Elimination of monocytes from cultures activated with recall antigens

Immunology Letters, 46 (1995) 229-235 01652478/95/%09.50 0 1995 Elsevier Science B.V. All rights reserved IMLET 2388

Elimination of monocytes from cultures activated with recall antigens J. Pryjma

a,

*, M.

Zembala

a, J. Baran

a, M. Ernst

b and H.-D.

Flad b

aInstitute of Paediatrics, Department of Clinical Immunology and Microbiology, Jagielionian Universiry Medical College, Wielicka 265, PL-30-663 Cracow, Poland b Department of Immunology and Cell Biology, Forschungsinstitut Borstel, Parkallee 22, D-23845 Borstel, Germany (Received

12 July 1994; revised 28 February

Key words: Monocyte killing; Cell death; Cytotoxic

1. Summary Recent data provide evidence that antigen-specific CD4+ T-cell clones or antigen-activated T-cell lines can kill antigen-presenting cells @PC). We focused our studies on monocytes acting as APC in cultures of T cells freshly isolated from peripheral blood. The presence of monocytes in culture was monitored by their ability to emit light during phagocytosis of latex particles (latex-induced chemiluminescence). Using this approach as well as flow cytometry, evidence is presented that monocytes are eliminated from cultures with T cells activated with recall antigens (PPD or TT). The mechanism of monocyte elimination involved apoptosis as judged from in situ detection of DNA strand breaks by the terminal deoxynucleotidyl transferase assay. The antigen- but not lectin-dependent monocyte elimination was MHC-restricted and mediated by CD4+ T lymphocytes. This finding supports the hypothesis that elimination of APC is a general phenomenon during T-cell activation and may represent an important immunoregulatory mechanism. 2. Introduction CD4’ cells which belong to Th, T lymphocytes possess the ability to kill antigen-presenting cells (APC) [l-5]. Antigen-specific T-cell clones or T lymphocytes activated with antigen for 7-8 days kill APC in a MHC-restricted manner [3,6]. Furthermore, there is evidence that cytotoxic cells that kill APC are not only generated in vitro but are also found in vivo [4]. Finally, CD4+ T lymphocytes kill APC by a mechanism which leads to cell apoptosis [5,7]. * Corresponding author: J. Pryjma, Institute of Paediatrics, Department of Clinical Immunology and Microbiology, Jagiellonian University Medical College, Wielicka 265, PL-30-663 Cracow, Poland. SSDI 0165.2478(95100051-S

1995; accepted 6 April 1995)

CD4+ T cell; Apoptosis;

Immunoregulation

In all these studies Ag-specific T-cell clones or T cells activated for several days in culture were used to kill macrophages or transformed cell lines serving as APC. In contrast, there is no information whether the same mechanism operates in antigen-activated cultures of cells freshly isolated from peripheral blood. This is the first report providing evidence that monocytes are eliminated from such cultures stimulated with recall antigens, purified protein derivative of tuberculin (PPD), or tetanus toxoid (TT). Furthermore, elimination of monocytes is shown to be mediated by CD4+ T cells and to involve monocyte apoptosis.

3. Materials and Methods 3.1. Cell populations Mononuclear cells were isolated from EDTA-treated peripheral blood of healthy donors by standard FicollPaque (Pharmacia, Uppsala, Sweden) gradient. From this population lymphocytes and monocytes were isolated by counterflow centrifugal elutriation and T cells by rosetting procedure, as previously described [S]. In some experiments elutriated lymphocytes were depleted of B lymphocytes, natural killer (NK) cells, HLA-DR+, and in some cases also CD8+ cells by using magnetic cell sorting (MACS, Miltenyi Biotech, Bergisch Gladbach, Germany) [9]. The depleted lymphocyte fraction contained less than 1% of CD20f, CD16+, or HLA-DR+ cells as monitored by flow cytometry and was regarded as T lymphocytes. CD4+-enriched T lymphocytes (when anti-CD8 monoclonal antibody (mAB) was included during separation) contained less than 2% of CD8+ cells. In some experiments CD4+ T lymphocytes were further separated into CD45RO and CD45RA subsets by negative 229

selection after labeling with UCHL-1 (Dako) or Leu-18 (Becton Dickinson) mAbs and cell sorting by using a FACStar Plus flow cytometer (Becton Dickinson). The populations obtained after additional labeling were up to 95% of expected phenotype and were contaminated with less than 2% of the reciprocal population. 3.2. Antigens and reagents Purified derivative of tuberculin (PPD) (Staten Serum Institute, Copenhagen, Denmark) was used in cultures at a final concentration of 10 pg/ml. Tetanus toxoid (TT) was obtained from Behring (Marburg, Germany) and used in cultures at a concentration of 1 Lf/ml. Pokeweed mitogen (PWM) was purchased from Gibco and used at a final of dilution 1: 200. Monoclonal anti-CD3 antibodies (Ortho) were dialyzed against PBS containing 0.1% BSA and used at a predetermined dilution to obtain maximal T-cell proliferation in the presence of 10% monocytes. Latex particles (diameter: 0.8-1.0 pm) were obtained from Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences (Cracow, Poland). These were washed twice with 70% ethanol and kept suspended in PBS at the concentration of 1.5 X 109/ml. 3.3. Cell cultures Monocytes (1 X 105) were cultured with 5 X 10’ T lymphocytes in the presence or absence of stimulus (antigen or mitogen). The culture medium used was RPMI-1640 (Gibco) supplemented with 10% of fetal calf serum (FCS, Seromed), 2 mM r_-glutamine, and antibiotics (all from Gibco, Grand Island, NY). Cells were placed in 47 X 12 mm polystyrene tubes (Berthold, Wildbad, Germany) in a total volume of 0.4 ml of culture medium. Tubes were kept loosely covered with aluminum foil at 37°C 100% humidity, and 5% CO,. For flow cytometry analysis and determination of apoptosis, cells were cultured and further processed in polystyrene tubes (Falcon 2054, Becton Dickinson). The total cell number was 1 X lo6 in 1 ml of culture medium. 3.4. Chemiluminescence (CL) measurement Krebs Ringer phosphate buffer containing glucose (11 mM) and bivalent cations (0.54 mM Ca*+ and 1.12 mM Mg*+ 1, 2 mM of luminol (5-amino-2-3-dihydro1,4-phthalanedione, Sigma), and 0.1 mM lucigenin (lO,lO’-dimethyl-9,9’-biacridinium dinitrate, Sigma) were used as a medium for CL measurement (CL medium). 230

Before measurement, two-thirds of culture medium were replaced by prewarmed CL medium, and tubes were placed in a 6-channel luminometer (Multi-Biolumat, LB9505C, Berthold) connected with a AT-compatible computer equipped with appropriate software provided by the manufacturer. Thereafter, 20 ~1 of latex suspension were added, and CL was recorded for 10 min. The combination of two chemiluminogenic probes was used, as luminol-dependent light emission by monocytes spontaneously decreases in culture due to decrease of myeloperoxidase [lo]. In preliminary studies the computed lo-min integral was shown to be directly proportional to the number of monocytes in the sample, and pretreatment of monocyte suspension with the lysosomotropic agent (L-leucine methyl ester [ll] resulted in a dose-dependent reduction of the signal (data not shown)). The results were calculated by the computer as lomin integrals of control and activated samples, which were always run in parallel in triplicates. The ‘number’ of monocytes in activated samples was calculated as: CL integral in analyzed

sample:

CL integral in the control X 100%. 3.5. Phenotypic cell analysis and detection of apoptotic

cells For flow cytometry cultured cells were harvested at indicated times and labeled with anti-CD14 (Leu-M3, Becton Dickinson) and in parallel with isotype control. In some experiments cells were double-labeled with FITC CD14 (Becton Dickinson) and PE CD3 (Dako) followed by 15-min incubation in the presence of 20 pg/ml of 7-amino-acinomycin D (Sigma), as desribed by Schmid et al. [12]. Cell samples stained with FITC and PE-conjugated mouse IgG were used as controls. To detect DNA strands by terminal deoxynucleotidyl transferase assay, the procedure described by Gorczyca et al. [13] was followed. Briefly, cells were fixed in 1% paraformaldehyde and at -20°C in 70% ethanol. After washing in PBS, cell samples were incubated for 20 min at 37°C in 50 ~1 of solution containing 0.1 M sodium cacodylate, 1 mM CoCl, 0.1 mM dithiothreitol (all from Sigma), 0.05 mg/ml bovine serum albumine, 10 U of terminal transferase, and 0.5 nM of biotin-16dUTP (both from Boehringer Mannheim). Thereafter, cells were rinsed in PBS and incubated (30 min, room temperature, in the dark) in 100 ~1 of staining solution (4 X saline-sodium citrate buffer (Sigma), 0.1% Triton X-100, 5% non-fat dry milk, and FITC-streptavidine (Jackson ImmuneResearch, final dilution 1 : 100)). Samples were analyzed using FACScan V or FACStar plus flow cytometers (Becton Dickinson).

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Fig. 1. Monocytes and T lymphocytes isolated from PPD-negative (A) and PPD-positive (B) donors were cultured in polystyrene tubes at an 1: 5 ratio in the presence of PPD (a), PWM ( T ), or anti-CD3 mAb ( v 1.At indicated time points, after replacement of culture medium with CL buffer, 3 tubes from each group were placed in a luminometer, and the light emission was measured after adding latex particles. Results (mean of 3 independent measurements) are presented as a percentage of the response (lo-min integral) recorded in the control non-stimulated culture considered as 100%. As additional control the CL response of monocytes cultured without T cells is also shown (0 1.

4. Results 4.1. Monocytes are eliminated lymphocyte cultures

from antigen-activated

Monocytes and T cells isolated from PPD-positive and PPD-negative donors were cultured in the presence of antigen. Unstimulated cultures, cells activated with either lectin (PWM) or anti-CD3 antibody, served as

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Fig. 2. Monocytes and T cells isolated from a PPD-responding donor were cultured at an 1: 4 ratio in a 24-well culture plate (1 X 106/well) in the presence of antigen or PWM. Control cultures were left non-stimulated. At indicated time points, cultures were harvested, cells labeled with anti-CD14 mAb, and analyzed by using flow cytometry. Figure shows the proportion of CD14+ cells.

controls. Cells were cultured in polystyrene tubes which could be directly used for CL measurement, and each culture was set up in several replicates. Every day a set of tubes was placed in the luminometer, and light emission was measured after the cells were challenged with latex particles in the presence of luminol and lucigenin. As shown in Fig. 1, the ability of cultured cells to respond by CL was considerably reduced in cultures activated with PWM and anti-CD3 mAb. The reduction of CL response in PWM- or anti-CD3-stimulated cultures occurred within 24 h of culture and was independent of the donor status. In contrast, CL response in antigen (PPD)-activated cultures was substantially reduced after 48-72 h and only when cells from PPD-responding donors were used. Similarly, the reduction of latex-triggered CL was seen in ‘IT-activated cultures only when cells were obtained from ‘IT-responding donors (not shown). The presence of unstimulated T-lymphocytes had no effect on the response, as CL signal recorded in cultures of monocytes incubated without T cells was comparable to the control (Fig. 1). In a separate experiment analyzed by flow cytometry it was found that the proportion of CD14+ cells was reduced in cultures activated with antigen and lectin although this process was slower in the former (Fig. 2). Furthermore, the proportion of dead cells which were stained with 7-AAD was greater in cultures activated with antigen or mitogen than in unstimulated cultures. As shown (Fig. 3 and Table 1) such cells were found predominantly in a population of CD3- cells. In activated cultures the proportion of CD14+ cells was reduced more quickly than the number of cells characterized by strong side scatter signals (SSC) which repre231

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Fig. 4. Lymphocytes depleted of CD20+, CD16+ and HLA-DR+ cells by Dynabeads magnetic cell sorting (T cells) or in addition depleted of CD8+ (CD4+ cells) were cultured together with monocytes in the presence of ‘IT. The proportion of viable monocytes in antigen-stimulated cultures was determined by CL in response to latex particles. Results (mean of 3 independent measurements) are presented as a percentage of the response (lo-min integral) recorded in the control, unstimulated cultures considered as 100%.

CD14 j

Donor

MO

of: Tcells

A

f-apoptotic

_

PPD

l--J

PWM

B A Fig. 3. Monocytes were cultured in polystyrene tubes for 24 h with T lymphocytes in the presence of PWM and labeled with antXD14, anti-CD3, and 7-AAD. All dot plots contain 10,000 cells. The dot plot A shows the staining with 7-AAD (y-axis) versus CD3 (x-axis); here the analysis of all cultured cells is depicted. Dot plot B shows the same sample as A with 7-AAD (y-axis) versus CD14 (x-axis). In dot plot C the same parameters of the same preparation as in B are shown, but here only cells with strong SSC (side scatter signals) were used for the analysis depicted.

B

C

I-

r

D D 0

sents the typical monocyte region in flow cytometry analysis. This may indicate that down-regulation of CD14 antigen precedes the death of monocytes. Such a conclusion is also suggested by the reduction of CD14 antigen expression, as judged from the fluorescence intensity (mean channel of fluorescence). Furthermore, in activated cultures, in contrast to unstimulated cultures, CD14+ cells and cells with higher SSC had lower FSC (forward scatter signals which relate to the cell 232

L

20

40

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60

80

(% of

100

120

control)

Fig. 5. T lymphocytes isolated from PPD-responding donors were cultured in polystyrene tubes in the presence of PPD or PWM and either autologous or heterologous monocytes. The CL response to latex after 48 h of culture in the presence of PWM and 72 h of culture in the presence of PPD is shown. Results (mean of 3 independent measurements) are presented as a percentage of the response (lo-min integral) recorded in the control, unstimulated cultures considered as 100%. Figure shows the results of two independent experiments using cells from donors A and B (exp. 1) and C and D (exp. 2).

TABLE

1

Exp.

Cultured cells

Proportion CD14+

CD3+

high SSC *

CD14-

M+T M+T +PWM M+T M+T + PPD M+PPD

16.6 42.8

4.7 3.5

25.4 38.8

11.2 58.8

8.5 59.8

4.8 3.8

10.2 68.8

35.2 76.2

4.1

8.5

1

If

2 *=*

5.8

of 7-ADD+

-

cells within gated cells CD3-

* Cells characterized by 90” scatter (SSC) higher than lymphocytes. * * CD4+ isolated by magnetic cell sorting were cultured with monocytes in the presence or absence of PWM for 48 h. * ’ T lymphocytes obtained by depletion of B, NK and HLA-DR+ cells. Culture was terminated after 96 h. l

size), i.e., were smaller (not shown). This observation as well as the presence of cells which stained weakly with 7-AAD (Fig. 3, dot-plot B) may suggest the presence of apoptotic cells [4].

Fig. 6. Flow cytometry analysis of cells with strong side scatter signals distributions of cells having incorporated biotinylated dUTP (which in turn from 48-h PPD-stimulated cultures of monocytes plus CD4+ CD45RO + monocytes plus CD4+ CD45RA+ lymphocytes CD). Control (A) shows the assay was omitted.

From the above data we conclude that CD14’ cells that give rise to latex-induced CL are eliminated from activated cultures of monocytes and T lymphocytes. 4.2. Antigen-dependent monocyte elimination from primary cultures is MHC-restricted and mediated by CD4 ’ cells In the experiments shown in Table 1, T lymphocytes and CD4+ cells isolated by magnetic cell sorting were used. Therefore, the obtained data provide evidence that the cytotoxic effect is mediated by CD4+ T lymphocytes and that it is not related to T-cell activation by CD2 antigen during rosetting procedure. Similarly, as shown in Fig. 4, monocytes were comparably eliminated by IT-activated T lymphocytes isolated by rosetting procedure and by CD4+ obtained by magnetic cell in antigen-activated cultures sorting. Furthermore, monocytes were eliminated only when T cells were activated in the presence of autologous monocytes.

(SSC) (analysis gate). The figure shows the fluorescence intensity was labeled with streptavidin-FITC). Samples for analysis were taken lymphocytes (B), monocytes plus total CD4+ lymphocytes (0, or same sample as B, but here addition of terminal transferase during the

233

Elimination of monocytes, as judged by reduction of CL, was observed in the presence of PPD only when T cells were cultured with autologous monocytes (Fig. 5). In contrast, in PWM-activated cultures the reduction of CL was similar when autologous or heterologous monocytes were used, although in the heterologous system killing of monocytes was slightly delayed. From these findings we conclude that in antigenstimulated lymphocyte cultures monocytes are eliminated by MHC-restricted antigen-activated CD4+ T lymphocytes. 4.3. Apoptosis of monocytes in the presence activated memory T lymphocytes

of antigen-

CD4+ T cells were shown to induce apoptosis of APC [5,7]. In our material, during flow cytometry analysis we noted that a part of the cells with monocyte characteristic (CD14+ and/or with strong SSC signal), obtained from antigen- or mitogen-activated cultures, had a reduced FSC signal which could indicate that they were apoptotic [12,14]. This was observed already after 24 h in cultures activated with PWM and not before 48 h when cultures were stimulated with PPD or TT. The presence of apoptotic cells was also suggested by the staining pattern with 7-AAD (Fig. 3, dot-plot C). To make sure that apoptotic cells are indeed present in antigen-activated cultures we used the terminal deoxynucleotidyl transferase assay to detect in situ cells with DNA strand breaks [13]. In this experiment monocytes were cultured either with CD4+ T lymphocytes or with their CD45RO and CD45RA subsets. As shown (Fig. 6) after 48 h of culture with PPD, a considerable proportion of cells incorporated biotin-labeled dUTP in cultures which contained cells of the CD45R0, but not CD45RA subset of CD4+ T lymphocytes. Only very few cells incorporating dUTP could be detected within a population of T cells (with lower SSC) (not shown). From this we conclude that monocyte apoptosis is indeed induced in the presence of antigen-activated T cells.

5. Discussion This paper shows that in PPD- or TT-activated cultures of monocytes and T lymphocytes freshly isolated from the peripheral blood antigen-activated CD4+ T cells eliminate monocytes. CD4+ T-cell lines or lymphocytes stimulated for a few days with antigen have already been reported to be cytotoxic for APC [l-7]. In contrast, we give evidence that presentation of recall antigens to freshly isolated T cells also results in elimination of APC. To show this, the presence of mono234

cytes in culture was monitored by latex-induced CL response. It is known that in a mononuclear cell population monocytes are the only cells able to emit light following latex phagocytosis [8,15], therefore the measured CL is directly related to the number of monocytes in culture. The use of the same tubes for cell culture and for CL measurement excluded the possibility that the results were affected by cell manipulation before use or by a preferential cell loss during harvesting. As it is known that during culture the ability of monocytes to respond by CL is declining [lo], all data were expressed as a percentage of CL response recorded in control cultures. CL response of monocytes cultured without T cells was comparable to that recorded in the control, indicating that the presence of T cells, by itself had no effect on monocytes survival. The observed reduction of CL was not due to the direct effect of antigen on monocytes, as no significant reduction of the CL response was noted in cultures of monocytes and T cells isolated from non-responders or when cultured monocytes and T cells were obtained from unrelated donors. Using this method we have demonstrated that monocytes were eliminated from cultures of monocytes and T cells activated with recall antigens in a MHC-restricted manner. The CL data were confirmed by the independent measurement of CD14+ cells by flow cytometry and by the detection in culture of dead or apoptotic cells with monocyte characteristics. This was done with the use of 7-AAD and the terminal transferase assay. The use of 7-amino-actinomycin D allowed, owing to its spectral characteristics, to correlate live, apoptotic, and dead cells with FITC and PE surface staining of T cells and monocytes. By this approach we were able to show that the dead and apoptotic cells which can be seen in antigen- or mitogen-activated samples are predominantly not lymphocytes. The presence of apoptotic cells was demonstrated in situ by the terminal transferase assay which allows to detect cells incorporating biotinlabeled dUTP into DNA brakes. These were present almost exclusively in the population of cells with high SSC signal. Therefore, these results collectively with the data mentioned above point at monocytes as the cells ‘programmed to die’ in antigen- and mitogenstimulated cultures. The effector cells were present in the CD8- T-cell population. In addition, T cells isolated by rosetting procedure or magnetic cell sorting by depletion of B and NK (CD16+) cells were equally effective in monocyte elimination, indicating that the phenomenon was not mediated by NK cells or related to pre-activation of CD2 antigen. Finally, apoptotic cells, most likely monocytes, were detected in antigen-stimulated cultures of monocytes and CD45R0, ‘memory’ cells, but not

CD45RA, ‘naive’ CD4’ T lymphocytes. All these data collectively strongly support the conclusion that monocytes are indeed eliminated from culture by recall antigen-activated CD4+ T lymphocytes. It has been postulated that killing of APC by CD4+ cytotoxic cells represents an important mechanism of elimination of macrophages infected with microorganisms and may play a role as immunoregulatory mechanism [1,3,7,16]. The time necessary to see monocyte elimination from antigen-activated cultures is much longer than in the case of lectin- or anti-CD3-stimulated cultures. This suggests that the effector T cells represent a separate cytotoxic population which is recruited and expanded or differentiates from memory cells. This view is in agreement with other data which show that antigen-specific T-cell lines differ in their ability to kill APC [2,6]. However, the possibility that during antigen presentation monocytes always receive an ‘off’ signal cannot be dismissed. In either case the presence of cells which can eliminate APC may play an important role in preventing overstimulation of the immune system by persisting antigen.

Acknowledgements We wish to thank Mrs. E. Isendorf and E. Kaltenhbser for skillful technical assistance. This work was supported in part by Grant KBN 2033/4/91 of the Polish Research Committee and by Grant HHS/79 of the U.S.-Polish M. Sklodowska-Curie Joint Fund.

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