 to 5 % were unidentifiable cells. PMC were adhered in complete medium containing 10% heat-inactivated FCS. The nonadherent cells were removed after 1 h, using three vigorous rinses of HBSS. Nonadherent cells represented half of the pulmonary mononuclear cells, among which are mostly lymphocytes and other, unidentifiable cells. The adherent cells were incubated for an additional14 to 16 h at 3]0 C in complete medium (RPMI) with 10% FCS. The cells released after three rinses with HBSS are referred to as loosely adherent mononuclear cells (LAM). Cell Analysis and Sorting Unstained LAM were separated, according to the presence or absence of autofluorescent inclusions, with a Coulter EP1CS· V. Data were processed on a Coulter MDADS computer system. Cell sizes were analyzed on a three log scale. Light source for fluorescence analysis was coherent INNOVA 90 using 488 wire length and 500 mW measured at 525 nm. Gates were set to remove cell debris and to select mature autofluorescent macrophages (AM) and nonphagocytic DC. The latter represents 3 to 7 % of the LAM and up to 70% of nonautofluorescent cells (11). Autofluorescence was analyzed using an excitation wavelength of 488 nm and emissions measured using a 585-nm filter. Preparation of Peripheral Blood T Lymphocytes and Monocytes Blood mononuclear cells were obtained from the buffy coat of normal volunteers from the blood transfusion center. After a Ficoll-Hypaque gradient, the interphase mononuclear cells were washed 3 times in HBSS and purified as previously described (10). In brief, after adhesion on plastic culture dishes, nonadherent cells were removed by washing.
The lymphocytes were passed through a nylon wool column and further depleted of accessory cells with 5.0nM L-leucine methylester (10). Monocytes were obtained by loose adherence similar to the process used to obtain loosely adherent pulmonary mononuclear cells (purity > 90 %). Mixed Leukocyte Reaction (MLR) LAM, DC, and AM were irradiated (3,500 rad) using a cesium source. Various numbers of accessory cells were incubated with purified allogeneic T cells (l05) in 200 pJ of medium containing 20% human serum in flat-bottomed microtiter plates (96 well tissue culture wells; Costar, Cambridge, MA) in triplicate for 6 d at 37° C in 95 % air/5 % CO2 , Cells were pulsed with 0.5-I-'Ci pH]thymidine (5.0 Ci/nmol; Amersham, Amersham, UK) 18 h prior to harvest. The cells were harvested onto filter paper, and their radioactivity analyzed in a liquid scintillation counter. Data were expressed in counts per minute ± 1 SEM. Dosage of IL-la and IL-l{1 by Enzymoimmunoassay IL-1 alpha and beta produced by various subsets of cells were measured by an enzymoimmunoassay (23). In brief, a mouse monoclonal antibody against IL-1a or IL-1{3 (2 I-'g/200 1-'1) was immobilized in 96-well microtiter plates (Nume; GIBCO). The plates were washed with 0.1 M phosphate buffer, pH 7.4, containing 0.1% Tween. Aliquots of culture supernatant were tested in the presence of a second monoclonal antibody against IL-1 coupled to acetylcholinesterase (1001-'1 each) (23). After an overnight incubation at 4° C, Ellmans reagent was used as a chromogen and absorbance at 405 nm and was read with automatic equipment (Embrach; Dynatech Produkte A.G., Zurich, Switzerland). Cell-associated IL-l was analyzed once the cells had been washed, frozen, and thawed 3 times in liquid nitrogen, and sonicated. Bioassay of IL-l/MCF IL-1 biologic activity was measured by the stimulation of PGE 2 production in human fibroblast, referred to as mononuclear cell factor (MCF) activity (24). Foreskin fibroblasts were exposed to the supernatant of lung cells or celllysates. After 72 h, cell-free supernatants from fibroblasts were stored at -20° C. PGE 2 was measured by radioimmunoassay using an antiserum kindly provided by Dr. L. Levine (Brandeis University, Waltham, MA), as previously described (24). Blocking Antibodies and Other Antibodies Rabbit antisera against IL-Ia and IL-1{3 (gift from A. Shaw, Glaxo 1MB, Geneva, CH) completely blocked the activity of 4 ng/ml or rIL-la or rIL-1{3, respectively, (kindly provided by Biogen S.A., Geneva, CH) as tested in the IL-I/MCF bioassay at 1:100 dilution. Anti-TNF-a, a goat antibody (gift from R. Ulevitch, La Jolla, CA) at 1:100 dilution, completely blocked the bioactivity of up to 4 ng/ml of rTNF-a but not of a rhTNF-{3 (gift from Biogen S.A., Geneva, Switzerland) (25). The IL-6 blocking antibody was obtained from the National Institute for Biological Standards and Control (Hertfordshire, UK). Lipopolysaccharide B was from Escherichia coli 0111:B4 (Difco, Detroit, MI).
Nicod, Galve-de Rochemonteix, and Dayer: Human Lung Dendritic Cells are Poor Producers of IL-l or TNF
517
TABLE 1
Phenotype of the lung dendritic cells* RFD1 (dendritic cells) up to 70% HLA-DR (OKIA1) >90% Cytokeratine
A small portion of human lung mononuclear cells are very potent stimulators of allogeneic resting T cells. Although several-fold more effective than phagocytic alveolar macrophages (AM) and blood monocytes (Mo), they do not produce more ofthe lymphocyte co-stimulators interleukin-I-alpha (IL-la), interleukinI-beta (IL-li3), or tumor necrosis factor-alpha (TNF-a) than did Mo. Blocking antibodies against IL-la, IL-li3, TNF-a, and IL-6 did not reduce T cell proliferation. These potent antigen-presenting cells (APC) are loosely adherent and do not have phagocytic inclusions. Most of them have the marker RFD, of dendritic cells (DC) rarely present on Mo or AM and have a strong tendency to form clusters with T cells like murine DC. Thus, we demonstrate an example in the human system of a dissociation between T cell activation and IL-l or TNF-a production by DC or Mo, implying a major role for other "co-stimulating signals" by lung APC with dendritic features. The presence of different APC with various co-stimulating signals may be of importance for T cell subsets modulation.
The majority of previous studies on lung immunity have focused on antigen presentation by alveolar macrophages (AM). However, several investigators demonstrated that human AM from bronchoalveolar lavage are usually poor accessory cells in in vitro lymphocyte proliferation assays (1-4) despite the presence of a high density of HLA-DR and HLADQ on more than 80% of AM (1). A decreased ability to produce interleukin-l (IL-l) (5, 6), an enhanced PG& production (7), a decreased density of LFA1 (8), or different carbohydrate composition of HLA-DR molecules on AM compared to peripheral blood monocytes (Mo) (9) are some of the reasons invoked to explain why AM are poorer than Mo in stimulating resting T cells. The presence of a potent accessory cell population with features of dendritic cells (DC) isolated from minced and enzyme-digested human lung has been described (10). More recently, after an adherence step, a high purity of DC was obtained when DC and a poorly functional accessory cell population were separated, based on the absence or presence, respectively, of autofluorescent phagocytic inclusions (11). The heterogeneity of lung accessory cells from bronchoalveolar lavage has previously been noted by other invesKey Words: blood monocytes, autofluorescent macrophages, dendritic cells, antigen-presenting cells (Received in original form October 30, 1989 and in revised form December 29, 1989)
Addresscorrespondence to: L. P. Nicod, Pulmonary Division, H6pital cantonal universitaire, 1211 Geneva 4, Switzerland. Abbreviations: autofluorescent macrophages, AM; antigen-presenting cells, APC; dendritic cells, DC; interleukin-1, IL-1; loosely adherent mononuclear cells, LAM; mononuclear cell factor, MCF; mixed leukocyte reaction, MLR; blood monocytes, Mo; pulmonary mononuclear cells, PMC; tumor necrosis factor, TNF. Am. J. Respir, Cell Mol. BioI. Vol. 2. pp. 515-522, 1990
tigators using cells from bronchoalveolar lavage (12, 13). The lack of specific markers has not allowed a good assessment of the location of the different antigen-presenting cells (APC) in the various compartments of the lung. T cell activation requires the interaction of the T cell receptor and the MHC complex (14). In addition, a "second signal;' or a co-stimulant, is necessary. IL-l is thought to be such a co-stimulant (15). However, additional signals may be provided by co-mitogenic factors such as IL-6 (16, 17), tumor necrosis factor-alpha (TNF-a) (18), or cell-cell interactions through membrane proteins (19). The variety of second signals from the different accessory cells may be of importance for the modulation of T cell subsets. Indeed, in a mouse model, it has been shown that T helper cells of type 1 are less dependent than T helper cells oftype 2 on IL-I expression by the APC (20). To study the mechanism of T cell activation by the small subset of pulmonary APC with dendritic features, we compared them with peripheral blood monocytes, precursors of most lung parenchymal macrophages (21). The majority of the potent lung APC are shown to have the marker RFD, of DC (22), not present in most blood monocytes and phagocytic macrophages. More importantly, the APC function of these dendritic-like cells appears unrelated to an enhanced capacity to produce IL-la, IL-l~, or TNF-a. This dissociation between T cell stimulation and IL-l or TNF-a secretion is a promising model to compare T cell modulation by dendritic cells and by monocyte-derived lung mononuclear cells.
Materials and Methods Reagents and Media Hanks' balanced salt solution with Ca2+ and Mg2+ (HBSS);
516
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990
fetal calf serum (FCS); and RPMI 1640 with 2.5 mM Hepes containing penicillin (l00 Vlml), streptomycin (100 Vim!), and glutamine (l %) were reagents all obtained from GIBCO (Paisley, Scotland). Preparation of Cells from Lung Normal pulmonary tissue was obtained from surgical specimens. Tissue distant from a limited primary lung carcinoma was collected by a pathologist. All surgical specimens were smokers, but no patient had clinical or histologic evidence of active pulmonary infection at the time of collection of the specimens. Pulmonary mononuclear cells were obtained from whole lung by mincing and enzyme digestion, as previously described (10). Briefly, lung specimens were rinsed with HBSS to remove residual blood. The minced tissue was digested with type I collagenase (150 Vlml) and type II elastase (10 Uzml), both from Sigma Chemical Co. (St. Louis, MO). The enzyme-digested fragments were taped through a stainless steel screen and separated on a Ficoll-Hypaque density gradient of 1.079 g/dl (Ficoll-Paque; Pharmacia, Piscataway, NJ) to obtain between 150 X 106 to 450 X 106 pulmonary mononuclear cells (PMC), from 20 to 30 g of lung tissue. Viability as assessed by trypan blue was> 90%. Differential counts were performed on May-Grunwald Giemsa-stained smears; 70 to 80% were monocytes or macrophage-like cells, 20 to 30 % were typical lymphocytes, and 2 to 5 % were unidentifiable cells. PMC were adhered in complete medium containing 10% heat-inactivated FCS. The nonadherent cells were removed after 1 h, using three vigorous rinses of HBSS. Nonadherent cells represented half of the pulmonary mononuclear cells, among which are mostly lymphocytes and other, unidentifiable cells. The adherent cells were incubated for an additional14 to 16 h at 3]0 C in complete medium (RPMI) with 10% FCS. The cells released after three rinses with HBSS are referred to as loosely adherent mononuclear cells (LAM). Cell Analysis and Sorting Unstained LAM were separated, according to the presence or absence of autofluorescent inclusions, with a Coulter EP1CS· V. Data were processed on a Coulter MDADS computer system. Cell sizes were analyzed on a three log scale. Light source for fluorescence analysis was coherent INNOVA 90 using 488 wire length and 500 mW measured at 525 nm. Gates were set to remove cell debris and to select mature autofluorescent macrophages (AM) and nonphagocytic DC. The latter represents 3 to 7 % of the LAM and up to 70% of nonautofluorescent cells (11). Autofluorescence was analyzed using an excitation wavelength of 488 nm and emissions measured using a 585-nm filter. Preparation of Peripheral Blood T Lymphocytes and Monocytes Blood mononuclear cells were obtained from the buffy coat of normal volunteers from the blood transfusion center. After a Ficoll-Hypaque gradient, the interphase mononuclear cells were washed 3 times in HBSS and purified as previously described (10). In brief, after adhesion on plastic culture dishes, nonadherent cells were removed by washing.
The lymphocytes were passed through a nylon wool column and further depleted of accessory cells with 5.0nM L-leucine methylester (10). Monocytes were obtained by loose adherence similar to the process used to obtain loosely adherent pulmonary mononuclear cells (purity > 90 %). Mixed Leukocyte Reaction (MLR) LAM, DC, and AM were irradiated (3,500 rad) using a cesium source. Various numbers of accessory cells were incubated with purified allogeneic T cells (l05) in 200 pJ of medium containing 20% human serum in flat-bottomed microtiter plates (96 well tissue culture wells; Costar, Cambridge, MA) in triplicate for 6 d at 37° C in 95 % air/5 % CO2 , Cells were pulsed with 0.5-I-'Ci pH]thymidine (5.0 Ci/nmol; Amersham, Amersham, UK) 18 h prior to harvest. The cells were harvested onto filter paper, and their radioactivity analyzed in a liquid scintillation counter. Data were expressed in counts per minute ± 1 SEM. Dosage of IL-la and IL-l{1 by Enzymoimmunoassay IL-1 alpha and beta produced by various subsets of cells were measured by an enzymoimmunoassay (23). In brief, a mouse monoclonal antibody against IL-1a or IL-1{3 (2 I-'g/200 1-'1) was immobilized in 96-well microtiter plates (Nume; GIBCO). The plates were washed with 0.1 M phosphate buffer, pH 7.4, containing 0.1% Tween. Aliquots of culture supernatant were tested in the presence of a second monoclonal antibody against IL-1 coupled to acetylcholinesterase (1001-'1 each) (23). After an overnight incubation at 4° C, Ellmans reagent was used as a chromogen and absorbance at 405 nm and was read with automatic equipment (Embrach; Dynatech Produkte A.G., Zurich, Switzerland). Cell-associated IL-l was analyzed once the cells had been washed, frozen, and thawed 3 times in liquid nitrogen, and sonicated. Bioassay of IL-l/MCF IL-1 biologic activity was measured by the stimulation of PGE 2 production in human fibroblast, referred to as mononuclear cell factor (MCF) activity (24). Foreskin fibroblasts were exposed to the supernatant of lung cells or celllysates. After 72 h, cell-free supernatants from fibroblasts were stored at -20° C. PGE 2 was measured by radioimmunoassay using an antiserum kindly provided by Dr. L. Levine (Brandeis University, Waltham, MA), as previously described (24). Blocking Antibodies and Other Antibodies Rabbit antisera against IL-Ia and IL-1{3 (gift from A. Shaw, Glaxo 1MB, Geneva, CH) completely blocked the activity of 4 ng/ml or rIL-la or rIL-1{3, respectively, (kindly provided by Biogen S.A., Geneva, CH) as tested in the IL-I/MCF bioassay at 1:100 dilution. Anti-TNF-a, a goat antibody (gift from R. Ulevitch, La Jolla, CA) at 1:100 dilution, completely blocked the bioactivity of up to 4 ng/ml of rTNF-a but not of a rhTNF-{3 (gift from Biogen S.A., Geneva, Switzerland) (25). The IL-6 blocking antibody was obtained from the National Institute for Biological Standards and Control (Hertfordshire, UK). Lipopolysaccharide B was from Escherichia coli 0111:B4 (Difco, Detroit, MI).
Nicod, Galve-de Rochemonteix, and Dayer: Human Lung Dendritic Cells are Poor Producers of IL-l or TNF
517
TABLE 1
Phenotype of the lung dendritic cells* RFD1 (dendritic cells) up to 70% HLA-DR (OKIA1) >90% Cytokeratine
