Clin. exp. Immunol (1992) 87, 485-492
Induction of lymphocyte proliferation by antigen-pulsed human neutrophils C. PRIOR, P. J. TOWNSEND, D. A. HUGHES & P. L. HASLAM Cell Biology Group, Department of Cardiothoracic Surgery, National Heart & Lung Institute, London, England
(Acceptedfor publication 10 September 1991)
SUMMARY We have investigated whether purified antigen-pulsed human neutrophils can induce a proliferative response in purified resting blood lymphocytes. Neutrophils were pulsed with soluble tetanus toxoid (dose range 25-250 Lf/ml) and co-cultured with autologous lymphocytes that had been depleted of MHC class II expressing cells. The antigen-pulsed neutrophils induced an increase of lymphocyte proliferation which was dependent on the antigen dose and the neutrophil/lymphocyte ratios. Neutrophils were less potent than autologous monocytes in stimulating lymphocyte proliferation. Blocking with a monoclonal antibody to a common determinant of the human MHC class II complex failed to reduce the lymphoproliferative effects and allogenic antigen-pulsed neutrophils were also able to elicit lymphocyte proliferation similar to autologous neutrophils. We conclude that antigenpulsed neutrophils are able to induce lymphocyte proliferation in a non-MHC-restricted fashion. Keywords neutrophils alloantigens lymphocyte proliferation MHC restriction antigen presentation
INTRODUCTION T helper lymphocytes require the assistance of appropriate 'antigen-presenting' cells in order to recognize and respond to protein antigens. The exact requirements are variable according to both the nature of the antigen and of the antigen-presenting cell type, and there are many facets of presentation which are still not fully understood. In the human host, cell types known to function as professional antigen-presenting cells include monocytes and macrophages [1], dendritic cells [2] and Langerhans cells [3]. In many cases an 'antigen-processing' step, involving ingestion and breakdown of protein antigen to immunogenic peptides, is required before 'antigen-presentation' can occur. After processing, antigenic determinants may then be expressed at the surface of antigen-presenting cells in association with products of the MHC class II molecules, in a form which allows their recognition and binding to T cells expressing the antigenspecific receptor. However, some cell types, for example B lymphocytes, can 'present' certain antigens without the need for processing' [4]; and the requirement for MHC class II association is another facet of antigen presentation which is still not
fully understood. Neutrophils share several features in common with macrophages. These include a common cell lineage, their phagocytic nature, similar surface receptors for opsonized antigens and the Correspondence: Dr Patricia L. Haslam, PhD MRCPath, Head of Cell Biology Group, Department of Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK.
485
ability to produce the mediator IL-l [5,6], which has the ability to prime lymphocytes for proliferation. However, the question whether neutrophils can function in processing and/or presenting antigens has received little attention. We are aware of only one recent study which has addressed this subject directly [7], showing that neutrophils can act as accessory cells in mitogeninduced stimulation of lymphocytes, although they did not appear to express HLA-D region antigens. Since there has been a report that antigen-induced T lymphocyte proliferation can be induced in certain MHC-unrestricted proliferation systems [8], we have sought to re-assess the role of neutrophils in presentation of antigens. A common problem in conducting antigen-presenting studies is the difficulty in obtaining absolutely pure cell populations, and the functional implications of cell contamination are difficult to assess. In this study, a modified and extended lymphocyte purification procedure has been employed and morphologic and phenotypic checks using flow cytometric analysis have been carried out on all purified cell populations in each experiment to avoid any bias resulting from occasional inadequacy of cell separation. Also, in contrast to former work [7], we have pulsed neutrophils with antigen so that only antigen taken up by, or bound to neutrophils was introduced into the culture system. Our results suggest that antigen-pulsed human neutrophils can induce a dose-dependent lymphoproliferative response. We propose that interactions between neutrophils and mononuclear cells can occur during inflammatory and immune processes which may result in further enhancement of the cellular immune response.
C. Prior et al.
486 MATERIALS AND METHODS
Study population The study population consisted of healthy adult volunteers, all of whom had been inoculated with tetanus toxoid 3-9 years (median 6 years) prior to the study. Blood lymphocytes, neutrophils and monocytes were purified from each volunteer, as described below, to employ defined numbers of each cell type in assays to investigate antigen-presenting function. Purification of Lymphocytes A 60-ml venous blood sample was taken from each volunteer, defibrinated in a siliconized glass vessel, then diluted in an equal volume of minimal essential medium containing 25 mm HEPES buffer (MEM; GIBCO, Uxbridge, UK). It was layered over lymphocyte separation medium (Flow Laboratories, Rickmansworth, UK), centrifuged at 600 g for 30 min at 20C, the interface layer of peripheral blood mononuclear cells (PBMC) recovered, washed twice in MEM, then adjusted to a concentration of 5 x 106 cells/ml in RPMI 1640 (GIBCO; containing 25 mm HEPES buffer, 30 mg/100 ml L-glutamine; 100 U/ml penicillin, and 100 ,ug/ml streptomycin) supplemented with 10% heat-inactivated (56'C, 30 min) autologous serum. The PBMC suspension was then depleted of monocytes using an adherence procedure. This was conducted in polystyrene tissue culture flasks (Flow Laboratories; 25 ml capacity) which were precoated by adding 5 ml of MEM containing 10% heat-inactivated fetal calf serum (FCS) (Flow Laboratories) to each flask, incubating at 370C for 1 h, then gently rinsing the coated surface with MEM. The PBM suspension was then added (4-5 ml containing 20-25 x 106 cells per flask), and the flasks were incubated for I h at 37 C to allow monocytes to adhere. Nonadherent cells (mainly lymphocytes) were washed from the coated surface using pre-warmed MEM (37 C) then panned using MoAb to remove any residual cells expressing products of the human class II MHC (HLA-D region antigens) as follows. In preparation for panning, the 'lymphocyte' population was transferred to a 15-ml graduated polypropylene tube (Falcon 2097, Becton Dickinson, Lincoln Park, NJ), sedimented by centrifugation at 300 g for 5 min at 4 C, then resuspended in PBS containing 10 yg/ml of CA2 MoAb (mouse IgG2a directed against a common determinant of the human class II MHC; [911] for 20 min at 4°C. The CA2 MoAb was kindly provided by Dr Julia Bodmer, Imperial Cancer Research Fund Laboratories, London, UK. After incubation, the cells were washed, resuspended at 1 x 107 cells/ml in RPMI 1640 containing 10% autologous serum then panned onto the surface of sterile 90-mm polystyrene Petri dishes (Sterilin, Teddington, UK) which had been pre-coated with affinity purified F(ab')2 rabbit anti-mouse IgG (Serotec, Kidlington, UK). For pre-coating, the dishes were incubated overnight at room temperature with 10 ml of a 10 pug/ ml solution of the rabbit anti-mouse antibody in Tris buffer, pH 9. Prior to panning, the antibody solution was discarded, the dishes were soaked in a I % solution of FCS in PBS (pH 7 2) at room temperature for 30 min, then the surface of the Petri dish was washed with MEM. For panning, 3 ml of the CA2-treated lymphocyte suspension were added to each dish and the dishes incubated at 4°C for 2 h to allow CA2-positive cells to adhere to the coated surface. Non-adherent lymphocytes were then collected, washed and re-suspended, and the panning step repeated. The final lymphocyte population was harvested by
gently washing with MEM and re-suspended at 1-25 x 106 cells/ ml in RPMI 1640 ready for use as the purified, HLA-D-regiondepleted responder lymphocytes in the assays of antigenpresenting function.
Purification of monocytes The adherent monocytes retained in the FCS-coated polystyrene flasks during lymphocyte purifications were detached from the surface of the flasks by gently scraping with a rubber policeman, recovered by vigorous washing with cold MEM, sedimented by centrifugation at 300 g for 10 min at 4°C, then resuspended at 1 x 106 cells/ml in RPMI 1640 ready for use as control known antigen-presenting cells in the functional assays. The monocyte populations were not exhaustively purified, as for the lymphocytes and neutrophils, due to limitations in obtaining sufficient numbers of all three cell types from a single individual. Purification of neutrophils At this point, further 20 ml of venous blood were taken from the same individual, defibrinated, and used to obtain purified neutrophils employing a modification of the method reported by Dooley, Simpson & Meryman [12]. This ensured that the shorter-lived neutrophil population was as fresh as possible when used in the assay. The 20 ml of defibrinated blood were mixed with 10 ml of 6% dextran (dextraven 1 10 in 0-9% sodium chloride) in a 50-ml graduated polypropylene tube (Falcon 2070, Becton Dickinson), the erythrocytes were allowed to sediment at room temperature for 20-30 min, then the leucocyte-rich supernatant was recovered and washed in MEM. The cell pellet was re-suspended in a solution of 55% Percoll (Pharmacia, Uppsala, Sweden). The solution of 55% Percoll, and also solutions of 70% and 81% Percoll, were prepared by mixing nine parts of concentrated stock Percoll with one part of 9% chloride to achieve isotonicity ('100% Percoll'), then further diluting the 100% Percoll by adding PBS. The densitities of the 81%, 70%/0 and 55% Percoll solutions were 1 1000, 1-0875 and 1-0697 g/ml, respectively. A gradient was then set up by first adding 5 ml of 81 % Percoll to a 1 5-ml polypropylene tube, then gently adding a layer of 3 ml 70% Percoll and finally a layer of 3 ml of the buffy coat suspension in 55% Percoll. The tube was spun at 600 g for 20 min at 20°C, and the neutrophil band was harvested from the interface of the 70% and 81 % layers. After washing in MEM, the purified neutrophils were re-suspended at a concentration of 1 x 106/ml in RPMI 1640 ready for use in the assays.
Methods to check the purity of the cell populations The purity of the populations of lymphocytes, neutrophils and monocytes was checked at every experiment. Morphology was assessed using cytocentrifuge slide preparations of the cells prepared by spinning 100-,l aliquots ofeach cell suspension (106 cells/ml RPMI 1640) onto glass slides for 10 min at 18 g in a cytocentrifuge (Cytospin, model 2; Shandon Southern Products, Runcorn, UK). After air drying, the cells were fixed in methanol and stained with May-Gruinwald-Giemsa stain. Two-hundred cells were counted and the percentage ofcontaminating cell types was recorded. In addition, flow cytometric checks were conducted to evaluate cell surface marker expression using the methods we have described in detail previously [13]. Briefly, aliquots of cells from each suspension were labelled with MoAbs directed
Induction of lymphocyte proliferation by neutrophils Density separation
Adherence
Double panning
CD14
A JM.JIP. A\
A
HLA DR
0)
4-
HLA DP
0
E z
HLA DQ
487
majority of the cells remaining, as used in the six main experiments were T lymphocytes (CD3+), mean 70+ 12% s.d., and NK cells (11 + 7% CD16+, and 9 +12% CD57+). Contamination was minimal (CD 14+, 2-4 + 2-0%; CD22+, 074 + 0 57%; HLA-DR+, 3-3+2.4%; HLA-DP+, 0 3+004%; and HLADQ+, 0-06 + 0-13%). By morphology, 98 + 2% had the appearance of lymphocytes while only 1 3+ 1-0% were monocytes, only 0-63+0 75o neutrophils, and only 025+0 50 eosinophils. Neutrophil populations By morphology, 97 +10/% of the cells used in the six main experiments were segmented neutrophils, 2-3 + 1 3% were eosinophils, and only 0 33 + 0 4% had the appearance of lymphocytes. Flow cytometric analysis confirmed these findings and showed that only 3 2+2 7% of cells expressed the monocyte marker CD 14.
Monocyte populations By morphology, the 'monocyte' populations used as positive controls in the functional studies contained 78+ 1400 monocytes and 20 + 15% lymphocytes, but only 1-8 + 2% neutrophils and 04 + 044% eosinophils. Flow cytometric analysis showed that 75 +220% expressed the monocyte marker CD14 and that the majority expressed HLA-DR antigens (69+26%). A few also expressed HLA-DP (14+9%) and/or HLA-DQ (3-4+2.9%).
CD22A
Log FITC fluorescence
Fig. 1. Histograms of FITC fluorescence emission from monoclonal antibody-stained cells showing progressive depletion of contaminating monocytes (CD14+ cells), HLA-D+ cells (HLA-DR, -DP, -DQ) and B lymphocytes (CD22+ cells) at different steps of the lymphocyte purification procedure. The analysis was undertaken using a FACS 440.
against a range of cell surface determinants (described below), stained with FITC-labelled anti-mouse immunoglobulin, then fixed and permeabilised using 5000 methanol. The nuclei of the cells were then stained with propidium iodide to acquire signals selectively from nucleated cells during analysis using a Becton Dickinson FACS 440. Percentages of cells positively stained with MoAbs were determined by comparison with controls set up without MoAbs. All the MoAbs were obtained from Becton Dickinson (Mountain View, CA), and T lymphocytes were detected using Leu 4 (CD3 MoAb), B lymphocytes using Leu 14 (CD22), monocytes using M3 (CD14) and neutrophils using Leu 1 b (CD 16) which also reacts with natural killer (NK) cells. NK cells were also evaluated using Leu 7 (CD57). To confirm that cells expressing class II MHC products had been depleted from the purified lymphocyte population, flow cytometric checks were also conducted using MoAbs to HLA-DR, -DP and -DQ (Becton Dickinson). These antibodies were also used to confirm that MHC class II region products were expressed on the monocyte populations and to study whether these products can be detected on human neutrophils.
Lymphocyte populations The modified purification method we employed led to the progressive depletion of contaminating monocytes (CD14+), cells expressing MHC class II region products (HLA-DR+, DP+ and DQ+), and B lymphocytes (CD22+) (Fig. 1). The
Method to prepare 'antigen-pulsed' neutrophils and monocytes The purified neutrophils and monocytes, suspended at 1 x 106 cells/ml in RPMI 1640 containing 10% autologous serum, were incubated with a range of doses of tetanus toxoid (TT) for 3 h at 37 iC. The TT (soluble TT, batch No. MWC5208/A/F), had a limited flocculation (Lf) value of 2567 Lf/ml and a purity of 1200 Lf/mg, and was kindly provided by Dr M. Hughes, Wellcome Biotechnology, Beckenham, UK. A dose range of 25-250 Lf/ml was employed. After incubation with TT, the monocytes or neutrophils were washed three times in MEM, then re-suspended at 106/ml in RPMI 1640 for use in the functional assays. Method to test the antigen-presenting Junction of the antigenpulsed cell populations To determine whether the antigen-pulsed neutrophils or monocytes could stimulate the proliferation of the purified autologous lymphocytes, co-cultures of the cells were set up in the following way: 800 yl of the purified lymphocyte suspension (containing 1 x 106 cells) in RPMI 1640 were added to 5-ml polypropylene tissue culture tubes (Falcon 2063, Becton Dickinson) and 100 I1 of antigen-pulsed neutrophil or monocyte suspension (containing I x 105 cells) were added to each tube. Each tube was then supplemented with 100 ,l (i.e. IO%) of heatinactivated autologous serum. The cultures were set up in triplicate. Controls were set up containing 106 lymphocytes alone to determine background levels. Tubes were also set up containing lymphocytes plus accessory cells which had been sham-pre-incubated in medium without antigen to check whether the accessory cells could stimulate an autologous mixed leucocyte reaction. The cell mixtures were co-cultured for 5 days, and 37 KBq of tritiated thymidine (methyl-3H thymidine < 37 MBq/ml; Amersham International, Amersham, UK) were added to each tube 18 h prior to harvesting. At the end of
C. Prior et al.
488 15,
P
Induction of lymphocyte proliferation by antigen-pulsed human neutrophils C. PRIOR, P. J. TOWNSEND, D. A. HUGHES & P. L. HASLAM Cell Biology Group, Department of Cardiothoracic Surgery, National Heart & Lung Institute, London, England
(Acceptedfor publication 10 September 1991)
SUMMARY We have investigated whether purified antigen-pulsed human neutrophils can induce a proliferative response in purified resting blood lymphocytes. Neutrophils were pulsed with soluble tetanus toxoid (dose range 25-250 Lf/ml) and co-cultured with autologous lymphocytes that had been depleted of MHC class II expressing cells. The antigen-pulsed neutrophils induced an increase of lymphocyte proliferation which was dependent on the antigen dose and the neutrophil/lymphocyte ratios. Neutrophils were less potent than autologous monocytes in stimulating lymphocyte proliferation. Blocking with a monoclonal antibody to a common determinant of the human MHC class II complex failed to reduce the lymphoproliferative effects and allogenic antigen-pulsed neutrophils were also able to elicit lymphocyte proliferation similar to autologous neutrophils. We conclude that antigenpulsed neutrophils are able to induce lymphocyte proliferation in a non-MHC-restricted fashion. Keywords neutrophils alloantigens lymphocyte proliferation MHC restriction antigen presentation
INTRODUCTION T helper lymphocytes require the assistance of appropriate 'antigen-presenting' cells in order to recognize and respond to protein antigens. The exact requirements are variable according to both the nature of the antigen and of the antigen-presenting cell type, and there are many facets of presentation which are still not fully understood. In the human host, cell types known to function as professional antigen-presenting cells include monocytes and macrophages [1], dendritic cells [2] and Langerhans cells [3]. In many cases an 'antigen-processing' step, involving ingestion and breakdown of protein antigen to immunogenic peptides, is required before 'antigen-presentation' can occur. After processing, antigenic determinants may then be expressed at the surface of antigen-presenting cells in association with products of the MHC class II molecules, in a form which allows their recognition and binding to T cells expressing the antigenspecific receptor. However, some cell types, for example B lymphocytes, can 'present' certain antigens without the need for processing' [4]; and the requirement for MHC class II association is another facet of antigen presentation which is still not
fully understood. Neutrophils share several features in common with macrophages. These include a common cell lineage, their phagocytic nature, similar surface receptors for opsonized antigens and the Correspondence: Dr Patricia L. Haslam, PhD MRCPath, Head of Cell Biology Group, Department of Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK.
485
ability to produce the mediator IL-l [5,6], which has the ability to prime lymphocytes for proliferation. However, the question whether neutrophils can function in processing and/or presenting antigens has received little attention. We are aware of only one recent study which has addressed this subject directly [7], showing that neutrophils can act as accessory cells in mitogeninduced stimulation of lymphocytes, although they did not appear to express HLA-D region antigens. Since there has been a report that antigen-induced T lymphocyte proliferation can be induced in certain MHC-unrestricted proliferation systems [8], we have sought to re-assess the role of neutrophils in presentation of antigens. A common problem in conducting antigen-presenting studies is the difficulty in obtaining absolutely pure cell populations, and the functional implications of cell contamination are difficult to assess. In this study, a modified and extended lymphocyte purification procedure has been employed and morphologic and phenotypic checks using flow cytometric analysis have been carried out on all purified cell populations in each experiment to avoid any bias resulting from occasional inadequacy of cell separation. Also, in contrast to former work [7], we have pulsed neutrophils with antigen so that only antigen taken up by, or bound to neutrophils was introduced into the culture system. Our results suggest that antigen-pulsed human neutrophils can induce a dose-dependent lymphoproliferative response. We propose that interactions between neutrophils and mononuclear cells can occur during inflammatory and immune processes which may result in further enhancement of the cellular immune response.
C. Prior et al.
486 MATERIALS AND METHODS
Study population The study population consisted of healthy adult volunteers, all of whom had been inoculated with tetanus toxoid 3-9 years (median 6 years) prior to the study. Blood lymphocytes, neutrophils and monocytes were purified from each volunteer, as described below, to employ defined numbers of each cell type in assays to investigate antigen-presenting function. Purification of Lymphocytes A 60-ml venous blood sample was taken from each volunteer, defibrinated in a siliconized glass vessel, then diluted in an equal volume of minimal essential medium containing 25 mm HEPES buffer (MEM; GIBCO, Uxbridge, UK). It was layered over lymphocyte separation medium (Flow Laboratories, Rickmansworth, UK), centrifuged at 600 g for 30 min at 20C, the interface layer of peripheral blood mononuclear cells (PBMC) recovered, washed twice in MEM, then adjusted to a concentration of 5 x 106 cells/ml in RPMI 1640 (GIBCO; containing 25 mm HEPES buffer, 30 mg/100 ml L-glutamine; 100 U/ml penicillin, and 100 ,ug/ml streptomycin) supplemented with 10% heat-inactivated (56'C, 30 min) autologous serum. The PBMC suspension was then depleted of monocytes using an adherence procedure. This was conducted in polystyrene tissue culture flasks (Flow Laboratories; 25 ml capacity) which were precoated by adding 5 ml of MEM containing 10% heat-inactivated fetal calf serum (FCS) (Flow Laboratories) to each flask, incubating at 370C for 1 h, then gently rinsing the coated surface with MEM. The PBM suspension was then added (4-5 ml containing 20-25 x 106 cells per flask), and the flasks were incubated for I h at 37 C to allow monocytes to adhere. Nonadherent cells (mainly lymphocytes) were washed from the coated surface using pre-warmed MEM (37 C) then panned using MoAb to remove any residual cells expressing products of the human class II MHC (HLA-D region antigens) as follows. In preparation for panning, the 'lymphocyte' population was transferred to a 15-ml graduated polypropylene tube (Falcon 2097, Becton Dickinson, Lincoln Park, NJ), sedimented by centrifugation at 300 g for 5 min at 4 C, then resuspended in PBS containing 10 yg/ml of CA2 MoAb (mouse IgG2a directed against a common determinant of the human class II MHC; [911] for 20 min at 4°C. The CA2 MoAb was kindly provided by Dr Julia Bodmer, Imperial Cancer Research Fund Laboratories, London, UK. After incubation, the cells were washed, resuspended at 1 x 107 cells/ml in RPMI 1640 containing 10% autologous serum then panned onto the surface of sterile 90-mm polystyrene Petri dishes (Sterilin, Teddington, UK) which had been pre-coated with affinity purified F(ab')2 rabbit anti-mouse IgG (Serotec, Kidlington, UK). For pre-coating, the dishes were incubated overnight at room temperature with 10 ml of a 10 pug/ ml solution of the rabbit anti-mouse antibody in Tris buffer, pH 9. Prior to panning, the antibody solution was discarded, the dishes were soaked in a I % solution of FCS in PBS (pH 7 2) at room temperature for 30 min, then the surface of the Petri dish was washed with MEM. For panning, 3 ml of the CA2-treated lymphocyte suspension were added to each dish and the dishes incubated at 4°C for 2 h to allow CA2-positive cells to adhere to the coated surface. Non-adherent lymphocytes were then collected, washed and re-suspended, and the panning step repeated. The final lymphocyte population was harvested by
gently washing with MEM and re-suspended at 1-25 x 106 cells/ ml in RPMI 1640 ready for use as the purified, HLA-D-regiondepleted responder lymphocytes in the assays of antigenpresenting function.
Purification of monocytes The adherent monocytes retained in the FCS-coated polystyrene flasks during lymphocyte purifications were detached from the surface of the flasks by gently scraping with a rubber policeman, recovered by vigorous washing with cold MEM, sedimented by centrifugation at 300 g for 10 min at 4°C, then resuspended at 1 x 106 cells/ml in RPMI 1640 ready for use as control known antigen-presenting cells in the functional assays. The monocyte populations were not exhaustively purified, as for the lymphocytes and neutrophils, due to limitations in obtaining sufficient numbers of all three cell types from a single individual. Purification of neutrophils At this point, further 20 ml of venous blood were taken from the same individual, defibrinated, and used to obtain purified neutrophils employing a modification of the method reported by Dooley, Simpson & Meryman [12]. This ensured that the shorter-lived neutrophil population was as fresh as possible when used in the assay. The 20 ml of defibrinated blood were mixed with 10 ml of 6% dextran (dextraven 1 10 in 0-9% sodium chloride) in a 50-ml graduated polypropylene tube (Falcon 2070, Becton Dickinson), the erythrocytes were allowed to sediment at room temperature for 20-30 min, then the leucocyte-rich supernatant was recovered and washed in MEM. The cell pellet was re-suspended in a solution of 55% Percoll (Pharmacia, Uppsala, Sweden). The solution of 55% Percoll, and also solutions of 70% and 81% Percoll, were prepared by mixing nine parts of concentrated stock Percoll with one part of 9% chloride to achieve isotonicity ('100% Percoll'), then further diluting the 100% Percoll by adding PBS. The densitities of the 81%, 70%/0 and 55% Percoll solutions were 1 1000, 1-0875 and 1-0697 g/ml, respectively. A gradient was then set up by first adding 5 ml of 81 % Percoll to a 1 5-ml polypropylene tube, then gently adding a layer of 3 ml 70% Percoll and finally a layer of 3 ml of the buffy coat suspension in 55% Percoll. The tube was spun at 600 g for 20 min at 20°C, and the neutrophil band was harvested from the interface of the 70% and 81 % layers. After washing in MEM, the purified neutrophils were re-suspended at a concentration of 1 x 106/ml in RPMI 1640 ready for use in the assays.
Methods to check the purity of the cell populations The purity of the populations of lymphocytes, neutrophils and monocytes was checked at every experiment. Morphology was assessed using cytocentrifuge slide preparations of the cells prepared by spinning 100-,l aliquots ofeach cell suspension (106 cells/ml RPMI 1640) onto glass slides for 10 min at 18 g in a cytocentrifuge (Cytospin, model 2; Shandon Southern Products, Runcorn, UK). After air drying, the cells were fixed in methanol and stained with May-Gruinwald-Giemsa stain. Two-hundred cells were counted and the percentage ofcontaminating cell types was recorded. In addition, flow cytometric checks were conducted to evaluate cell surface marker expression using the methods we have described in detail previously [13]. Briefly, aliquots of cells from each suspension were labelled with MoAbs directed
Induction of lymphocyte proliferation by neutrophils Density separation
Adherence
Double panning
CD14
A JM.JIP. A\
A
HLA DR
0)
4-
HLA DP
0
E z
HLA DQ
487
majority of the cells remaining, as used in the six main experiments were T lymphocytes (CD3+), mean 70+ 12% s.d., and NK cells (11 + 7% CD16+, and 9 +12% CD57+). Contamination was minimal (CD 14+, 2-4 + 2-0%; CD22+, 074 + 0 57%; HLA-DR+, 3-3+2.4%; HLA-DP+, 0 3+004%; and HLADQ+, 0-06 + 0-13%). By morphology, 98 + 2% had the appearance of lymphocytes while only 1 3+ 1-0% were monocytes, only 0-63+0 75o neutrophils, and only 025+0 50 eosinophils. Neutrophil populations By morphology, 97 +10/% of the cells used in the six main experiments were segmented neutrophils, 2-3 + 1 3% were eosinophils, and only 0 33 + 0 4% had the appearance of lymphocytes. Flow cytometric analysis confirmed these findings and showed that only 3 2+2 7% of cells expressed the monocyte marker CD 14.
Monocyte populations By morphology, the 'monocyte' populations used as positive controls in the functional studies contained 78+ 1400 monocytes and 20 + 15% lymphocytes, but only 1-8 + 2% neutrophils and 04 + 044% eosinophils. Flow cytometric analysis showed that 75 +220% expressed the monocyte marker CD14 and that the majority expressed HLA-DR antigens (69+26%). A few also expressed HLA-DP (14+9%) and/or HLA-DQ (3-4+2.9%).
CD22A
Log FITC fluorescence
Fig. 1. Histograms of FITC fluorescence emission from monoclonal antibody-stained cells showing progressive depletion of contaminating monocytes (CD14+ cells), HLA-D+ cells (HLA-DR, -DP, -DQ) and B lymphocytes (CD22+ cells) at different steps of the lymphocyte purification procedure. The analysis was undertaken using a FACS 440.
against a range of cell surface determinants (described below), stained with FITC-labelled anti-mouse immunoglobulin, then fixed and permeabilised using 5000 methanol. The nuclei of the cells were then stained with propidium iodide to acquire signals selectively from nucleated cells during analysis using a Becton Dickinson FACS 440. Percentages of cells positively stained with MoAbs were determined by comparison with controls set up without MoAbs. All the MoAbs were obtained from Becton Dickinson (Mountain View, CA), and T lymphocytes were detected using Leu 4 (CD3 MoAb), B lymphocytes using Leu 14 (CD22), monocytes using M3 (CD14) and neutrophils using Leu 1 b (CD 16) which also reacts with natural killer (NK) cells. NK cells were also evaluated using Leu 7 (CD57). To confirm that cells expressing class II MHC products had been depleted from the purified lymphocyte population, flow cytometric checks were also conducted using MoAbs to HLA-DR, -DP and -DQ (Becton Dickinson). These antibodies were also used to confirm that MHC class II region products were expressed on the monocyte populations and to study whether these products can be detected on human neutrophils.
Lymphocyte populations The modified purification method we employed led to the progressive depletion of contaminating monocytes (CD14+), cells expressing MHC class II region products (HLA-DR+, DP+ and DQ+), and B lymphocytes (CD22+) (Fig. 1). The
Method to prepare 'antigen-pulsed' neutrophils and monocytes The purified neutrophils and monocytes, suspended at 1 x 106 cells/ml in RPMI 1640 containing 10% autologous serum, were incubated with a range of doses of tetanus toxoid (TT) for 3 h at 37 iC. The TT (soluble TT, batch No. MWC5208/A/F), had a limited flocculation (Lf) value of 2567 Lf/ml and a purity of 1200 Lf/mg, and was kindly provided by Dr M. Hughes, Wellcome Biotechnology, Beckenham, UK. A dose range of 25-250 Lf/ml was employed. After incubation with TT, the monocytes or neutrophils were washed three times in MEM, then re-suspended at 106/ml in RPMI 1640 for use in the functional assays. Method to test the antigen-presenting Junction of the antigenpulsed cell populations To determine whether the antigen-pulsed neutrophils or monocytes could stimulate the proliferation of the purified autologous lymphocytes, co-cultures of the cells were set up in the following way: 800 yl of the purified lymphocyte suspension (containing 1 x 106 cells) in RPMI 1640 were added to 5-ml polypropylene tissue culture tubes (Falcon 2063, Becton Dickinson) and 100 I1 of antigen-pulsed neutrophil or monocyte suspension (containing I x 105 cells) were added to each tube. Each tube was then supplemented with 100 ,l (i.e. IO%) of heatinactivated autologous serum. The cultures were set up in triplicate. Controls were set up containing 106 lymphocytes alone to determine background levels. Tubes were also set up containing lymphocytes plus accessory cells which had been sham-pre-incubated in medium without antigen to check whether the accessory cells could stimulate an autologous mixed leucocyte reaction. The cell mixtures were co-cultured for 5 days, and 37 KBq of tritiated thymidine (methyl-3H thymidine < 37 MBq/ml; Amersham International, Amersham, UK) were added to each tube 18 h prior to harvesting. At the end of
C. Prior et al.
488 15,
P
