Immunological Reviews 1991, No. 119 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from tbe author(s)
Detection of Human Cytokine-Secreting Cells in Distinct Anatomical Compartments CECIL CZERKINSKY', GLTDRUN ANDERSSON^, BERNARD F E R R U A ^ INGER NORDSTROM', MARIANNE Q U I D I N G ' , K R I S H N A ERIKSSON', LISBETH LARSSON', KRISTOFFER HELLSTRAND'* & HANS-PETER
INTRODUCTION A major regulatory role responsible for modulating the vertebrate immune response has been ascribed to soluble factors or cytokines. The term "cytokine" is collectively employed to designate a heterogeneous group of proteins which are produced by a wide variety of cells, lympboid and non-lymphoid, and that induce similar, distinct, synergistic, or even contradictory signals. This heterogeneous group of proteins has a number of common characteristics: (1) Cytokines are of low molecular weight (ernatants from MNC cultures were determined by ELISA methods described by Andersson et
Figure 2. Typical appearance of a well developed by nicans; ol" Ihc reverse IILISPOT assay for detection of interferon-y-secretlng cells (x 180).
CYTOKINE-SECRETING CELLS
13
al. (1989) and by Ferrua et al. (1988), respectively. Recombinant cytokines were used to generate calibration curves. SPONTANEOUS AND MITOGEN-INDUCED IFN-y SECRETION BY HUMAN MNC Peripheral blood Nitrocellulose membranes coated with mAb IFN-y antibodies as solid-phase capture system and biotinylated mAb IFN-y antibodies as a developing reagent, were used to detect human peripheral blood lymphocytes secreting immune IFN as spot-forming cells (SFC). The reactions were visualized as red spots which appeared within a few minutes following stepwise addition of HRP-conjugated avidin and AEC-H2O2 chromogen substrate (Fig. 2). These reactions, which differed in diameter, were smaller and generally less distinct than the spots which we routinely observe when using plastic surfaces exposed to a gel overlay containing paraphenylenediamine-HjOj substrate (Czerkinsky et al. 1983). The enumeration of these reactions necessitated the systematic use of the stereomicroscope in order to distinguish true, homogeneously-stained spots from small, dense, artifactual dots that were frequently observed. The latter reactions were considered as artifacts since they could be observed even in wells that had not been exposed to MNC. TABIE I Frequency of gamma-interferon-secreting cells in cultures of milogen-stimulated and unstimulaied human blood mononuclear cells as detected by reverse ELISPOT assay Number of cells assayed per assay well lO" 5x10^ 10^
Experiment
Stimulation with PHA (72 h)
1
+
>100 12±4 ND" 4±2
44+11 3±3 21±5 0
II±3 0 5±2 0
3
+
ND I3±4
29 + 6 6±2
12 + 5 0
4
+
ND 4±1
63 ±16 ND
19 + 5 ND
5
-1-1- (with cycloheximide'^)
ND ND 15±4 0
63±16 0 ND ND
19 + 5 0 ND ND
t
— (with cycloheximide)
' Peripheral blood MNC were assayed at the indicated cell densities for numbers of IFN)'-secreting cells; results represent mean SFC numbers + SEM of 3—4 replicate wells. " Not done. " Cycloheximide (2 mM) was added during the assay culture period.
14
CZERKINSKY ET AL.
IFN-y-secreting cells were first detected by approximately 8 h of incubation in mAb IFN-y-coated wells. Maximum numbers of SFC were observed between 12and 14-h of cell incubation. The frequency of these cells remained constant during the following 10-12 h. For convenience, a duration of 20 h was selected as a standard cell incubation time. Under these conditions, as many as 7082 ±3175 SFC per 1 x 10* PHA-stimulated peripheral blood MNC, and 974±231 SFC per 1 X 10^ unstimulated MNC were detected in five consecutive experiments (Table Active secretion of IFN-y in the assay was revealed by the addition of cycloheximide during the cell incubation period, a treatment which abolished spot formation not only by mitogen-stimulated MNC but also by unstimulated MNC (Table I). The latter observation is consistent with previous reports demonstrating the presence of cells spontaneously secreting IFN-y in normal human peripheral blood (Palacios et al. 1983, Martinez-Maza et al. 1984). It should, however, be pointed out that reverse ELISPOT assays performed directly after isolation of blood MNC without preculture resulted in the detection of considerably lower numbers of IFN-y-secreting cells as compared to "unstimulated" MNC maintained in culture for 48-72 h (see also Table II). Omission of either capture mAb IFN-)' antibodies, cells., or biotinylated mAb IFN-y antibodies prevented subsequent spot formation. Addition of graded amounts of free soluble mAb IFN-y (7-B6-1) during the cell incubation period inhibited, in a dose-dependent manner, spot formation by human MNC assayed in wells coated with the same monoclonal antibody reagent (Fig. 3). Furthermore, addition of unlabelled mAb IFN-y (1-DlK) after the cell incubation period prevented the development of spots following subsequent exposure of treated wells to biotinylated mAb IFN-y of the same preparation (Fig. 3). In contrast, treatment of replicate wells with mAb IFN-y (7-B6-7) before the addition of biotinylated mAb IFN-y (1-DlK) had no effect on the subsequent development of spots (Fig. 3). The latter observations attest to the high degree of specificity of this assay and demonstrate that the two mAb IFN-y antibodies recognize distinct antigenic epitopes on the IFN-y molecule.
TABLE II Frequency of spontaneous gamma-interferon-secreting cells in various anatomical sites in humans Duodenal mucosa SFC/10* MNCa (range)
97 (20-240) n^7
Spleen
Bood
Bone marrow
Tonsils
Gingiva
278
13 (2^0) n=13
8 (0-11) n=9
116 (40-350) n=13
21 (0-64) n=7
n=l
* Results are expressed as geometric mean (where applicable) numbers of IFN-y-spot forming cells (SFC) per 10^ MNC.
15
CYTOKINE-SECRETING CELLS
inhibitor added
[jg/weii
mAb iFN-
20 10 5
r-B6-i
1-DIK niAb IFN-
20 ZZ2 10
7-B6-1 mAt) IFN-
5 1 2.5 1
777777777777
-
20
40
60
80
100
maximum SFC numbers
Figure 3. Specificity of the reverse ELISPOT assay for detection of human IFN-y-secreting cells. Monoclonal anti-IFN-antibodies were added during (closed bars) or after (hatched bars) incubation of PHA stimulated peripheral blood MNC in mAb IFN-y 7-B6-I coated weils.
Spontaneous secretion of IFN-y by human MNC isolated from parenchymal tissues Reverse ELISPOT analyses performed on enzymatically or mechanically dispersed tissues revealed that the human intestinal mucosa is a major source of cells producing IFN-y. When compared to peripheral blood, inflammed gingival tissue and bone marrow, the duodenal mucosa contained considerably more IFN}'-secreting cells (Table II). In addition, the amount of IFN-y produced during short-term (24 h) cultures of duodenal cells was particularly impressive, since immunoassayable quantities (>0.05 i.u. IFN-y/ml) of IFN-y could be detected by ELISA in supernatants of cultures containing as few as 8 x IC* intestinal MNC in 200 /z medium; in contrast, IFN-y production by high density (10*' MNC per 200 /il) cultures could not be detected in culture supernatants of unstimulated peripheral blood MNC, even after prolonged (96 h) incubation period (data not shown). The observation that treatment with thermolysine lowers the frequency of detectable IFN-y-secreting cells by mitogen-stimulated peripheral blood MNC (data not shown) indicates that the intestinal mucosa may be even richer in IFNy-secreting cells than recorded in this study. Tonsillar IFN-y-secreting cells were also detected in substantial numbers (116 + 23 IFN-y-secreting cells/10'' MNC, n = 14). No significant differences were
16
CZERKINSKY ET AL.
observed when comparing the frequencies of IFN-y-secreting cells between clinically healthy tonsils and inflamed ones (data not shown). Unexpectedly, and in contrast to the situation seen with MNC obtained from most other organs examined, the relation between numbers of tonsillar MNC assayed and numbers of detectable IFN-y-secreting cells was not linear, but decreased dramatically below a threshold cell density of 10^ MNC plated (Fig. 4). This lack of linearity was also observed with a cell suspension obtained from a human spleen specimen. This observation was confirmed by quantitative ELISA analyses of IFN-y levels in supernatants from tonsillar MNC cultures performed at various cell densities (Fig. 4A). Depletion of plastic adherent cells from the tonsillar MNC culture decreased the number of detectable IFN-y-secreting cells by almost 50%, even though the original cell density was restored (Fig. 4A). Addition of picogram amounts of purified IL-1^ (and for that matter of IL-la) to non-fractionated, as well as adherent cell-depleted tonsillar MNC suspensions, partially restore*.! the predicted linearity (Fig. 4B). These observations demonstrate that adherent cells (presumably macrophages or dendritic cells) and/or soluble IL-1 provide essential signals for supporting the secretion of IFN-y. In fact, the paucity of macrophages and dendritic cells present in our tonsillar suspensions, and the comparatively low levels of IL-1-secreting cells detected in these suspensions (unpublished observations) are compatible with this interpretation.
10 5
Detection of Human Cytokine-Secreting Cells in Distinct Anatomical Compartments CECIL CZERKINSKY', GLTDRUN ANDERSSON^, BERNARD F E R R U A ^ INGER NORDSTROM', MARIANNE Q U I D I N G ' , K R I S H N A ERIKSSON', LISBETH LARSSON', KRISTOFFER HELLSTRAND'* & HANS-PETER
INTRODUCTION A major regulatory role responsible for modulating the vertebrate immune response has been ascribed to soluble factors or cytokines. The term "cytokine" is collectively employed to designate a heterogeneous group of proteins which are produced by a wide variety of cells, lympboid and non-lymphoid, and that induce similar, distinct, synergistic, or even contradictory signals. This heterogeneous group of proteins has a number of common characteristics: (1) Cytokines are of low molecular weight (ernatants from MNC cultures were determined by ELISA methods described by Andersson et
Figure 2. Typical appearance of a well developed by nicans; ol" Ihc reverse IILISPOT assay for detection of interferon-y-secretlng cells (x 180).
CYTOKINE-SECRETING CELLS
13
al. (1989) and by Ferrua et al. (1988), respectively. Recombinant cytokines were used to generate calibration curves. SPONTANEOUS AND MITOGEN-INDUCED IFN-y SECRETION BY HUMAN MNC Peripheral blood Nitrocellulose membranes coated with mAb IFN-y antibodies as solid-phase capture system and biotinylated mAb IFN-y antibodies as a developing reagent, were used to detect human peripheral blood lymphocytes secreting immune IFN as spot-forming cells (SFC). The reactions were visualized as red spots which appeared within a few minutes following stepwise addition of HRP-conjugated avidin and AEC-H2O2 chromogen substrate (Fig. 2). These reactions, which differed in diameter, were smaller and generally less distinct than the spots which we routinely observe when using plastic surfaces exposed to a gel overlay containing paraphenylenediamine-HjOj substrate (Czerkinsky et al. 1983). The enumeration of these reactions necessitated the systematic use of the stereomicroscope in order to distinguish true, homogeneously-stained spots from small, dense, artifactual dots that were frequently observed. The latter reactions were considered as artifacts since they could be observed even in wells that had not been exposed to MNC. TABIE I Frequency of gamma-interferon-secreting cells in cultures of milogen-stimulated and unstimulaied human blood mononuclear cells as detected by reverse ELISPOT assay Number of cells assayed per assay well lO" 5x10^ 10^
Experiment
Stimulation with PHA (72 h)
1
+
>100 12±4 ND" 4±2
44+11 3±3 21±5 0
II±3 0 5±2 0
3
+
ND I3±4
29 + 6 6±2
12 + 5 0
4
+
ND 4±1
63 ±16 ND
19 + 5 ND
5
-1-1- (with cycloheximide'^)
ND ND 15±4 0
63±16 0 ND ND
19 + 5 0 ND ND
t
— (with cycloheximide)
' Peripheral blood MNC were assayed at the indicated cell densities for numbers of IFN)'-secreting cells; results represent mean SFC numbers + SEM of 3—4 replicate wells. " Not done. " Cycloheximide (2 mM) was added during the assay culture period.
14
CZERKINSKY ET AL.
IFN-y-secreting cells were first detected by approximately 8 h of incubation in mAb IFN-y-coated wells. Maximum numbers of SFC were observed between 12and 14-h of cell incubation. The frequency of these cells remained constant during the following 10-12 h. For convenience, a duration of 20 h was selected as a standard cell incubation time. Under these conditions, as many as 7082 ±3175 SFC per 1 x 10* PHA-stimulated peripheral blood MNC, and 974±231 SFC per 1 X 10^ unstimulated MNC were detected in five consecutive experiments (Table Active secretion of IFN-y in the assay was revealed by the addition of cycloheximide during the cell incubation period, a treatment which abolished spot formation not only by mitogen-stimulated MNC but also by unstimulated MNC (Table I). The latter observation is consistent with previous reports demonstrating the presence of cells spontaneously secreting IFN-y in normal human peripheral blood (Palacios et al. 1983, Martinez-Maza et al. 1984). It should, however, be pointed out that reverse ELISPOT assays performed directly after isolation of blood MNC without preculture resulted in the detection of considerably lower numbers of IFN-y-secreting cells as compared to "unstimulated" MNC maintained in culture for 48-72 h (see also Table II). Omission of either capture mAb IFN-)' antibodies, cells., or biotinylated mAb IFN-y antibodies prevented subsequent spot formation. Addition of graded amounts of free soluble mAb IFN-y (7-B6-1) during the cell incubation period inhibited, in a dose-dependent manner, spot formation by human MNC assayed in wells coated with the same monoclonal antibody reagent (Fig. 3). Furthermore, addition of unlabelled mAb IFN-y (1-DlK) after the cell incubation period prevented the development of spots following subsequent exposure of treated wells to biotinylated mAb IFN-y of the same preparation (Fig. 3). In contrast, treatment of replicate wells with mAb IFN-y (7-B6-7) before the addition of biotinylated mAb IFN-y (1-DlK) had no effect on the subsequent development of spots (Fig. 3). The latter observations attest to the high degree of specificity of this assay and demonstrate that the two mAb IFN-y antibodies recognize distinct antigenic epitopes on the IFN-y molecule.
TABLE II Frequency of spontaneous gamma-interferon-secreting cells in various anatomical sites in humans Duodenal mucosa SFC/10* MNCa (range)
97 (20-240) n^7
Spleen
Bood
Bone marrow
Tonsils
Gingiva
278
13 (2^0) n=13
8 (0-11) n=9
116 (40-350) n=13
21 (0-64) n=7
n=l
* Results are expressed as geometric mean (where applicable) numbers of IFN-y-spot forming cells (SFC) per 10^ MNC.
15
CYTOKINE-SECRETING CELLS
inhibitor added
[jg/weii
mAb iFN-
20 10 5
r-B6-i
1-DIK niAb IFN-
20 ZZ2 10
7-B6-1 mAt) IFN-
5 1 2.5 1
777777777777
-
20
40
60
80
100
maximum SFC numbers
Figure 3. Specificity of the reverse ELISPOT assay for detection of human IFN-y-secreting cells. Monoclonal anti-IFN-antibodies were added during (closed bars) or after (hatched bars) incubation of PHA stimulated peripheral blood MNC in mAb IFN-y 7-B6-I coated weils.
Spontaneous secretion of IFN-y by human MNC isolated from parenchymal tissues Reverse ELISPOT analyses performed on enzymatically or mechanically dispersed tissues revealed that the human intestinal mucosa is a major source of cells producing IFN-y. When compared to peripheral blood, inflammed gingival tissue and bone marrow, the duodenal mucosa contained considerably more IFN}'-secreting cells (Table II). In addition, the amount of IFN-y produced during short-term (24 h) cultures of duodenal cells was particularly impressive, since immunoassayable quantities (>0.05 i.u. IFN-y/ml) of IFN-y could be detected by ELISA in supernatants of cultures containing as few as 8 x IC* intestinal MNC in 200 /z medium; in contrast, IFN-y production by high density (10*' MNC per 200 /il) cultures could not be detected in culture supernatants of unstimulated peripheral blood MNC, even after prolonged (96 h) incubation period (data not shown). The observation that treatment with thermolysine lowers the frequency of detectable IFN-y-secreting cells by mitogen-stimulated peripheral blood MNC (data not shown) indicates that the intestinal mucosa may be even richer in IFNy-secreting cells than recorded in this study. Tonsillar IFN-y-secreting cells were also detected in substantial numbers (116 + 23 IFN-y-secreting cells/10'' MNC, n = 14). No significant differences were
16
CZERKINSKY ET AL.
observed when comparing the frequencies of IFN-y-secreting cells between clinically healthy tonsils and inflamed ones (data not shown). Unexpectedly, and in contrast to the situation seen with MNC obtained from most other organs examined, the relation between numbers of tonsillar MNC assayed and numbers of detectable IFN-y-secreting cells was not linear, but decreased dramatically below a threshold cell density of 10^ MNC plated (Fig. 4). This lack of linearity was also observed with a cell suspension obtained from a human spleen specimen. This observation was confirmed by quantitative ELISA analyses of IFN-y levels in supernatants from tonsillar MNC cultures performed at various cell densities (Fig. 4A). Depletion of plastic adherent cells from the tonsillar MNC culture decreased the number of detectable IFN-y-secreting cells by almost 50%, even though the original cell density was restored (Fig. 4A). Addition of picogram amounts of purified IL-1^ (and for that matter of IL-la) to non-fractionated, as well as adherent cell-depleted tonsillar MNC suspensions, partially restore*.! the predicted linearity (Fig. 4B). These observations demonstrate that adherent cells (presumably macrophages or dendritic cells) and/or soluble IL-1 provide essential signals for supporting the secretion of IFN-y. In fact, the paucity of macrophages and dendritic cells present in our tonsillar suspensions, and the comparatively low levels of IL-1-secreting cells detected in these suspensions (unpublished observations) are compatible with this interpretation.
10 5
