Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom
Received December 9, 2002; accepted January 24, 2003
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ABSTRACT |
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Key Words: allergens; irritants; local lymph node assay; nonradioisotopic; flow cytometry.
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INTRODUCTION |
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There has been some interest in the development of alternative end points for the LLNA, including those that might obviate the requirement for radioactive labeling, particularly in certain regions where the use of radioisotopes is regulated strictly. Among the alternative methods that have been examined are the assessment of lymph node activation as a function of changes in lymph node cellularity or lymph node weight (Homey et al., 1998) and analysis of changes in the relative frequencies in lymphocyte subpopulations in lymph nodes draining the site of topical exposure. Although contact hypersensitivity is a T lymphocyte-mediated reaction, it has been demonstrated that the draining lymph nodes of mice exposed topically to contact allergens display an increased frequency of B lymphocytes measured as a function of B220+ cells and IgG/IgM+ cells (Gerberick et al., 1997
, 1999
, 2002
; Manetz and Meade, 1999
; Sikorski et al., 1996
). It has been proposed, therefore, that analysis of induced changes in the frequency of B220+ cells within draining lymph nodes may provide an adjunct or supplementary read-out for the LLNA (Gerberick et al., 2002
). Another alternative end point that has been investigated is the use of a nonradioisotopic analogue of thymidine, bromodeoxyuridine (BrdU), that can be detected using an anti-BrdU antibody (Takeyoshi et al., 2001
). Limited experience of this method suggests that it may have some potential for the identification of contact allergens, although the sensitivity with respect to allergen-induced fold changes in BrdU incorporation is less than that achieved using radiolabeled thymidine.
A method for the analysis of cell turnover that is gaining some popularity in the fields of immunology and cell biology is tracking cell division using the membrane permeable molecule 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSASE) (Givan et al., 1999; Lyons, 1999
; Parish, 1999
). The nonfluorescent highly membrane-permeable diacetate form is taken up readily by cells (Parish, 1999
). Once inside the cell, however, intracellular esterases cleave the diacetate groups and the resultant fluorescent moiety, 5,6-carboxyfluorescein succinimidyl ester (CFSE), is retained within the cell. Due to the high reactivity of the succinimidyl group with amines, a proportion of these molecules couple to cytosolic proteins that cannot easily escape from the cell, such that the cells are labeled stably. Indeed, the fluorescent labeling is so stable that cells labeled using this methodology for migration studies can be detected up to 6 months after labeling (Lyons, 1999
). Of relevance to the potential application of this technique to monitoring cell division was the observation that upon division of CFSE-labeled lymphocytes, the marker was divided equally between daughter cells in each successive division. Thus, it has been confirmed by BrdU staining that having undergone 1 and 2 divisions, cells express 50% and 25%, respectively, of the CFSE label compared with parental cells (Lyons, 1999
). Up to 10 discrete populations can be detected on the basis of decreasing fluorescence intensity, identifying cells that have undergone up to 10 successive divisions. Again, the mean fluorescence intensity of the cells has been shown to decrease by 50% for each successive division (Lyons and Parish, 1994
). One of the major advantages of this method is that not only can proliferating cells be identified in complex cell mixtures, but also the use of two or three color immunofluorescence allows immunophenotyping of the dividing cells using monoclonal antibodies conjugated to other fluorescent dyes.
Despite the fact that this technique has been established for some time, it has as yet found little application in toxicology. In the current investigations we have examined the potential utility of this method for the identification of cells proliferating in response to chemical allergens. Dose-response studies have been performed in BALB/c strain mice with two potent contact allergens, oxazolone (ox) and 2,4-dinitrochlorobenzene (DNCB). Additional studies have been performed with hexyl cinnamic aldehyde (HCA), a chemical that is currently recommended as a positive control substance in tests for contact sensitization hazard identification (Steiling et al., 2001), and is described variously as a mild to moderately sensitizing chemical or a weak contact allergen (Basketter et al., 2001
; Steiling et al., 2001
). The selectivity of the proliferative response has been assessed by analysis of the response to the nonsensitizing skin irritant, methyl salicylate (MS; Kligman, 1966
). The frequency of CD4+ and CD8+ proliferating cells within the total intact lymphocyte pool and the percentage of total proliferating lymphocytes have been analyzed. These studies have been conducted according to the standard LLNA exposure protocol; however, BALB/c strain mice, rather than the recommended CBA strain mice, have been used. This strain of mouse has been shown previously to give comparable proliferative responses to those achieved in CBA strain mice using reference contact allergens, including HCA (Woolhiser et al., 2000
). The results have been compared with incorporation of radiolabeled thymidine in vitro in cells cultured concurrently.
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MATERIALS AND METHODS |
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Chemicals.
-HCA (85% pure) was obtained from Aldrich Chemical Co. (Gillingham, Dorset, UK). DNCB (97% pure), 4-ethoxymethylene-2-phenyloxazol-5-one (ox; 80% pure), and methyl salicylate (MS, 99% pure) were all purchased from Sigma Chemical Co. (Poole, Dorset, U.K.). All chemicals were dissolved in 4:1 v/v acetone:olive oil (AOO) and were prepared freshly, prior to dosing. Chemicals were dispensed in a fume cupboard, with gloves for dermal protection.
Sensitization regimen.
Animals (n = 3 to 5 per group for chemical; n = 5 per group for vehicle) received 25 µl of various concentrations of chemical in vehicle (AOO), or AOO alone, on the dorsum of both ears, daily for three consecutive days. In some experiments, animals (n = 3) received a single topical application of 25 µl of chemical in vehicle (AOO) on the dorsum of both ears. Protective clothing for licensees handling animals included mask, gloves, and safety spectacles.
Preparation of draining lymph node cells.
Three or five days after the initiation of exposure, draining auricular lymph nodes were excised into RPMI 1640 (GibcoBRL, Renfrewshire, U.K.) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine (GibcoBRL), 400 µg/ml streptomycin, and 400 µg/ml ampicillin (Sigma) (RPMI-FCS) and pooled on an experimental group basis. A single cell suspension of lymph node cells (LNC) was prepared under aseptic conditions by mechanical disaggregation through 200-mesh stainless steel gauze. Viable cell counts were performed by exclusion of 0.5% trypan blue and LNC resuspended in phosphate-buffered saline (PBS) at 107 cells/ml.
CFSE labeling.
Single cell suspensions of LNC in PBS were seeded into Falcon tubes (107 cells/tube). To each 1-ml aliquot of cells, 10 µl of 500 µM CFSASE diluted in PBS (Cambridge Bioscience, Cambridge, U.K.) was added and cells were incubated at 37°C for 15 min. In order to stop the reaction, 100 µl of FCS was added per ml of cells, the cells washed twice in RPMI-FCS, and resuspended at 107 cells/ml in RPMI-FCS. In some experiments, allergen-activated LNC were cultured in the presence of 2 µg/ml of the T-cell mitogen concanavalin A (con A; Sigma). Cell preparations were cultured in the dark at 37°C in a humidified atmosphere of 5% CO2 in air at 107 cells/ml for 96 h and harvested by centrifugation for FACS analysis.
Thymidine incorporation in vitro.
Parallel cell cultures were established for measurement of proliferation by radiolabeled thymidine incorporation. Cells were seeded in quintuplicate into 96-well, round-bottomed microtiter plates (106 cells/well) and cultured for 96 h at 37°C in a humidified atmosphere of 5% CO2 in air. For the final 24 h of culture, cells were pulsed with 2 µCi [3H]-methyl thymidine (specific activity 2 Ci/mmol Amersham International, UK). Culture was terminated by automated cell harvesting and incorporation of thymidine was measured by ß-scintillation counting.
Flow cytometric analyses.
Fluorescence staining for CD4+ and CD8+ cells was carried out in 96-well, round-bottomed microtiter plates. Aliquots of 2 x 106 cells were incubated with optimal (typically, 1 µg per 106 cells) concentrations of Tri-color (TC) conjugated rat monoclonal antimouse CD4 or CD8 antibodies (anti-CD4 antibody, clone S3.5; anti-CD8 antibody, clone 3B5; Caltag, Burlingame, CA). It was not possible to dual stain proliferating cells for CD4 and CD8 expression as CFSE has such a wide emission spectrum and range of intensities that the use of the FL2 channel is technically very difficult. Cells were incubated for 30 min on ice, washed, and resuspended in FACS buffer to a concentration of 107 cells/ml. Cells were fixed in 1% formaldehyde in PBS prior to analysis by flow cytometry. Labeled populations (CFSE labeled cells stained for either CD4 or CD8 expression) were analyzed, using a FACsCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and CellQuest Pro software. An intact lymphocyte cell gate was set on the basis of cell size (forward scatter; FSC) and cell granularity (side scatter; SSC) (Chrest et al., 1993). This region was unaltered between samples analyzed on the same day. These events were displayed on an FL1CFSE versus FL3CD4/CD8 log dot plot. For each sample, data from 1.5 x 105 cells contained within the intact lymphocyte gate were acquired. Regions were drawn to define CD4+ and CD4- and CD8+ and CD8- populations, based on FL3 (TC intensity). Cells proliferating in response to con A, which formed 10 discrete populations according to number of cell divisions completed, and measured as a function of reducing CFSE intensity, were used to define regions representing allergen proliferating cells through divisions 1 to 9. In addition, cells were analyzed on the basis of CFSE F1 intensity versus FSC (total lymphocyte pool), in order to enumerate the percentage of all proliferating lymphocytes (T cell and non-T cell). Data were expressed as the percentage of CD4+ or CD8+ proliferating cells in the total lymphocyte population, or as the percentage of all proliferating cells in the total lymphocyte population. Data are expressed as the percentage of cells per division or as a summation of the percentage of cells that had passed through each division. To derive the latter data set, the percentage of cells that had passed through one division was calculated by summation of the cells found in divisions 1, 2, 3, 4, 5, 6, 7, 8, and 9; the percentage of cells which had passed through 2 divisions was calculated by summation of the cells found in divisions 2, 3, 4, 5, 6, 7, 8, and 9; the percentage of cells which had passed through 3 divisions was calculated by summation of the cells found in divisions 3, 4, 5, 6, 7, 8, and 9, and so on. Allergen-induced changes in percentages of proliferating cells in each division were considered significant if differences were greater than mean plus 3 x SD for the recorded values for cells derived from vehicle-treated animals.
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RESULTS |
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FACS profiles of CD4+ and CD8+ proliferating cells: Comparisons of ox and MS.
In subsequent experiments, dose-response analyses were conducted to compare the frequency of proliferating CD4+ and CD8+ cells in lymph nodes isolated following topical exposure of mice to the potent contact allergen ox, or to the nonsensitizing skin irritant MS. In these experiments, and in all subsequent experiments, mice were dosed according to a standard LLNA-type protocol (Kimber and Basketter, 1992), with animals receiving 25 µl of ox (0.01% to 0.25%), or MS (1.25% to 20%) (both delivered in AOO) on the dorsum of both ears, daily, for three consecutive days, with lymph nodes isolated 5 days after the initiation of exposure. Control mice were exposed to vehicle (AOO) alone. Lymph nodes were pooled for each experimental group: a single cell suspension of LNC prepared, labeled with CFSE, and cultured for 96 h in the absence of further restimulation in vitro. Aliquots of cells were stained with anti-CD4 and anti-CD8 TC labeled antibodies for the enumeration of CD4+ and CD8+ proliferating cells by flow cytometry. Cells in each population have been assigned to cell divisions 1 to 9 using the gates derived for mitogen-activated LNC as described previously (Fig. 1A
). FACS profiles for CD4+ and CD8+ proliferating cells in ox-stimulated LNC and in those derived following treatment with MS are displayed in Figures 2
and 3
, respectively. For ox-treated LNC, a clear dose response effect was observed with respect to increasing percentages of CD4+ and CD8+ cells passing through cell divisions 1 to 9, compared with the relatively low levels of proliferating cells recorded for LNC isolated from vehicle (AOO)-treated mice (Fig. 2
). In contrast, despite exposure to high doses of MS (up to a maximum of 20%) at all concentrations tested, similar profiles of CD4+ and CD8+ cells were detected reaching divisions 1 to 9, as were those observed for LNC derived from vehicle-exposed mice (Fig. 3
).
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DISCUSSION |
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Measurement of cell division as a function of CFSE incorporation correctly identified all three contact allergens, with marked increases in the percentages of both CD4+ and CD8+ proliferating T lymphocytes observed in each case when compared with vehicle-treated controls. For DNCB and HCA, the sensitivity of this technique was comparable with that achieved using the standard LLNA protocol, with significant increases in proliferating cells recorded at 0.05% DNCB and 5% HCA; doses close to the EC3 values obtained in the standard LLNA using CBA strain mice. For ox, however, the measurement of cell turnover using CFSE incorporation appeared to be rather less sensitive, with the numbers of proliferating cells returning to background levels (similar to those observed for cells derived from concurrent vehicle-treated control animals) following treatment of mice with 0.01% ox, particularly for CD8+ proliferating cells. This concentration of chemical is some orders of magnitude higher than the EC3 value derived using a conventional LLNA (Loveless et al., 1996). The selectivity of this end point was determined using the nonsensitizing skin irritant MS (Kligman, 1966
). At all concentrations tested, this chemical failed to stimulate increased cell division measured as a function of CFSE incorporation. In international interlaboratory trials of the standard LLNA, the same application concentrations of MS were also uniformly negative (5 out of 5 laboratories) with respect to failing to induce an SI of 3 or above at any test concentration (Kimber et al., 1995
). However, 3 out of 5 laboratories utilized a protocol where individual animals lymph nodes were processed, rather than lymph nodes pooled on an experimental group basis, and we were therefore able to conduct statistical analyses on LLNA responses. Two out of these three laboratories found a statistically significant increase in thymidine incorporation at the maximum dose of MS tested (20%); in contrast, there was no evidence for any increase in proliferation measured as a function of CFSE incorporation.
One of the advantages of this flow cytometric method for the measurement of cell turnover is that the phenotype of proliferating cells can be assessed simultaneously. Interestingly, all three contact allergens stimulated increased cell division in both the CD4+ and the CD8+ T-lymphocyte populations. These data are consistent with previous experience in which the relative contributions of CD4+ and CD8+ cells to ox-induced proliferative responses were assessed using negative selection (complement depletion) (Kimber et al., 1991). In those experiments, in vitro incorporation of radiolabeled thymidine was measured following depletion of CD4+ or CD8+ cell populations, revealing that both populations contributed to the proliferative response induced by ox. The fact that in the current experiments fewer proliferating CD8+ cells were recorded than CD4+ cells is presumably a reflection of the fact that in resting and in allergen-activated lymph nodes derived from BALB/c strain mice, approximately two thirds of the total T-lymphocyte population are CD4+ cells (Dearman et al., 1996
). Regardless of the fact that more proliferating CD4+ T cells than CD8+ T cells were recorded, and considering the current debate as to phenotype of the cellular effectors of contact allergy (Kimber and Dearman, 2002
), these data clearly demonstrate that both cell populations are dividing in response to both potent and relatively weak allergens. It is of interest that for DNCB- and HCA-stimulated LNC, the total percentage of proliferating cells within the intact lymphocyte pool was approximately equivalent to the summation of the proliferating CD4+ and CD8+ cells, suggesting that these cells accounted for the majority of proliferating cells within the draining lymph node. These data suggest therefore that the reported increases in the frequency of B cells (B220+/IgG+/IgM+) observed in the draining lymph node following topical exposure to similar concentrations of these chemicals (0.25% DNCB and 50% HCA) may be due largely to the preferential accumulation of B lymphocytes within the allergen-activated lymph node, rather than B-cell division (Gerberick et al., 2002
). However, independent experiments in which the numbers of proliferating B220+ cells in resting lymph nodes are compared with those found in allergen-activated lymph nodes are required to formally demonstrate this hypothesis. Following longer exposure protocols where there is the opportunity for secondary follicles and germinal centers to develop, then undoubtedly B lymphocytes will contribute significantly to overall LNC turnover (Kimber et al., 1991
).
With respect to the possible utility of this end point in the LLNA, it is likely that the most sensitive (and relatively simple) configuration would be the assessment of the percentage of proliferating cells in the total intact lymphocyte pool. This would obviate the requirement for immunophenotyping, which, although potentially a powerful tool for exploring mechanistic aspects of skin sensitization, adds considerably to the complexity of the method. Clearly there is a need for cell culture and for relatively sophisticated analytical techniques for the successful application of this method. Furthermore, LNC proliferation using the end point of CFSE incorporation must be assessed in the freshly isolated viable cell pool; thus, there is no opportunity for longitudinal comparisons and batch analyses, as there is when the end point is the production of a soluble factor by cultured draining LNC, such as various cytokines including interleukin (IL) 2, interferon or IL-12 (Dearman et al., 1999
; Hatao et al., 1995
).
In common with other methods that rely upon measurement of ex vivo parameters of lymph node activation, the assessment of cell turnover as a function of CFSE incorporation is of somewhat lesser sensitivity compared with measurement of thymidine incorporation in vivo. Thus, exposure to potent contact allergens such as DNCB provoked a maximal three-fold increase in the frequency of B220+ cells (Gerberick et al., 2002). A somewhat larger dynamic range was recorded for the end point of CFSE expression; thus, maximal increases in lymphocyte division of between 12 and 20 fold were observed for DNCB and ox, respectively. In a standard LLNA with thymidine incorporation, maximal SIs in excess of 75 have been recorded for potent allergens including DNCB (Loveless et al., 1996
). One of the reasons why this latter method yields more vigorous responses is because activity is assessed on a whole lymph node basis. Thus, not only is the fact that there is an increase in the frequency of proliferating cells taken into account, but also the fact that there is a marked increase in total lymph node cellularity following exposure to allergen as described above. In some methods where in vitro parameters of lymph node activation, such as incorporation of radiolabeled thymidine, are assessed, a factor for increases in total lymph node cellularity is included in the calculation of the SI (de Jong et al., 2002
). If this approach is used, however, it is very important to ensure that the total cell yields of LNC populations are measured accurately and consistently in both allergen-treated and -untreated groups.
In summary, these data demonstrate that it is possible to measure allergen-induced lymphocyte proliferation by flow cytometry using the stable cytosolic fluorescent dye CFSE. This method can be used also to provide information as to the phenotype of the proliferating LNC, although in the context of a potential supplementary end point for the LLNA, assessment of the total percentage of proliferating cells within the intact lymphocyte pool probably represents the most sensitive and relatively simple end point. Further evaluation will be required, to confirm or otherwise, the sensitivity and selectivity of this technique using a range of allergens and nonallergens, although current experience suggests that this method is sufficiently sensitive to identify potent skin sensitizing chemicals.
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NOTES |
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