Toxicology, 69 ( 1991 ) 209-218 Elsevier Scientific Publishers Ireland Ltd.
209
Draining lymph node cell activation in guinea pigs: Comparisons with the murine local lymph node assay T h o m a s M a u r e r a and Ian K i m b e r b "Toxicolqgy Services, Ciba-Geigy Ltd. 4002 Basel (Switzerland) and l'lCl Central To.vicologv LahoratolT. A lderley Park. Maccle,~)qeld. Cheshire SKIO 4TJ (U. K. ). (Received April 15th, 1991: accepted August 12th, 1991 )
Summary The local lymph node assay in the mouse is a novel predictive test for the identification of contact sensitizing chemicals. The purpose of the studies described was to determine whether a similar local lymph node assay could be performed successfully in guinea pigs; currently the species of choice for assessment of sensitizing potential for regulatory purposes. Ten sensitizing chemicals (oxazolone, picryl chloride, 2,4-dinitrofluorobenzene, benzocaine, cinnamic aldehyde, 2,4,-dinitrothiocyanobenzene, p-nitrosodimethylaniline, formaldehyde, p-phenylenediamine and cyanuric chloride) and equal concentrations of sodium lauryl sulphate were examined in a guinea pig local lymph node assay. Animals received three consecutive daily applications of various concentrations of the test chemical on the dorsum of both ears. Control animals were untreated. Five days following the initiation of exposure, draining auricular lymph nodes were excised and weighed. Suspensions of lymph node cells (LNC) were prepared and cultured for 24 or 48 h and proliferation measured by incorporation of [3H]thymidine. Exposure to at least one concentration of all sensitizing chemicals, other than benzocaine, induced proliferation by draining LNC. Responses were higher at 24 h rather than 48 h. Evidence is presented that guinea pig LNC proliferation may be enhanced or maintained by addition to culture of an exogenous source of the T cell growth factor interleukin 2 (IL-2). Draining lymph node weight was increased following exposure to some sensitizing chemicals but, compared with LNC proliferation, provided a less sensitive correlate of lymph node activation. Exposure to sodium lauryl sulphate failed to induce changes in either lymph node weight or LNC proliferation. Data are compared with three-day murine local lymph node assays performed concurrently. The available information indicates that the local lymph node assay may be performed in guinea pigs.
Key words." Contact sensitization: Predictive tests: Guinea pig and mouse; Local lymph node assay: Lymphocyte proliferation
Introduction In recent years there has been considerable interest in the development of novel murine test methods for the identification of contact allergens. The elicitation of contact sensitization in the mouse can be measured as a function of challengeCorre,spondence to. Thomas Maurer 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
210
induced increases in ear thickness [1] and several assays based upon this technique have been described. Included among these are the mouse ear swelling test (MEST) [21, the mouse ear sensitization assay (MESA) [3] and a variant of these in which mice are fed on a diet supplemented with vitamin A [4]. An alternative approach developed in one of our laboratories (ICI), the murine local lymph node assay, has as its theoretical basis the fact that topical exposure to skin allergens results in the activation of draining lymph nodes [5-8]. In practice, sensitizing activity is evaluated by measurement of draining lymph node cell (LNC) proliferation. The results of recent comparative studies indicate that the local lymph node assay is robust and provides an accurate and cost-effective alternative for the identification of at least moderate and strong skin allergens [7,81. Despite new approaches such as these, the guinea pig continues to be the species of choice for the formal assessment of contact allergic potential. A variety of test methods are available [9,10], the most widely applied of these being the guinea pig maximization test [ 11,12] and the occluded patch test of Buehler [13,14]. In all such methods an evaluation of skin sensitizing activity is derived from visual assessment of challenge-induced erythema. As the local lymph node assay offers a number of important advantages compared with currently available guinea pig tests, while historically, the majority of information available on predictive analysis is derived from guinea pig studies, we considered it relevant to examine whether the local lymph node assay could also be performed successfully in this species. Comparative studies of 11 chemicals, in which local lymph node assays have been performed in both the mouse and guinea pig, are described. Materials and methods
Chemicals Ethyl-p-aminobenzoate (benzocaine), 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone), 2,4,6-trinitrochlorobenzene (picryl chloride) and p-phenylenediamine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cinnamic aldehyde, 2,4-dinitrofluorobenzene (DNFB), p-nitrosodimethylaniline and 2,4,6-trichloro1,3,5-triazine (cyanuric chloride) were obtained from Aldrich Chemical Co. (Gillingham, U.K.). Formaldehyde and sodium lauryl sulphate were purchased from BDH (Poole, Dorset, U.K.). 2,4-Dinitrothiocyanobenzene (DNTB) was supplied by ICN Pharmaceuticals (Plainview, New York, U.S.A.). Chemicals were dissolved in 4:1 acetone:olive oil (AOO) or in dimethylformamide (sodium lauryl sulphate) for mouse studies, and in DAE433 (dimethylacetamide:acetone:ethanol; 4:4:3) for guinea pig studies. Animals Young adult (6-8 weeks old) CBA/Ca strain mice (Barriered Animal Breeding Unit, Alderley Park, U.K.) were used. Adult Dunkin-Hartley-Pirbright (Tif:DHP) guinea pigs (300-350 g; Animal Production, Ciba-Geigy, 4332 Stein, Switzerland) were used.
211
Murine local lymph node assay Assays were performed as described previously [5]. Briefly, groups of mice (n = 3) received 25txl of various concentrations of the test chemical, or of vehicle alone, on the dorsum of both ears. Treatment was performed daily for 3 consecutive days. One day following the final application, mice were killed and the draining auricular nodes removed under aseptic conditions. A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200-mesh stainless steel gauze. Lymphocyte suspensions were washed once in phosphate-buffered saline (PBS; pH 7.2) and resuspended in RPMI-1640 culture medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 400 t~g/ml ampiciilin, 400 t~g/ml streptomycin and 10% heat-inactivated (56°C for 30 min) foetal calf serum (RPMI-FCS). Viable cell counts were performed by exclusion of 0.5% trypan blue and the cell concentration adjusted to working values (2.5 x 106 cells/ml) in RPMI-FCS. Lymphocyte suspensions were seeded into 96-well microtitre plates at a concentration of 1.2 x 106 cells/well (5 wells per group) and cultured for 24 h at 37°C in a humidified atmosphere of 5% CO 2 in air with 2 /~Ci of [methyl-3H]thymidine (Amersham International, Amersham, U.K.). Culture was terminated by automatic cell harvesting and [3H]TdR incorporation was determined by ~-scintillation counting. Guinea pig local lymph node assay Assays were performed as described above but with the following modifications. Guinea pigs (n = 3) received 50/~1 of the test material on the dorsum of each ear. Treatment was performed daily for 3 consecutive days and animals killed 5 days following the initiation of exposure. Draining lymph nodes were excised and weighed. LNC cultures (2 × 105 cells/well) were maintained for 24 h and 48 h. Fortyeight-hour cultures were established in duplicate and maintained in the presence or absence of an exogenous source of human recombinant IL-2 (3 t~g/ml; Amersham International, Amersham, U.K.). In the case of 48-h cultures [3H]thymidine was added after 24 h. Cultures were terminated by automatic cell harvesting and thymidine incorporation measured by/3-scintillation counting. In preliminary experiments it was observed that exposure to the vehicle (AOO) alone under these conditions caused no change in any parameter measured compared with naive animals. For this reason, in subsequent experiments described here control values are derived from untreated guinea pigs. Results
Guinea pig studies Ten chemicals known to possess skin sensitizing activity were used to evaluate local lymph node assay responses in guinea pigs. Each of these chemicals was also assessed in a standard guinea pig maximization test (Laboratory A) using the method described by Magnusson and Kligman [11,12]. In the case of oxazolone, DNFB, DNTB, picryl chloride, cyanuric chloride, p-nitroso-dimethylaniline and pphenylenediamine, all sensitized guinea pigs exhibited elicitation reactions following
2,4,6-trichloro- 1,3,5-triazine (cyanuric chloride)
(picryl chloride)
2,4,6-trinitrochlorobenzene
2,4-dinitrothiocyanobenzene (DNTB)
2,4-dinitrofluorobenzene (DNFB)
4-ethoxymethylene-2-phenyloxazol-5-one ( O x a z o l o n e )
Chemical
19.72 41.69 + 12.37 420.74 4- 12.30 18.54 ± 4.29 10.90 4- 2.96
5 0 0.5 1 2 5 2.10 ± 7.62 ± 8.81 ± 4.81 ± 8.77 + 4.70 + 26.90 ± 27.14 ± 36.45 ± 25.46 ±
0 0.5 I 2 5 0 0.5 1 2 5
4± 4±
3.08 8.85 20.86 16.60
0 0.5 1 2
5.68 2.78 1.84
1.15 3.94
0.93
1.72 1.89 0.65
0.25
0.48 3.88
2.99
0.59 4.14 7.83 3.08
1.94 2.60 2.86
19.58 422.93 440.89 ±
1 2 5
2.42 1.53
3.73 ± 5.45 4-
5~77 7.76 5.42
5.72
4.18
3.63 4.20 2.29
-
12.27 10.97 6.45
7.32
6.40
2.87 6.77 5.39
5.25 6.15 10.96
1.46
± ± 4±
0.34 1.55 2.94 0.18
1.49 ± 0.45 6.08 ± 3.54 4.99 ± 2.59
0.59 ± 0.16 1.98 + 0.56
2.21 4- 0.73
1.29 + 0.68 n.d. 1.75 ± 0.44
1.10 + 0.14
0.51 ± 0.19 0.40 4- 0.14 0.13 4- 0.03
0.30 ± 0.13 0.22 ± 0.09
3.75 4- 1.44
0.88 5.05 7.24 3.11
4.77 4- 1.92 7.19 ± 2.54 17.57 ± 5.52
0.95 ± 0.05 1.10 ± 0.16
2.53 10.31 8.46
3.36
2.01
1.17 1.59
-
1.70 1.33 0.43
0.73
4.26
5.74 8.23 3.53
5.02 7.57 18.49
1.15
M e a n ct/min SI ± S.D. x 10 -3
M e a n * ct/min + S . D . x 10 -3
SI
48 h
[3H]TdR incorporation
± ± ± ±
0.32 7.73 6.14 0.67
10.53 ± 1.23 21.89 ± 2.82 13.50 ± 2.89
1.33 ± 1.10 8.71 ± 1.84
4.99 4- 0.57
1.06 ± 0.25 n.d. 2.73 ± 0.16
1.24 ± 0.19
0.94 4- 0.18 0.71 ± 0.24 0.30 ± 0.18
0.26 ± 0.09 0.32 ± 0.18
8.87 4- 1.80
1.01 31.03 21.36 13.42
13.42 ± 5.31 24.60 4- 5.05 71.52 ± 8.47
1.11 ± 0.49 2.21 ± 1.24
M e a n ct/min S.D. × 10 -3
7.92 16.46 10.15
6.55
4.02
0.85 2.20
-
3.62 2.73 1.15
1.23
8.78
30.72 21.15 13.29
12.09 22.16 64.43
1.99
SI
AND CHANGES
48 h + IL-2
LNC PROLIFERATION
24 h
L N C proliferation
0 0.5
Concentration (% w/v)
L O C A L L Y M P H N O D E A S S A Y R E S P O N S E S IN T H E G U I N E A P I G : D R A I N I N G
TABLE I
4± 4±
1.2 3.2 8.7 3.2
1.1 1.3
28.1 ± 2.8 34.8 ± 2.9 21.7 ± 0.9
11.1 ± 0.9 16.5 ± 1.8
32.2 + 2.9
13.7 ± 0.9 16.6 ± 0.7 23.3 ± 1.0
14.5 ± 0.8
26.1 4- 1.6 13.2 ± 0.6 16.8 ± 1.5
10.2 ± 22.9 ±
15.2 ± 1.2
15.2 36.7 60.7 36.7
18.5 ± 1.5 20.9 ± 1.0 38.1 ± 6.6
16.6 ± 1.4 15.2 ± 1.0
Mean LN weight (mg) ± S.E.
IN N O D E W E I G H T
tJ
n.d.
1.49 1.69
4.93 43.83 -42.59 ± 0.97
0.89 1.53
4.04 44.71 +
0.26 40.37 40.33 ±
2.10 4-
2.02
0.71
3.23 ±
1.22 0.95 0.64
0.42 0.40 0.69
444-
0.42 40.45 4-
0.44
-4-
1.70 1.32 1.35 1.86
0.78 1.61 1.63
0.04 0.24 1.41 0.31
± ± ± 4-
1.17
2.54 1.52 2.11
6.11 48.78 413.77 ±
2.67 5.27 7.06
16.12 4- 3.56 31.79 ± 8.62 42.59 ± 13.88
0.24
0.13 0.09
0.17 0.30
0.53
0.84 2.22
6.74 46.96 +
2.75
1.60 1.24 2.58 2.60
12.91
30.12 ±
± 2.73 ± 0.96 4- 2.37 4- 11.40
15.55
n.d.
n.d.
n.d.
n.d.
4.92 5.16 6.63 30.23
1.64 1.74 0.89
2.41
1.82 4- 0.77
± 0.28 4- 0.22 4- 1.45
11.52
± 0.07
0.62 0.90 0.74 2.40
3.94 9.79 9.48
3.44 4.10 9.25
LNC, l y m p h node cells. * M e a n values derived f r o m at least 8 culture wells.
n.d., not d e t e r m i n e d . n.a., not applicable.
0
s o d i u m lauryl sulphate 0.5
0 0.5
formaldehyde
1.78 8.39
6.03 ± 16.61 4-
0 0.5
8.01
32.65 .4-
p-phenylenediamine
0.04 1.23 3.75 4.87
2.10 7.22 8.62 19.42
444.4-
0 0.5
1.25 0.54 0.56
2.61 42.76 41.42 4-
1 2 5
0.28 1.09
1.59 + 3.83 4-
5.62
16.13 4-
5 0 O. 5
5.58 5.84
0.21 1.45
1.40 45.51 ± 13.71 .413.27 ±
0 0.5 1 2
p-nitroso-dimethylaniline
et h y l - p - a m i n o b e n z o a t e (benzocaine)
cinnamic aldehyde
0.62 0.88 0.79
1.07
1.24
0.78 0.79 1.09
0.91 1.32 2.04
1.03
6.12
1.05 1.35 6.14
2.94
1.45 1.19 3.87 ±
± 4441.33
0.09 1.00 0.55 2.83
1.51 2.04
0.17 ± 0.13 ± 0.29 ±
0.33 40.17 ±
2.92 4-
1.81 44.29 45.35 4-
1.94 4-
0.08 0.12 0.19
0.09 0.05
0.38
0.84 1.36 1.48
1.09
6.73 ± 2.45 51.47 .4- 10.55 67.83 ± 9.02
6.96 46.51 4-
73.27 + 28.83
16.78 4- 4.92 25.30 .4- 5.07 97.50 4- 19.95
n.d.
n.d. n.d. n.d.
n.d.
ll.d.
1.02 1.53 2.00 3.27 3.49
0.88
0.52 0.39
0.52
1.51
0.93 2.21 2.76
0.97 7.40 9.75
0.94
n.a.
n.a.
n.a.
n.a.
[1.a.
3.42
1.50 1.96 3.21
.4± .4±
0.9 2.7 1.2 0.6
+ + 444-
0.7 4.4 2.3 7.4 0.4
8.4 + 0.5
10.6 4- 0.4 8.7 4- 0.7
9.2 + 0.2 8.0 + 0.5
10.3 4- 0.7
10.9 4- 0.3 8.2 4- 0.6 8.8 + 0.4
9.5 4- 0.6
12.4 4- 0.4 21.5 + 1.7 21.6 ± 3.9
I 1.9 4- 1.0 13.9 .4- 1.2
12.6 24.5 20.8 36.3 49.9
10.4 .4- O.l 8.8 + 0.7 5.9 4- 0.5
8.5 4- 0.5 8.1 .4-0.5
11.8 4- 0.8
9.8 15.4 12.5 11.3
214 challenge. Formaldehyde and cinnamic aldehyde induced reactions in 9 out of 10 and 8 out of 10 test guinea pigs, respectively. Responses to benzocaine were somewhat weaker with 6 out of 20 animals positive (data not presented). In addition, local lymph node assays were performed with sodium lauryl sulphate which is considered not to cause allergic contact dermatitis. In local lymph node assays guinea pigs received three consecutive daily applications of various concentrations of the test chemical. Animals were killed 5 days following the initiation of exposure and draining LNC cultured for 24 h or 48 h. In 48 h assays culture was performed in the presence or absence of an exogenous source of IL-2. Results are presented as the mean [3H]TdR incorporation for each experimental group and in the form of stimulation indices relative to control values (Table I). The mean lymph node weight for each experimental group is also recorded. With the exception of benzocaine, at least one concentration of each sensitizing chemical tested induced an elevation of proliferation in 24 h assays. The vigour of responses varied. Substantial activity was observed with the majority of allergens examined; stimulation indices of greater than four were recorded with at least one concentration of all sensitizers other than benzocaine and formaldehyde. In most instances dose-response relationships were evident, although with DNTB and cyanuric chloride there was some indication of inhibition with higher test concentrations. An examination of the results recorded for LNC isolated from untreated guinea pigs reveals some variability among control values. In 24 h cultures the mean [3H]TdR incorporation for control populations varied from 1.59-6.03 ct/min × 10 -3. It is clear, therefore, that, in this series of experiments, the use of stimulation indices for comparative evaluation of responses requires care. In most instances reactivity measured following 48 h culture was lower and in some cases, such as with DNTB, considerably lower. However, proliferative activity was maintained or enhanced in cultures supplemented with IL-2. The exception was cultures of LNC derived from DNTB-treated animals where 48 h values were low in either the presence or absence of IL-2, possibly as the result of a technical failure. Interleukin 2 failed to influence materially [3H]TdR incorporation by LNC prepared from untreated animals. In no circumstances did topical exposure to sodium lauryl sulphate at equivalent concentrations induce proliferative activity by draining LNC. As the data in Table I reveal, in most instances the induction of LNC proliferative activity was accompanied by an increase in draining lymph node weight. With some sensitizers, however, increases in node weight were either modest (cinnamic aldehyde) or absent (benzocaine, formaldehyde). Exposure to sodium lauryl sulphate failed to influence draining lymph node weight. Mouse studies
All chemicals, with the exception of benzocaine, were also evaluated using the murine lymph node assay. In this series of experiments mice received three consecutive daily applications of the test material and the proliferative activity of draining LNC was evaluated 1 day following the final exposure. Responses following culture for 24 h in the absence of IL-2 were measured.
215
TABLE II LOCAL LYMPH N O D E ASSAY IN THE MOUSE. D R A I N I N G LNC P R O L I F E R A T I O N Chemical
Concentration (% w/v)
LNC proliferation [3H]TdR incorporation mean* ct/min ± S.D. x 10 -3
4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone)
0 0.5
2,4-dinitrofluorobenzene (DNFB)
2 0 0.5
2,4-dinitrothiocyanobenzene (DNTB)
2 0 0.5
2,4,6-trinitrochlorobenzene (picryl chloride)
2 0 0.5
2,4,6-trichloro-1,3,5-triazine (cyanuric chloride)
2 0 0.5
1.12 97.92 104.21 93.39 1.43 18.21 22.04 n.d. 1.75 10.08 15.22 18.01 0.98 37.66 64.24 54.71 1.72 22.41 30.78 43.21 1.19 0.98 1.76 3.93 1.47 7.12 15.16 28.98 0.78 2.69 4.11 3.72 1.02 n.d. 0.78 2.39 0.69 |.01 0.74 0.72
cinnamic aldehyde
p-nitroso-dimethylaniline
p-phenylenediamine
formaldehyde
sodium lauryl sulphate
n.d., not determined. LNC, lymph node cells. *Mean values from 5 culture wells.
2 0 0.5 2 0 0.5 2 0 0.5 2 0 0.5 1 2 0 0.5 1 2
± ± ± ± ± ± ±
0.10 4.71 5.89 6.41 0.09 1.14 1.02
± ± ± ± ± ± ± ± ± ± + ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.31 I.I1 0.09 1.78 0.08 2.16 8.41 1.37 0.24 1.72 4.11 3.76 0.08 0.12 0.21 0.62 0.12 0.83 1.05 1.17 0.06 0.14 0.78 0.55 0.13
± ± ± ± ± ±
0.08 0.31 0.04 0.07 0.12 0.03
SI
87.43 93.04 83.38 12.73 15.41
5.76 8.70 10.29 38.43 65.55 55.83 13.03 17.90 25.12 0.82 1.48 3.30 4.84 10.31 19.71 3.45 5.27 4.77
0.76 2.34 1.46 1.07 1.04
216
Thymidine incorporation by LNC isolated from vehicle-treated mice varied between 0.69-1.75 ct/min x 10 -3 (Table II). Exposure of mice to sodium lauryl sulphate failed to elicit a response. With all other chemicals there was evidence of induced proliferative activity following treatment with at least one concentration. Compatible with the results of previous experiments [5], some chemicals, such as oxazolone, picryl chloride and cyanuric chloride, induced high levels of [3H]TdR incorporation at all test concentrations. Less vigorous responses were observed with formaldehyde, cinnamic aldehyde and p-phenylenediamine where maximum stimulation indices of 2.34, 3.30 and 5.27 respectively, were recorded. Discussion
The primary objective of the studies described was to establish whether in practice it is possible to measure local lymph node responses in guinea pigs. In summary the data indicate that responses are maximal following 24 h culture and that they may be enhanced further by the addition of IL-2. Of the sensitizing chemicals examined in the local lymph node assay all except benzocaine induced increased proliferative activity. The failure of benzocaine to elicit a response is of some interest as in initial studies of the murine local lymph node assay this material induced activity in both the in vivo and in vitro versions of the test [5,6]. Subsequently, however, in the context of an interlaboratory comparison, each of four participating laboratories failed to identify benzocaine as a sensitizer on the basis of murine local lymph node assay responses [8]. The reasons for these apparently contradictory results are currently the subject of further investigations. Changes in lymph node weight were also measured in the guinea pig but, in common with experience gained using the murine local lymph node assay [5], proved a less sensitive correlate of lymph node activation. The data recorded, particularly with regard to proliferation by control lymph node cells, suggest that some further optimization of the test method will be necessary, especially if sensitizing potential is to be measured as a function of stimulation indices. Compared with the mouse, little information is available regarding the culture of guinea pig lymphocytes and judicious modification of the method may provide a more precise assessment of proliferative activity. Despite the possible need for further method development, results to date indicate that the local lymph node assay can be performed in the guinea pig. Although this is the first systematic investigation of LNC proliferation in the guinea pig as a means of identifying sensitizing agents, another study in this species did address the question of lymph node activation [15]. In that study the authors sought to assess skin sensitizing potential as a function of pyroninophilia in draining lymph nodes. Using a series of acrylates, a correlation between the frequency of large pyroninophilic cells in sections of draining lymph nodes and contact sensitization was reported [15]. Although, in the context of routine analysis, direct measurement of lymphocyte proliferative activity by thymidine incorporation has clear advantages compared with visual assessment of the number of pyroninophilic cells, the study by Bull et al. [15] does provide confirmatory evidence that lymphocyte activation in the guinea pig correlates with sensitizing potential.
2t7 With the exception of benzocaine, all chemicals examined in the guinea pig were also evaluated in the murine local lymph node assay. While, on the basis of the limited data available, it is premature to draw firm conclusions with respect to relative sensitivity, it would appear that, in general terms, induced proliferative responses in the mouse are somewhat more vigorous. This need not, of course, necessarily reflect differential biological activity and may be attributable to the fact that the measurement of guinea pig LNC proliferative responses has yet to be fully optimized. It may also be relevant that while lymph node responses in guinea pigs were assessed 5 days following the initiation of exposure, in mice a 3-day assay was employed. It is known that chemicals vary with respect to the kinetics of the LNC proliferative response they induce [6] and it is possible that such kinetics also vary between species. It is nevertheless the case that chemicals such as oxazolone, cyanuric chloride, the dinitrobenzene derivatives, picryl chloride and pnitrosodimethylaniline, each induced marked proliferative responses in both the guinea pig and mouse. In both species formaldehyde caused only modest activity. Some differences were apparent. Thus, picryl chloride caused more vigorous proliferation in the mouse than in the guinea pig, while responses to p-phenylenediamine and cinnamic aldehyde were stronger in the guinea pig. A more extensive study with a wider range of chemicals will be required for an accurate comparative analysis of sensitivity. The conclusion that may be drawn from these investigations is that the local lymph node assay can be performed successfully in the guinea pig and that with further optimization of the method this might prove as robust as the murine equivalent. It is not, however, our intention to propose that local lymph node assays are performed in the guinea pig rather than the mouse. The primary advantage of developing this method in the guinea pig is to facilitate comparisons between the local lymph node assay and assessment of sensitizing activity by currently available guinea pig test methods. To date, the interpretation of comparative studies of the sensitivity of the local lymph node assay and conventional predictive test methods has been hampered by the fact that differences may be attributable to either fundamental methodological differences (proliferation at sensitization versus erythema at challenge), or differences in the relative sensitivity of the two species to different chemicals. Now with the development of the guinea pig local lymph node assay there is an opportunity, within a single species, to compare the sensitivity of assays such as the guinea pig maximization test with the local lymph node assay.
References 1 G.L. Asherson and W. Ptak, Contact and delayed hypersensitivity in the mouse. 1. Activesensitization and passive transfer. Immunology, 15 (1968) 405. 2 S.C. Gad, B.J. Dunn, D.W. Dobbs, C. Reilly and R.D. Walsh, Development and validation of an alternative dermal sensitization test: the mouseear swelling test (MEST). Toxicol. Appl. Pharmacol.. 84 (1986) 93. 3 J. Descotes, Identification of contact allergens. The mouse ear sensitization assay. J. Toxicol. Cut. Ocul. Toxic., 7 (1988) 263. 4 J. Maisey and K. Miller, Assessment of the ability of mice fed on vitamin A-supplemented diet to respond to a variety of potential contact sensitizers. Contact Dermatitis, 15 (1986) 17.
218 5 6
7
8
9 10 I1 12 13 14
15
I. Kimber and C. Weisenberger, A murine local lymph node assay for the identification of contact allergens: assay development and results of an initial validation study. Arch. Toxicol. 63 (1989) 274. I. Kimber, J. Hilton and C. Weisenberger, The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis, 21 (1989) 215. I. Kimber, J. Hilton and P.A. Botham, Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler occluded patch test in guinea pigs. J. Appl. Toxicol., 10 (1990) 173. 1. Kimber, J. Hilton, P.A. Botham, D.A. Basketter, E.W. Scholes, K. Miller, M.C. Robbins, P.T.C. Harrison, T.J.B. Gray and S.J. Waite, The murine local lymph node assay: results of an interlaboratory trial. Toxicol. Lett., 5 (1991) 203. T. Maurer, Contact and Photocontact Allergens. A Manual of Predictive Test Methods, Marcel Dekker, Basle, 1983. K.E. Andersen and H.1. Maibach, Contact Allergy-Predictive Tests in Guinea Pigs. Current Problems in Dermatology, Vol. 14, Karger, Basle, 1985. B. Magnusson and A.M. Kligman, The identification of contact allergens by animal assay, the guinea pig maximization test method. J. Invest. Dermatol., 52 (1969) 268. B. Magnusson and A.M. Kligman, Allergic Contact Dermatitis in the Guinea Pig, Charles C Thomas, Springfield, IL, 1970. E.V. Buehler, Delayed contact hypersensitivity in the guinea pig. Arch. Dermatol., 91 (1965) 171. E.V. Buehler, A rationale for the selection of occlusion to induce and elicit delayed contact hypersensitivity in the guinea pig, in K.E. Andersen and H.I. Maibach (Eds.), Contact Allergy Predictive Tests in Guinea Pigs. Current Problems in Dermatology, Vol. 14, Karger, Basle, 1985, p. 39. J.E. Bull, D. Parker and J.L. Turk, Predictive value of assessment of lymph node weight and Tlymphocyte proliferation in contact sensitivity in acrylates. J. Invest. Dermatol., 85 (1985) 403.
209
Draining lymph node cell activation in guinea pigs: Comparisons with the murine local lymph node assay T h o m a s M a u r e r a and Ian K i m b e r b "Toxicolqgy Services, Ciba-Geigy Ltd. 4002 Basel (Switzerland) and l'lCl Central To.vicologv LahoratolT. A lderley Park. Maccle,~)qeld. Cheshire SKIO 4TJ (U. K. ). (Received April 15th, 1991: accepted August 12th, 1991 )
Summary The local lymph node assay in the mouse is a novel predictive test for the identification of contact sensitizing chemicals. The purpose of the studies described was to determine whether a similar local lymph node assay could be performed successfully in guinea pigs; currently the species of choice for assessment of sensitizing potential for regulatory purposes. Ten sensitizing chemicals (oxazolone, picryl chloride, 2,4-dinitrofluorobenzene, benzocaine, cinnamic aldehyde, 2,4,-dinitrothiocyanobenzene, p-nitrosodimethylaniline, formaldehyde, p-phenylenediamine and cyanuric chloride) and equal concentrations of sodium lauryl sulphate were examined in a guinea pig local lymph node assay. Animals received three consecutive daily applications of various concentrations of the test chemical on the dorsum of both ears. Control animals were untreated. Five days following the initiation of exposure, draining auricular lymph nodes were excised and weighed. Suspensions of lymph node cells (LNC) were prepared and cultured for 24 or 48 h and proliferation measured by incorporation of [3H]thymidine. Exposure to at least one concentration of all sensitizing chemicals, other than benzocaine, induced proliferation by draining LNC. Responses were higher at 24 h rather than 48 h. Evidence is presented that guinea pig LNC proliferation may be enhanced or maintained by addition to culture of an exogenous source of the T cell growth factor interleukin 2 (IL-2). Draining lymph node weight was increased following exposure to some sensitizing chemicals but, compared with LNC proliferation, provided a less sensitive correlate of lymph node activation. Exposure to sodium lauryl sulphate failed to induce changes in either lymph node weight or LNC proliferation. Data are compared with three-day murine local lymph node assays performed concurrently. The available information indicates that the local lymph node assay may be performed in guinea pigs.
Key words." Contact sensitization: Predictive tests: Guinea pig and mouse; Local lymph node assay: Lymphocyte proliferation
Introduction In recent years there has been considerable interest in the development of novel murine test methods for the identification of contact allergens. The elicitation of contact sensitization in the mouse can be measured as a function of challengeCorre,spondence to. Thomas Maurer 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
210
induced increases in ear thickness [1] and several assays based upon this technique have been described. Included among these are the mouse ear swelling test (MEST) [21, the mouse ear sensitization assay (MESA) [3] and a variant of these in which mice are fed on a diet supplemented with vitamin A [4]. An alternative approach developed in one of our laboratories (ICI), the murine local lymph node assay, has as its theoretical basis the fact that topical exposure to skin allergens results in the activation of draining lymph nodes [5-8]. In practice, sensitizing activity is evaluated by measurement of draining lymph node cell (LNC) proliferation. The results of recent comparative studies indicate that the local lymph node assay is robust and provides an accurate and cost-effective alternative for the identification of at least moderate and strong skin allergens [7,81. Despite new approaches such as these, the guinea pig continues to be the species of choice for the formal assessment of contact allergic potential. A variety of test methods are available [9,10], the most widely applied of these being the guinea pig maximization test [ 11,12] and the occluded patch test of Buehler [13,14]. In all such methods an evaluation of skin sensitizing activity is derived from visual assessment of challenge-induced erythema. As the local lymph node assay offers a number of important advantages compared with currently available guinea pig tests, while historically, the majority of information available on predictive analysis is derived from guinea pig studies, we considered it relevant to examine whether the local lymph node assay could also be performed successfully in this species. Comparative studies of 11 chemicals, in which local lymph node assays have been performed in both the mouse and guinea pig, are described. Materials and methods
Chemicals Ethyl-p-aminobenzoate (benzocaine), 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone), 2,4,6-trinitrochlorobenzene (picryl chloride) and p-phenylenediamine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Cinnamic aldehyde, 2,4-dinitrofluorobenzene (DNFB), p-nitrosodimethylaniline and 2,4,6-trichloro1,3,5-triazine (cyanuric chloride) were obtained from Aldrich Chemical Co. (Gillingham, U.K.). Formaldehyde and sodium lauryl sulphate were purchased from BDH (Poole, Dorset, U.K.). 2,4-Dinitrothiocyanobenzene (DNTB) was supplied by ICN Pharmaceuticals (Plainview, New York, U.S.A.). Chemicals were dissolved in 4:1 acetone:olive oil (AOO) or in dimethylformamide (sodium lauryl sulphate) for mouse studies, and in DAE433 (dimethylacetamide:acetone:ethanol; 4:4:3) for guinea pig studies. Animals Young adult (6-8 weeks old) CBA/Ca strain mice (Barriered Animal Breeding Unit, Alderley Park, U.K.) were used. Adult Dunkin-Hartley-Pirbright (Tif:DHP) guinea pigs (300-350 g; Animal Production, Ciba-Geigy, 4332 Stein, Switzerland) were used.
211
Murine local lymph node assay Assays were performed as described previously [5]. Briefly, groups of mice (n = 3) received 25txl of various concentrations of the test chemical, or of vehicle alone, on the dorsum of both ears. Treatment was performed daily for 3 consecutive days. One day following the final application, mice were killed and the draining auricular nodes removed under aseptic conditions. A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200-mesh stainless steel gauze. Lymphocyte suspensions were washed once in phosphate-buffered saline (PBS; pH 7.2) and resuspended in RPMI-1640 culture medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 400 t~g/ml ampiciilin, 400 t~g/ml streptomycin and 10% heat-inactivated (56°C for 30 min) foetal calf serum (RPMI-FCS). Viable cell counts were performed by exclusion of 0.5% trypan blue and the cell concentration adjusted to working values (2.5 x 106 cells/ml) in RPMI-FCS. Lymphocyte suspensions were seeded into 96-well microtitre plates at a concentration of 1.2 x 106 cells/well (5 wells per group) and cultured for 24 h at 37°C in a humidified atmosphere of 5% CO 2 in air with 2 /~Ci of [methyl-3H]thymidine (Amersham International, Amersham, U.K.). Culture was terminated by automatic cell harvesting and [3H]TdR incorporation was determined by ~-scintillation counting. Guinea pig local lymph node assay Assays were performed as described above but with the following modifications. Guinea pigs (n = 3) received 50/~1 of the test material on the dorsum of each ear. Treatment was performed daily for 3 consecutive days and animals killed 5 days following the initiation of exposure. Draining lymph nodes were excised and weighed. LNC cultures (2 × 105 cells/well) were maintained for 24 h and 48 h. Fortyeight-hour cultures were established in duplicate and maintained in the presence or absence of an exogenous source of human recombinant IL-2 (3 t~g/ml; Amersham International, Amersham, U.K.). In the case of 48-h cultures [3H]thymidine was added after 24 h. Cultures were terminated by automatic cell harvesting and thymidine incorporation measured by/3-scintillation counting. In preliminary experiments it was observed that exposure to the vehicle (AOO) alone under these conditions caused no change in any parameter measured compared with naive animals. For this reason, in subsequent experiments described here control values are derived from untreated guinea pigs. Results
Guinea pig studies Ten chemicals known to possess skin sensitizing activity were used to evaluate local lymph node assay responses in guinea pigs. Each of these chemicals was also assessed in a standard guinea pig maximization test (Laboratory A) using the method described by Magnusson and Kligman [11,12]. In the case of oxazolone, DNFB, DNTB, picryl chloride, cyanuric chloride, p-nitroso-dimethylaniline and pphenylenediamine, all sensitized guinea pigs exhibited elicitation reactions following
2,4,6-trichloro- 1,3,5-triazine (cyanuric chloride)
(picryl chloride)
2,4,6-trinitrochlorobenzene
2,4-dinitrothiocyanobenzene (DNTB)
2,4-dinitrofluorobenzene (DNFB)
4-ethoxymethylene-2-phenyloxazol-5-one ( O x a z o l o n e )
Chemical
19.72 41.69 + 12.37 420.74 4- 12.30 18.54 ± 4.29 10.90 4- 2.96
5 0 0.5 1 2 5 2.10 ± 7.62 ± 8.81 ± 4.81 ± 8.77 + 4.70 + 26.90 ± 27.14 ± 36.45 ± 25.46 ±
0 0.5 I 2 5 0 0.5 1 2 5
4± 4±
3.08 8.85 20.86 16.60
0 0.5 1 2
5.68 2.78 1.84
1.15 3.94
0.93
1.72 1.89 0.65
0.25
0.48 3.88
2.99
0.59 4.14 7.83 3.08
1.94 2.60 2.86
19.58 422.93 440.89 ±
1 2 5
2.42 1.53
3.73 ± 5.45 4-
5~77 7.76 5.42
5.72
4.18
3.63 4.20 2.29
-
12.27 10.97 6.45
7.32
6.40
2.87 6.77 5.39
5.25 6.15 10.96
1.46
± ± 4±
0.34 1.55 2.94 0.18
1.49 ± 0.45 6.08 ± 3.54 4.99 ± 2.59
0.59 ± 0.16 1.98 + 0.56
2.21 4- 0.73
1.29 + 0.68 n.d. 1.75 ± 0.44
1.10 + 0.14
0.51 ± 0.19 0.40 4- 0.14 0.13 4- 0.03
0.30 ± 0.13 0.22 ± 0.09
3.75 4- 1.44
0.88 5.05 7.24 3.11
4.77 4- 1.92 7.19 ± 2.54 17.57 ± 5.52
0.95 ± 0.05 1.10 ± 0.16
2.53 10.31 8.46
3.36
2.01
1.17 1.59
-
1.70 1.33 0.43
0.73
4.26
5.74 8.23 3.53
5.02 7.57 18.49
1.15
M e a n ct/min SI ± S.D. x 10 -3
M e a n * ct/min + S . D . x 10 -3
SI
48 h
[3H]TdR incorporation
± ± ± ±
0.32 7.73 6.14 0.67
10.53 ± 1.23 21.89 ± 2.82 13.50 ± 2.89
1.33 ± 1.10 8.71 ± 1.84
4.99 4- 0.57
1.06 ± 0.25 n.d. 2.73 ± 0.16
1.24 ± 0.19
0.94 4- 0.18 0.71 ± 0.24 0.30 ± 0.18
0.26 ± 0.09 0.32 ± 0.18
8.87 4- 1.80
1.01 31.03 21.36 13.42
13.42 ± 5.31 24.60 4- 5.05 71.52 ± 8.47
1.11 ± 0.49 2.21 ± 1.24
M e a n ct/min S.D. × 10 -3
7.92 16.46 10.15
6.55
4.02
0.85 2.20
-
3.62 2.73 1.15
1.23
8.78
30.72 21.15 13.29
12.09 22.16 64.43
1.99
SI
AND CHANGES
48 h + IL-2
LNC PROLIFERATION
24 h
L N C proliferation
0 0.5
Concentration (% w/v)
L O C A L L Y M P H N O D E A S S A Y R E S P O N S E S IN T H E G U I N E A P I G : D R A I N I N G
TABLE I
4± 4±
1.2 3.2 8.7 3.2
1.1 1.3
28.1 ± 2.8 34.8 ± 2.9 21.7 ± 0.9
11.1 ± 0.9 16.5 ± 1.8
32.2 + 2.9
13.7 ± 0.9 16.6 ± 0.7 23.3 ± 1.0
14.5 ± 0.8
26.1 4- 1.6 13.2 ± 0.6 16.8 ± 1.5
10.2 ± 22.9 ±
15.2 ± 1.2
15.2 36.7 60.7 36.7
18.5 ± 1.5 20.9 ± 1.0 38.1 ± 6.6
16.6 ± 1.4 15.2 ± 1.0
Mean LN weight (mg) ± S.E.
IN N O D E W E I G H T
tJ
n.d.
1.49 1.69
4.93 43.83 -42.59 ± 0.97
0.89 1.53
4.04 44.71 +
0.26 40.37 40.33 ±
2.10 4-
2.02
0.71
3.23 ±
1.22 0.95 0.64
0.42 0.40 0.69
444-
0.42 40.45 4-
0.44
-4-
1.70 1.32 1.35 1.86
0.78 1.61 1.63
0.04 0.24 1.41 0.31
± ± ± 4-
1.17
2.54 1.52 2.11
6.11 48.78 413.77 ±
2.67 5.27 7.06
16.12 4- 3.56 31.79 ± 8.62 42.59 ± 13.88
0.24
0.13 0.09
0.17 0.30
0.53
0.84 2.22
6.74 46.96 +
2.75
1.60 1.24 2.58 2.60
12.91
30.12 ±
± 2.73 ± 0.96 4- 2.37 4- 11.40
15.55
n.d.
n.d.
n.d.
n.d.
4.92 5.16 6.63 30.23
1.64 1.74 0.89
2.41
1.82 4- 0.77
± 0.28 4- 0.22 4- 1.45
11.52
± 0.07
0.62 0.90 0.74 2.40
3.94 9.79 9.48
3.44 4.10 9.25
LNC, l y m p h node cells. * M e a n values derived f r o m at least 8 culture wells.
n.d., not d e t e r m i n e d . n.a., not applicable.
0
s o d i u m lauryl sulphate 0.5
0 0.5
formaldehyde
1.78 8.39
6.03 ± 16.61 4-
0 0.5
8.01
32.65 .4-
p-phenylenediamine
0.04 1.23 3.75 4.87
2.10 7.22 8.62 19.42
444.4-
0 0.5
1.25 0.54 0.56
2.61 42.76 41.42 4-
1 2 5
0.28 1.09
1.59 + 3.83 4-
5.62
16.13 4-
5 0 O. 5
5.58 5.84
0.21 1.45
1.40 45.51 ± 13.71 .413.27 ±
0 0.5 1 2
p-nitroso-dimethylaniline
et h y l - p - a m i n o b e n z o a t e (benzocaine)
cinnamic aldehyde
0.62 0.88 0.79
1.07
1.24
0.78 0.79 1.09
0.91 1.32 2.04
1.03
6.12
1.05 1.35 6.14
2.94
1.45 1.19 3.87 ±
± 4441.33
0.09 1.00 0.55 2.83
1.51 2.04
0.17 ± 0.13 ± 0.29 ±
0.33 40.17 ±
2.92 4-
1.81 44.29 45.35 4-
1.94 4-
0.08 0.12 0.19
0.09 0.05
0.38
0.84 1.36 1.48
1.09
6.73 ± 2.45 51.47 .4- 10.55 67.83 ± 9.02
6.96 46.51 4-
73.27 + 28.83
16.78 4- 4.92 25.30 .4- 5.07 97.50 4- 19.95
n.d.
n.d. n.d. n.d.
n.d.
ll.d.
1.02 1.53 2.00 3.27 3.49
0.88
0.52 0.39
0.52
1.51
0.93 2.21 2.76
0.97 7.40 9.75
0.94
n.a.
n.a.
n.a.
n.a.
[1.a.
3.42
1.50 1.96 3.21
.4± .4±
0.9 2.7 1.2 0.6
+ + 444-
0.7 4.4 2.3 7.4 0.4
8.4 + 0.5
10.6 4- 0.4 8.7 4- 0.7
9.2 + 0.2 8.0 + 0.5
10.3 4- 0.7
10.9 4- 0.3 8.2 4- 0.6 8.8 + 0.4
9.5 4- 0.6
12.4 4- 0.4 21.5 + 1.7 21.6 ± 3.9
I 1.9 4- 1.0 13.9 .4- 1.2
12.6 24.5 20.8 36.3 49.9
10.4 .4- O.l 8.8 + 0.7 5.9 4- 0.5
8.5 4- 0.5 8.1 .4-0.5
11.8 4- 0.8
9.8 15.4 12.5 11.3
214 challenge. Formaldehyde and cinnamic aldehyde induced reactions in 9 out of 10 and 8 out of 10 test guinea pigs, respectively. Responses to benzocaine were somewhat weaker with 6 out of 20 animals positive (data not presented). In addition, local lymph node assays were performed with sodium lauryl sulphate which is considered not to cause allergic contact dermatitis. In local lymph node assays guinea pigs received three consecutive daily applications of various concentrations of the test chemical. Animals were killed 5 days following the initiation of exposure and draining LNC cultured for 24 h or 48 h. In 48 h assays culture was performed in the presence or absence of an exogenous source of IL-2. Results are presented as the mean [3H]TdR incorporation for each experimental group and in the form of stimulation indices relative to control values (Table I). The mean lymph node weight for each experimental group is also recorded. With the exception of benzocaine, at least one concentration of each sensitizing chemical tested induced an elevation of proliferation in 24 h assays. The vigour of responses varied. Substantial activity was observed with the majority of allergens examined; stimulation indices of greater than four were recorded with at least one concentration of all sensitizers other than benzocaine and formaldehyde. In most instances dose-response relationships were evident, although with DNTB and cyanuric chloride there was some indication of inhibition with higher test concentrations. An examination of the results recorded for LNC isolated from untreated guinea pigs reveals some variability among control values. In 24 h cultures the mean [3H]TdR incorporation for control populations varied from 1.59-6.03 ct/min × 10 -3. It is clear, therefore, that, in this series of experiments, the use of stimulation indices for comparative evaluation of responses requires care. In most instances reactivity measured following 48 h culture was lower and in some cases, such as with DNTB, considerably lower. However, proliferative activity was maintained or enhanced in cultures supplemented with IL-2. The exception was cultures of LNC derived from DNTB-treated animals where 48 h values were low in either the presence or absence of IL-2, possibly as the result of a technical failure. Interleukin 2 failed to influence materially [3H]TdR incorporation by LNC prepared from untreated animals. In no circumstances did topical exposure to sodium lauryl sulphate at equivalent concentrations induce proliferative activity by draining LNC. As the data in Table I reveal, in most instances the induction of LNC proliferative activity was accompanied by an increase in draining lymph node weight. With some sensitizers, however, increases in node weight were either modest (cinnamic aldehyde) or absent (benzocaine, formaldehyde). Exposure to sodium lauryl sulphate failed to influence draining lymph node weight. Mouse studies
All chemicals, with the exception of benzocaine, were also evaluated using the murine lymph node assay. In this series of experiments mice received three consecutive daily applications of the test material and the proliferative activity of draining LNC was evaluated 1 day following the final exposure. Responses following culture for 24 h in the absence of IL-2 were measured.
215
TABLE II LOCAL LYMPH N O D E ASSAY IN THE MOUSE. D R A I N I N G LNC P R O L I F E R A T I O N Chemical
Concentration (% w/v)
LNC proliferation [3H]TdR incorporation mean* ct/min ± S.D. x 10 -3
4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone)
0 0.5
2,4-dinitrofluorobenzene (DNFB)
2 0 0.5
2,4-dinitrothiocyanobenzene (DNTB)
2 0 0.5
2,4,6-trinitrochlorobenzene (picryl chloride)
2 0 0.5
2,4,6-trichloro-1,3,5-triazine (cyanuric chloride)
2 0 0.5
1.12 97.92 104.21 93.39 1.43 18.21 22.04 n.d. 1.75 10.08 15.22 18.01 0.98 37.66 64.24 54.71 1.72 22.41 30.78 43.21 1.19 0.98 1.76 3.93 1.47 7.12 15.16 28.98 0.78 2.69 4.11 3.72 1.02 n.d. 0.78 2.39 0.69 |.01 0.74 0.72
cinnamic aldehyde
p-nitroso-dimethylaniline
p-phenylenediamine
formaldehyde
sodium lauryl sulphate
n.d., not determined. LNC, lymph node cells. *Mean values from 5 culture wells.
2 0 0.5 2 0 0.5 2 0 0.5 2 0 0.5 1 2 0 0.5 1 2
± ± ± ± ± ± ±
0.10 4.71 5.89 6.41 0.09 1.14 1.02
± ± ± ± ± ± ± ± ± ± + ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.31 I.I1 0.09 1.78 0.08 2.16 8.41 1.37 0.24 1.72 4.11 3.76 0.08 0.12 0.21 0.62 0.12 0.83 1.05 1.17 0.06 0.14 0.78 0.55 0.13
± ± ± ± ± ±
0.08 0.31 0.04 0.07 0.12 0.03
SI
87.43 93.04 83.38 12.73 15.41
5.76 8.70 10.29 38.43 65.55 55.83 13.03 17.90 25.12 0.82 1.48 3.30 4.84 10.31 19.71 3.45 5.27 4.77
0.76 2.34 1.46 1.07 1.04
216
Thymidine incorporation by LNC isolated from vehicle-treated mice varied between 0.69-1.75 ct/min x 10 -3 (Table II). Exposure of mice to sodium lauryl sulphate failed to elicit a response. With all other chemicals there was evidence of induced proliferative activity following treatment with at least one concentration. Compatible with the results of previous experiments [5], some chemicals, such as oxazolone, picryl chloride and cyanuric chloride, induced high levels of [3H]TdR incorporation at all test concentrations. Less vigorous responses were observed with formaldehyde, cinnamic aldehyde and p-phenylenediamine where maximum stimulation indices of 2.34, 3.30 and 5.27 respectively, were recorded. Discussion
The primary objective of the studies described was to establish whether in practice it is possible to measure local lymph node responses in guinea pigs. In summary the data indicate that responses are maximal following 24 h culture and that they may be enhanced further by the addition of IL-2. Of the sensitizing chemicals examined in the local lymph node assay all except benzocaine induced increased proliferative activity. The failure of benzocaine to elicit a response is of some interest as in initial studies of the murine local lymph node assay this material induced activity in both the in vivo and in vitro versions of the test [5,6]. Subsequently, however, in the context of an interlaboratory comparison, each of four participating laboratories failed to identify benzocaine as a sensitizer on the basis of murine local lymph node assay responses [8]. The reasons for these apparently contradictory results are currently the subject of further investigations. Changes in lymph node weight were also measured in the guinea pig but, in common with experience gained using the murine local lymph node assay [5], proved a less sensitive correlate of lymph node activation. The data recorded, particularly with regard to proliferation by control lymph node cells, suggest that some further optimization of the test method will be necessary, especially if sensitizing potential is to be measured as a function of stimulation indices. Compared with the mouse, little information is available regarding the culture of guinea pig lymphocytes and judicious modification of the method may provide a more precise assessment of proliferative activity. Despite the possible need for further method development, results to date indicate that the local lymph node assay can be performed in the guinea pig. Although this is the first systematic investigation of LNC proliferation in the guinea pig as a means of identifying sensitizing agents, another study in this species did address the question of lymph node activation [15]. In that study the authors sought to assess skin sensitizing potential as a function of pyroninophilia in draining lymph nodes. Using a series of acrylates, a correlation between the frequency of large pyroninophilic cells in sections of draining lymph nodes and contact sensitization was reported [15]. Although, in the context of routine analysis, direct measurement of lymphocyte proliferative activity by thymidine incorporation has clear advantages compared with visual assessment of the number of pyroninophilic cells, the study by Bull et al. [15] does provide confirmatory evidence that lymphocyte activation in the guinea pig correlates with sensitizing potential.
2t7 With the exception of benzocaine, all chemicals examined in the guinea pig were also evaluated in the murine local lymph node assay. While, on the basis of the limited data available, it is premature to draw firm conclusions with respect to relative sensitivity, it would appear that, in general terms, induced proliferative responses in the mouse are somewhat more vigorous. This need not, of course, necessarily reflect differential biological activity and may be attributable to the fact that the measurement of guinea pig LNC proliferative responses has yet to be fully optimized. It may also be relevant that while lymph node responses in guinea pigs were assessed 5 days following the initiation of exposure, in mice a 3-day assay was employed. It is known that chemicals vary with respect to the kinetics of the LNC proliferative response they induce [6] and it is possible that such kinetics also vary between species. It is nevertheless the case that chemicals such as oxazolone, cyanuric chloride, the dinitrobenzene derivatives, picryl chloride and pnitrosodimethylaniline, each induced marked proliferative responses in both the guinea pig and mouse. In both species formaldehyde caused only modest activity. Some differences were apparent. Thus, picryl chloride caused more vigorous proliferation in the mouse than in the guinea pig, while responses to p-phenylenediamine and cinnamic aldehyde were stronger in the guinea pig. A more extensive study with a wider range of chemicals will be required for an accurate comparative analysis of sensitivity. The conclusion that may be drawn from these investigations is that the local lymph node assay can be performed successfully in the guinea pig and that with further optimization of the method this might prove as robust as the murine equivalent. It is not, however, our intention to propose that local lymph node assays are performed in the guinea pig rather than the mouse. The primary advantage of developing this method in the guinea pig is to facilitate comparisons between the local lymph node assay and assessment of sensitizing activity by currently available guinea pig test methods. To date, the interpretation of comparative studies of the sensitivity of the local lymph node assay and conventional predictive test methods has been hampered by the fact that differences may be attributable to either fundamental methodological differences (proliferation at sensitization versus erythema at challenge), or differences in the relative sensitivity of the two species to different chemicals. Now with the development of the guinea pig local lymph node assay there is an opportunity, within a single species, to compare the sensitivity of assays such as the guinea pig maximization test with the local lymph node assay.
References 1 G.L. Asherson and W. Ptak, Contact and delayed hypersensitivity in the mouse. 1. Activesensitization and passive transfer. Immunology, 15 (1968) 405. 2 S.C. Gad, B.J. Dunn, D.W. Dobbs, C. Reilly and R.D. Walsh, Development and validation of an alternative dermal sensitization test: the mouseear swelling test (MEST). Toxicol. Appl. Pharmacol.. 84 (1986) 93. 3 J. Descotes, Identification of contact allergens. The mouse ear sensitization assay. J. Toxicol. Cut. Ocul. Toxic., 7 (1988) 263. 4 J. Maisey and K. Miller, Assessment of the ability of mice fed on vitamin A-supplemented diet to respond to a variety of potential contact sensitizers. Contact Dermatitis, 15 (1986) 17.
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I. Kimber and C. Weisenberger, A murine local lymph node assay for the identification of contact allergens: assay development and results of an initial validation study. Arch. Toxicol. 63 (1989) 274. I. Kimber, J. Hilton and C. Weisenberger, The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis, 21 (1989) 215. I. Kimber, J. Hilton and P.A. Botham, Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler occluded patch test in guinea pigs. J. Appl. Toxicol., 10 (1990) 173. 1. Kimber, J. Hilton, P.A. Botham, D.A. Basketter, E.W. Scholes, K. Miller, M.C. Robbins, P.T.C. Harrison, T.J.B. Gray and S.J. Waite, The murine local lymph node assay: results of an interlaboratory trial. Toxicol. Lett., 5 (1991) 203. T. Maurer, Contact and Photocontact Allergens. A Manual of Predictive Test Methods, Marcel Dekker, Basle, 1983. K.E. Andersen and H.1. Maibach, Contact Allergy-Predictive Tests in Guinea Pigs. Current Problems in Dermatology, Vol. 14, Karger, Basle, 1985. B. Magnusson and A.M. Kligman, The identification of contact allergens by animal assay, the guinea pig maximization test method. J. Invest. Dermatol., 52 (1969) 268. B. Magnusson and A.M. Kligman, Allergic Contact Dermatitis in the Guinea Pig, Charles C Thomas, Springfield, IL, 1970. E.V. Buehler, Delayed contact hypersensitivity in the guinea pig. Arch. Dermatol., 91 (1965) 171. E.V. Buehler, A rationale for the selection of occlusion to induce and elicit delayed contact hypersensitivity in the guinea pig, in K.E. Andersen and H.I. Maibach (Eds.), Contact Allergy Predictive Tests in Guinea Pigs. Current Problems in Dermatology, Vol. 14, Karger, Basle, 1985, p. 39. J.E. Bull, D. Parker and J.L. Turk, Predictive value of assessment of lymph node weight and Tlymphocyte proliferation in contact sensitivity in acrylates. J. Invest. Dermatol., 85 (1985) 403.
