Toxicology 257 (2009) 113–116
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Increased cell proliferation in spleen and lymph nodes peripheral to contact
allergen application site
Itai Chipinda∗, Stacey E. Anderson, Leon F. Butterworth, Donald Beezhold, Paul D. Siegel
Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health,
1095 Willowdale Road, Morgantown, WV 26505-2888, United States
a r t i c l ei n f o
Received 3 December 2008
Received in revised form 15 December 2008
Accepted 16 December 2008
Available online 30 December 2008
Local lymph node assay
Peripheral lymph nodes
a b s t r a c t
The local lymph node assay (LLNA) is widely used to identify chemicals that are contact sensitizers.
The assay involves dosing mice with the chemical on both ears and pooling the superficial parotid lymph
potential reduction in animal usage by dosing one ear with the allergen and the other with vehicle-only.
The respective draining lymph nodes were processed separately for tritiated thymidine (3H-TdR) incor-
poration. Cell proliferation in proper axillary and renal nodes, as well as in the spleen was also assessed.
Cross-contamination of the chemicals from the dosed ears to other parts of the body via preening was
prevented by dosing restrained animals and washing off the residual chemical with saline after 4h. Dos-
ing the left ear with 0.02% oxazolone (OX) on unrestrained animals resulted in marked cell proliferation
vehicle-dosed ear (SI=6), as well as the proper axillary lymph nodes (SI=3.3). Increased3H-TdR incor-
poration was not observed in the renal lymph nodes (SI=1.1). Similar stimulation of cells was observed
in the lymph node draining the ear contra-lateral to the 30% hexylcinnamaldehyde (HCA)-dosed ear.
Increased proliferative activity was observed in contra-lateral draining lymph nodes of restrained mice
demonstrating that these results cannot be attributed to cross-contamination of adjacent skin. A signifi-
cant increase in proliferation of splenocytes was also observed. It is concluded that dermal application of
a contact allergen, as exemplified by OX and HCA, may induce cell proliferation in the neighboring lymph
nodes and spleen indicative of hapten and/or haptenated proteins diffusing through the skin to periph-
eral nodes and the blood to produce systemic sensitization. It is also possible that lymphatic capillaries
may communicate between the left and right side of the mouse head. Thus the contra-lateral draining
superficial parotid node cannot be used as a control for application of contact allergen to a single ear in
a modified LLNA.
Published by Elsevier Ireland Ltd.
The local lymph node assay (LLNA) (Kimber et al., 1994) has sur-
passed traditional methods such as the Guinea Pig Maximization
Test (GPMT) or the Occluded Patch Test of Buehler (Buehler, 1965;
Magnusson and Kligman, 1970) to become the widely accepted
as a function of lymph node cell proliferative responses induced
in mice following repeated topical exposure to the test chemi-
The formal adoption of the LLNA as a validated method after
rigorous evaluations and comparisons (Dean et al., 2001; Kimber
et al., 2002) has made it a stand-alone method for chemical hazard
∗Corresponding author. Tel.: +1 304 285 5721x2; fax: +1 304 285 6126.
E-mail address: IChipinda@cdc.gov (I. Chipinda).
identification and assessment. Although the LLNA has been shown
to reduce assay time and animal numbers required to provide a
robust assessment of chemical hazard, and to provide a reduction
in dermal trauma to which animals are potentially subject, recent
studies have explored ways to further reduce or eliminate the need
for experimental animals in skin-sensitization safety assessments
(Kimber et al., 2006; Ryan et al., 2008).
The present study examined the potential use of a modified
LLNA in which the contact allergen/sensitizer is applied to one
ear with the control vehicle being applied on the other ear of
the mouse. This would allow reduction of animal use by elim-
ination of separate vehicle control groups. It was hypothesized
that regional lymphatic drainage is unidirectional, with lymphat-
ics from the left and right side of the head emptying into the
venous system independently (similar to human anatomy), and
that chemical diffusion/absorption would be insignificant with
respect to antigenic stimulation at peripheral lymph nodes and the
0300-483X/$ – see front matter. Published by Elsevier Ireland Ltd.
I. Chipinda et al. / Toxicology 257 (2009) 113–116
2. Materials and methods
Oxazolone (OX), hexylcinnamic aldehyde (HCA), phosphate buffered saline
(PBS), trichloroacetic acid (TCA), and acetone (A) were acquired from Sigma–Aldrich
(St. Louis, MO). Tritiated thymidine (3H-TdR, specific activity 2 Ci/mmol) was from
Dupont NEN (Waltham, MA) and scintillation fluid from PerkinElmer (Waltham,
Female BALB/c mice were purchased from Taconic (Hudson, NY). Animals were
6–8 weeks old upon arrival and allowed to acclimate for a minimum of 10 days. Ani-
Animal Care International-accredited animal facility at NIOSH, Morgantown, WV.
Animals were housed under controlled environmental conditions in HEPA-filtered
ventilated polycarbonate cages on autoclaved hardwood Beta-chip bedding with
cotton fiber nesting materials (Nestlets) and were provided Teklad 7913 food and
by the NIOSH Animal Care and Use Committee.
2.3. Lymph node nomenclature
The lymph nodes examined in this study are named in accordance with the
standard nomenclature proffered by Van den Broeck et al. (2006). These include the
superficial parotid lymph nodes (Ln. parotideus superficialis), proper axillary lymph
nodes (Ln. axillaris proprius) and renal lymph nodes (Ln. renalis). The superficial
parotid lymph nodes drain the ears/head region and are the lymph nodes assayed in
the LLNA. They have also been referred in the literature by various names including
the cranial, cervical or auricular lymph nodes.
2.4. Experimental design
The LLNA was performed according to the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM) standard method (Dean et al.,
2001) with modification as follows: after grouping mice into homogenous groups
(n=3–5) based on their weight, mice were dosed with 25?L of a test chemical (OX
(0%, 0.0025%, 0.005%, and 0.02%) or HCA (0%, 7.5%, 15%, and 30%)) applied to the
dorsum of the left ear of each mouse. The vehicle (25?L acetone) was applied on
the right ear of each mouse. This was repeated for 3 consecutive days. A separate
control group (n=4) in which vehicle was dosed on both ears was run concurrently.
Two hundred microliters of 0.01M PBS containing 20?Ci3H-TdR was injected into
the tail vein on day 6, and after 5h the mice were euthanized via CO2asphyxiation.
Left and right draining superficial parotid, proper axillary and renal lymph nodes
were excised for each animal. The left and right superficial parotid nodes were pro-
cessed separately and so were the other nodes. The spleen was also collected. For
each tissue, single cell suspensions were made and following overnight incubation
in 5% TCA, samples were counted using a Packard Tri-Carb 2500TR (Meriden, CT)
liquid scintillation counter with subtraction of the background. Stimulation indices
(SIs) were calculated by dividing the mean disintegrations per minute (DPM) per
test group by the mean DPM for the vehicle control group. In one experiment both
ears were dosed with 0.02% OX for comparison of proliferative activity of superficial
parotid, proper axillary and renal lymph node cells.
Custom made Teflon mouse restrainers were used to examine the potential con-
able to retract their head into the restrainer or preen during the exposure period.
2.5. Statistical analysis
Jose, CA). Data were analyzed by one-way ANOVA followed by a post hoc Bonferroni
test. Comparisons between superficial parotid nodes from the same animal were
done using a paired t-test. Data were considered significant at p<0.05.
OX and HCA are extreme and moderate sensitizers, respectively.
Dosing mice with OX (0.0025%, 0.005%, and 0.02%) or HCA (7.5%,
15%, and 30%) on the left ear and acetone-only concurrently on the
right ear resulted in dose-dependent cell stimulation in superficial
parotid lymph nodes from both right and left ears (Figs. 1 and 2)
cell proliferation in the superficial parotid lymph node contra-
in the superficial parotid lymph node directly draining that ear.
Fig. 1. Lymph node cell (LNC) proliferation measured as stimulation index (SI). Mice
were dosed with OX on the left ear and the corresponding right ear was dosed with
the acetone vehicle-only. A separate control group was dosed with the acetone vehi-
cle on both ears. Dose-dependent LNC proliferation was observed in lymph nodes
draining both allergen exposed and contra-lateral ears. Statistically significant stim-
ulation (*p<0.05, **p<0.01) over the vehicle (both ears) control was observed with
the proliferation in the LNC draining the left ear (dosed with 0.02% OX) and the right
ear that was dosed with vehicle (#p<0.01).
For example,3H-TdR incorporation in the contra-lateral superfi-
cial parotid lymph node cells was 46.8±9.1% for 0.02% OX and
44.6±8.5% for 30% HCA, of the activity in the cells from lymph
nodes directly draining the allergen-dosed ears (SI=12.8 for OX
the acetone-only control group (acetone dosed on both ears). The
results clearly indicate that both contact allergens (OX and HCA)
induced cell proliferation in draining lymph nodes contra-lateral to
the allergen-dosed ear.
Allergen induced lymph node cell proliferation was next
assessed in lymph nodes caudal to the cranium. The high dose
(0.02%) OX applied to both ears resulted in marked cell prolifer-
ation in the proper axillary lymph nodes (SI=3.3±1.0). Increased
proliferative activity was not observed in the renal lymph nodes
(SI=1.1±0.6) as exhibited in Table 1. A similar trend was observed
Fig. 2. LNC proliferation measured as stimulation index (SI). Mice were dosed with
HCA on the left ear and the corresponding right ear was dosed with the vehicle-
only. A separate control group was dosed with the acetone vehicle on both ears.
gen exposed and contra-lateral ears. Statistically significant stimulation (*p<0.05,
**p<0.01) over the vehicle (both ears acetone) control was observed with all HCA
doses. All the HCA doses resulted in significant difference between the proliferation
in the lymph nodes draining the left ears (dosed with HCA) and the right ears that
were dosed with vehicle (#p<0.05,##p<0.01).
I. Chipinda et al. / Toxicology 257 (2009) 113–116
Oxazolone induced lymphocyte proliferation estimated as stimulation index (SI)
relative to the vehicle control group which had an SI=1. The groups with restrained
animals were used.
Lymphatic organAllergen treatment
0.02% OX on
left ear only SI
both ears SI
Left superficial parotid lymph node
Right superficial parotid lymph node
Proper axillary lymph node
Renal lymph node
12.1 ± 3.9*
7.6 ± 2.4*
2.3 ± 0.9*
1.3 ± 0.6
2.5 ± 1.1*
10.0 ± 2.0*
11.3 ± 3.2*
3.3 ± 1.0*
1.1 ± 0.6
3.0 ± 0.1*
*Significant increase in cell proliferation in comparison to mice dosed with ace-
tone vehicle on both ears (p<0.05).
Fig. 3. Lymphocyte proliferation in the superficial parotid lymph nodes draining
the 0.02% OX treated and vehicle-dosed ears of restrained animals. The correspond-
ing controls were mice dosed with vehicle on both ears. The nodes draining the
vehicle-dosed ears had lymph node cell proliferative activity 10.8 times the control
the vehicle-dosed ears.
when only the left ear was dosed with OX and the right ear with
acetone (Table 1). The proper axillary lymph node cell proliferation
than the activity for the group dosed on one ear (SI=2.3±0.9).
The group dosed on both ears with OX was exposed to twice
the amount of chemical per mouse than the group dosed on one
ear. The data suggest that there is some lymphatic drainage to
the axillary region potentially bypassing superficial parotid lymph
The potential for physical cross-contamination within the ani-
mal from preening or contact with cage mates was investigated
by dosing restrained animals (in holding tubes with only the head
exposed) for 4h and then washing off any residual chemical from
the ear’s surface. Fig. 3 shows the comparative cell proliferative
activity in the superficial parotid nodes for the restrained ani-
mals that were dosed on one ear with 0.02% OX. A significant
increase in proliferative activity in the contra-lateral lymph node
which averaged 21% of the activity of the superficial parotid lymph
nodes directly draining the OX dosed ears was observed.
When the spleen from the restrained animals was collected and
processed for cell proliferation, a statistically significant (p=0.01)
increase in3H-TdR incorporation in splenocytes from OX-dosed
mice was observed relative to the vehicle control. The splenocytes
stimulation is shown in Table 1.
The initial objective of the present study was to investigate the
potential of modifying the LLNA to dose a single ear and use the
draining lymph node from the contra-lateral ear as a control. It
was hypothesized that lymph drainage is unidirectional from the
left side of the head to the thoracic duct as in humans, and aller-
gen or activated antigen presenting cells would be greatly diluted
and/or filtered through lymphatic organs distal to the thoracic duct
before reaching the contra-lateral superficial parotid lymph node.
The time-course of the LLNA is such that it is a measure of sen-
sitization, not allergic contact dermatitis induction. Clearly, our
hypothesis was disproven by the very high, dose-dependent prolif-
erative cell response observed in the lymph node draining the ears
contra-lateral to the allergen-dosed ears. This observation raised
sequent stimulation of peripheral lymph node and spleen cells.
(Van den Broeck et al., 2006), the drainage pattern and distribution
of lymphatic vessels is relatively unknown.
The study was extended to include lymph nodes distant to
the superficial parotid lymph nodes to the thoracic duct. Allergen
induced lymph node cell proliferation was observed in the proper
axillary lymph node, which receives lymphatic drainage from the
forelimb. No increase in proliferative activity was observed in the
renal lymph node, which is the furthest removed from the appli-
cation area of the lymph nodes collected in this study. The most
probable explanation of the high proliferative activity observed in
the superficial parotid lymph node draining the ear contra-lateral
to the allergen-dosed ear is that there is significant diffusion of
chemical allergen through interstitial spaces and/or dendritic cell
migration in the lymphatic drainage from the head of the mouse
which crosses the midline to communicate through lymphatic ves-
sels on the opposite side.
Increased proliferation was also observed in the spleen suggest-
ing sufficient allergen (either as unbound hapten or protein bound)
was absorbed into the blood to induce a splenic immune medi-
ated response. After skin absorption the chemical molecules may
be absorbed into either the lymphatic system or the capillaries of
the blood. Blood capillaries supplying the interstitium are charac-
terized by the presence of tight interendothelial junctions and an
(<1–2kDa) moderately lipophilic molecules (Porter et al., 2001).
The uptake of molecules into the blood and/or lymph is subject to
the molecules passing through the extra-cellular matrix (Swartz,
2001). The evidence of allergen stimulation in the spleen after top-
ical application is of particular interest, but an often ignored aspect
of the LLNA. There is a possibility of local and systemic toxicity with
attendant effects on the immune response observed with appli-
cation of allergenic chemicals to the skin. For instance, local and
systemic toxicity of the test chemical must be considered in the
justification for using high LLNA doses (≥10%) (Kimber et al., 2006).
It must be noted, that the definition of a high dose is highly depen-
dent on the specific chemical with respect to both allergenicity and
direct local and systemic toxicity.
The potential confounders of preening or cross-contamination
between animals were eliminated by preventing physical transfer
gen after a 4-h exposure. Allergen induced proper axillary lymph
node stimulation indexes were comparable between the restrained
and unrestrained groups demonstrating that cross-contamination
the immune stimulation in sites peripheral to the allergen applica-
demonstrated the importance of vehicle selection in the assess-
ment of the effect of carbon chain length on LLNA measurement
of dermal sensitization by bromoalkanes (Siegel et al., 2009). The
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I. Chipinda et al. / Toxicology 257 (2009) 113–116
ies due to (1) solubility of test chemicals and (2) its high vapor
pressure. The volatility of acetone should limit its absorption into
the skin and limit solvent induced allergen migration through the
In conclusion, epicutaneous application of contact allergen, as
exemplified by OX and HCA, induces antigen induced cell prolifer-
ation in lymph nodes that do not directly drain the dosed area, and
area in an individual animal cannot be used as controls. This study
demonstrates that local dermal application of allergen can spread,
most likely through absorption and diffusion potentially producing
Conflict of interest statement
No competing interests.
The authors received funding source from CDC/NIOSH.
The findings and conclusion in this report are those of the
authors and do not necessarily represent the views of the National
Institute for Occupational Safety and Health.
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