MucosalImmunology | VOLUME XX NUMBER X | MONTH 2012
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The incidence of asthma is on the rise worldwide, resulting in
the increased morbidity and mortality and making it an eco-
nomic burden to society. 1,2 The pathophysiology of asthma is
characterized by eosinophil-rich inflammatory cell infiltrates,
mucus hypersecretion, airway hyperresponsiveness, and airway
wall remodeling. 3,4 Despite a number of currently used therapies
such as glucocorticoids, ? -agonists, antihistamines, phosphodi-
esterase inhibitors, and anticholinergic agents, many patients
continue to suffer from the disease exacerbation. Moreover,
many side effects of these medicines, which involve bone, met-
abolic, and cardiovascular systems, also limit their efficacy. 5,6
Therefore, novel molecular targets are being investigated 7 and
novel approaches are being developed 8 that may have a profound
impact on improving the diagnostic, prevention, and treatment
of the allergic asthmatic diseases.
Semaphorin 4A (Sema4A) belongs to a large family of secreted
and membrane-bound glycoproteins that were originally found
to be expressed in the nervous system and function as axon
guidance molecules. 9 Recently, Sema4A was located in the lym-
phoid tissues where its expression was preferentially restricted to
the antigen-presenting cells (APCs) such as dendritic cells (DCs)
and B cells. 10 It was reported to act as a costimulatory molecule,
enhancing the activation and differentiation of T cells in vitro
and potentiating the generation of antigen (Ag)-specific T cells
in vivo. 10,11 Sema4A functionally interacts with Tim-2 (T-cell
immunoglobulin and mucin domain 2) expressed on T cells,
T helper type 2 (Th2) cells predominantly. 10,11 In nonlymphoid
tissues, Sema4A functionally interacts with plexin family mem-
bers, namely Plexin D1 and Plexin B1. 12 – 14 These interactions
have been shown to play important roles in several physiologi-
cal and pathological conditions such as angiogenesis, 14 neuron
growth cone collapse, 13 and retina formation. 12
In vitro , Sema4A-Fc has been reported to enhance T-cell
activation, proliferation, and interleukin 2 (IL-2) production,
whereas anti-Sema4A antibody (Ab) inhibited allogeneic
T-cell / DC mixed lymphocyte reaction. 10 Mice with a targeted
disruption of Sema4A developed normally but showed defects
in the DC-mediated T-cell response. 15 Whereas Sema4A − / −
DCs matured and responded normally to lipopolysaccharide
or agonistic anti-CD40 Ab in vitro , the in vivo production of Ag-
specific T cells and cytokines was downregulated in Sema4A − / −
Neuroimmune semaphorin 4A downregulates the
severity of allergic response
EH Nkyimbeng-Takwi 1 , 2 , K Shanks 1 , E Smith 1 , A Iyer 1 , MM Lipsky 3 , LJ DeTolla 3 , H Kikutani 4 ,
AD Keegan 1 , 2 , 5 and SP Chapoval 1 , 2 , 5
To define the role of semaphorin 4A (Sema4A) in allergic response, we employed Sema4A − / − and wild-type (WT) mice in
the experimental model of ovalbumin (OVA)-induced allergic airway inflammation. We observed a selective increase in
eosinophilic airway infiltration accompanied by bronchial epithelial cell hyperplasia in allergen-treated Sema4A − / − mice
relative to WT mice. This enhanced inflammatory response was associated with a selective increase in bronchoalveolar
lavage (BAL) interleukin 13 (IL-13) content, augmented airway hyperreactivity, and lower regulatory T cell (Treg) numbers.
In vivo allergen-primed Sema4A − / − CD4 + T cells were more effective in transferring T helper type 2 (Th2) response to
naive mice as compared with WT CD4 + T cells. T-cell proliferation and IL-13 productions in OVA 323 – 339 -restimulated
Sema4A − / − cell cultures were upregulated. Generated bone marrow chimeras showed an equal importance of both
lung-resident cell and inflammatory cell Sema4A expression in optimal disease regulation. These data provide a new
insight into Sema4A biology and define Sema4A as an important regulator of Th2-driven lung pathophysiology.
1 Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine , Baltimore , Maryland , USA . 2 Department of Microbiology and Immunology,
University of Maryland School of Medicine , Baltimore , Maryland , USA . 3 Department of Pathology, University of Maryland School of Medicine , Baltimore , Maryland , USA .
4 Department of Molecular Immunology, Osaka University , Osaka , Japan . 5 Program in Oncology, Greenebaum Cancer Center, University of Maryland School of Medicine ,
Baltimore , Maryland , USA . Correspondence: SP Chapoval ( email@example.com )
Received 8 August 2011; accepted 17 February 2012; advance online publication 4 April 2012. doi: 10.1038/mi.2012.18
VOLUME XX NUMBER X | MONTH 2012 | www.nature.com/mi
mice after challenge with various Ags. 15 Mice deficient in one
of Sema4A functional receptors, Tim-2, exhibited increased
lung inflammation and Th2 cytokine production in response
to allergen. 16 Based on all of the above, we hypothesized that
Sema4A may have a critical nonredundant regulatory role in
In this research, using the experimental model of allergen-
induced asthma, we show that Sema4A downregulates the sever-
ity of allergic airway response. Ovalbumin (OVA)-challenged
Sema4A − / − mice showed an increased number of bronchoalveolar
lavage (BAL) eosinophils and a wider spread of multiple inflam-
matory sites in the airways as compared with similarly treated
wild-type (WT) mice. In addition, employing bone marrow (BM)
chimeric mice, we demonstrate that Sema4A expression by lung-
resident cells as well as by inflammatory cells is critically impor-
tant for the regulation of the disease severity. Using the in vivo
cell transfer technique, we show here that allergen-primed spleen-
derived Sema4A − / − CD4 + T cells are equally effective in either
WT or Sema4A − / − host to induce the substantial lung infiltra-
tion with eosinophils in response to OVA challenges, whereas WT
CD4 + T cells were much less efficient. This could be explained,
in part, by a lower number of regulatory T cells (Tregs) found in
Sema4A − / − mice in addition to their higher local and systemic IL-
13 response to allergen. Our findings establish the cellular mecha-
nisms of Sema4A function in the allergic response regulation and
its potential as a novel therapeutic agent for asthma.
Sema4A downregulates allergic airway response
Although the role of Sema4A in experimental myocarditis 17
and experimental autoimmune encephalomyelitis 10 was estab-
lished previously, its regulation of a Th2 response has not been
investigated. Therefore, to detail Sema4A function in the aller-
gic response, WT and Sema4A − / − mice were subjected to
the allergen priming, boost, and challenges as depicted in
Figure 1a . Control mice were treated with Alum alone and neb-
ulized with phosphate-buffered saline (PBS). A classical allergic
airway response was observed in WT mice after OVA treatment
( Figure 2a,b ). This response consisted of prominent BAL and
airway eosinophilic infiltration where > 50 % of BAL cells were
eosinophils ( Figure 2a ). Eosinophils were also the predominant
cells in the lung tissue multiple peribronchial and perivascular
inflammatory infiltrates ( Figure 2b ). Allergic inflammatory
response was accompanied by mucus hypersecretion ( Figure 2c ).
All these features of allergic response were significantly upregu-
lated in Sema4A − / − mice ( Figure 2a,c ).
Sema4 deficiency was associated with higher serum levels of
allergen-specific IgG1 and IgG2b, but not IgG2a ( Figure 2d and
data not shown). No significant differences in the total serum
IgE levels between OVA-treated WT and Sema4A − / − mice
were found (648.0 ± 89.2 and 714.6 ± 130.7 ng ml – 1 , respectively),
whereas OVA-specific IgE concentrations differed significantly
(409.2 ± 81.4 and 725.0 ± 89.9 ng ml – 1 , respectively, P < 0.01). It
is noteworthy that the levels of total IgE and OVA-specific IgE
were measured in the sera samples obtained in separate in vivo
experiments ( n = 4 – 5 mice per experiment).
It is well known that allergic asthma is a Th2-driven disease
where Th2 cytokines play critical roles in the disease induction
and exacerbation. 18,19 To evaluate a possible local lung cytokine
Bone marrow transfer
DCs or T cells from
–6 Days 05
12 14 1516
Figure 1 Experimental protocols used in this study. ( a ) Experimental
protocols of the allergen treatment and ( b ) the in vivo adoptive cell
transfer followed by antigen (Ag) nebulizations. Models are detailed in
the Methods section. AHR, airway hyperreactivity; BAL, bronchoalveolar
lavage; DC, dendritic cell; FACS, fluorescence-activated cell sorting;
Figure 2 Semaphorin 4A (Sema4A) deficiency increases the severity of allergic airway response in mice. ( a ) Wild-type (WT) and Sema4A − / −
mice were immunized with ovalbumin (OVA). Control mice were challenged with phosphate-buffered saline (PBS). ( a ) The average numbers
( n = 3 – 5) of bronchoalveolar lavage (BAL) total cells (white bars) macrophages (black bars), eosinophils (forward hatched bars), lymphocytes
(reverse hatched bars), and neutrophils (gray bars) ± s.e.m. in one of three representative experiments are shown. * , # P < 0.05, differences in
absolute numbers of total cells and eosinophils in BAL between OVA-challenged WT and Sema4A − / − mice. ( b ) Lung tissue sections were stained
with hematoxylin and eosin (H & E) for inflammation and ( c ) periodic acid-Schiff stain (PAS) for mucous cell hyperplasia evaluation. Magnification
used for pictures is × 40 and for inserts is × 100. ( d ) Anti-OVA specific IgG antibodies (Abs) in serum samples ( n = 4 mice per group) were measured
using corresponding enzyme-linked immunosorbent assay (ELISA) kits as described in the Methods. * P < 0.05, OVA-treated WT vs. Sema4A − / −
mice. ( e ) The levels of cytokines in BAL fluids (BALFs) of OVA-challenged WT (open bars) and Sema4A − / − (black bars) mice measured by either
ELISA or protein array were combined. Data are shown as mean ± s.e.m. ( n = 3 – 6 mice). * P < 0.05, OVA-challenged WT vs. Sema4A − / − mice.
( f ) For cytokine Cytometric Bead Array (CBA) in lung lysates and 5 × concentrated BALF, data were acquired by BD FACS Calibur and shown
are FlowJo-generated dot plots for individual proteins for an individual mouse in one of two representative experiments ( n = 2 per group). For a
numeric representation of cytokine concentration, CBA data were calculated using the BD CBA software. * P < 0.02 and * P < 0.004, monocyte
chemotactic protein-1 (MCP-1) and interleukin (IL)-17A in WT vs. Sema4A − / − mice, respectively. ( g ) Airway reactivity is shown as the percent
increase in PenH over a baseline ( n = 6 – 8 mice per group in the separate 3 experiments). * P < 0.05, OVA-treated WT mice vs. Sema4A − / − mice for
the corresponding doses of methacholine. Forced oscillation measurements were done in anesthetized mice employing FlexiVent (SCIREQ) and
airway resistance is graphed as cm H 2 O – 1 ml – 1 s – 1 ( n = 2 mice per group). IFN- ? , interferon- ? ; TGF- ? , transforming growth factor- ? ; TNF- ? , tumor
necrosis factor- ? .
MucosalImmunology | VOLUME XX NUMBER X | MONTH 2012
dysregulation induced by the Sema4A deficiency, we compared
the levels of Th1 / Th2 cytokines in the BAL and lung lysates
obtained from allergen-treated WT and Sema4A − / − mice.
As shown in Figure 2e , we found a selective increase in IL-13
levels in the BAL of Sema4A − / − mice when compared with WT
mice, whereas other Th2 cytokines (IL-4 and IL-5) were not
affected by Sema4A deficiency. In addition to that, the increased
BAL levels of IL-12p40 were detected in allergen-challenged
Sema4A − / − mice. It is noteworthy that no substantial
differences in the BAL contents of eotaxin, RANTES (Regulated
upon Activation, Normal T-cell Expressed, and Secreted),
macrophage inflammatory protein (MIP)-1 ? , MIP-1 ? , and
vascular endothelial growth factor were detected between WT
and Sema4A − / − mice (data not shown).
Anti-OVA Ab, mg ml–1
Absolute number of
cells in BAL, × ×103
pg ml–1 BALF
pg ml–1 BALF
pg ml–1 of BALF
0 6.25 12.5 25 50 100
Methacholine (mg ml–1)
% Baseline PenH
BL PBS 6.25 12.5 25
Methacholine (mg ml–1)
RL (cm H2O–1 ml–1 s–1)
VOLUME XX NUMBER X | MONTH 2012 | www.nature.com/mi
Proinflammatory cytokine Cytometric Bead Array (CBA)
data showed decreased monocyte chemotactic protein-1 lev-
els in the whole lung lysates and BAL fluid (BALF) obtained
from OVA-treated Sema4A − / − mice as compared with similarly
treated WT mice ( Figure 2f ). It is noteworthy that lung lysates
used in this analysis were equalized for the total protein content.
In contrast, similar levels of tumor necrosis factor- ? and IL-6
measured with the CBA Th1 / Th2 / Th17 kit were found in the
5 × concentrated BAL fluids obtained from both lines of OVA-
challenged mice. Only IL-17A levels, although very low, were
significantly increased in Sema4A − / − mouse BALF.
To assess a direct effect of Sema4A on the airway physiol-
ogy, we performed the airway hyperreactivity (AHR) meas-
urements by a noninvasive technique ( Figure 2g ). We found
a significant augmentation in % baseline PenH, an index of
airway obstruction, for four increasing doses of methacholine
used (from 12.5 to 100 mg ml – 1 ) in allergen-treated Sema4A − / −
mice as compared with similarly treated WT counterparts. The
baseline PenH numbers differed significantly between OVA-
treated experimental groups (0.75 ± 0.02 vs. 0.92 ± 0.03, WT vs.
Sema4A − / − mice, respectively, P < 0.0002), whereas no differ-
ence was observed with the lowest dose of methacholine for
challenge. Surprisingly, we also found that PBS-challenged
Sema4A − / − mice have a significantly higher airway reactivity to
methacholine challenges when compared with PBS-challenged
WT mice. The results obtained employing an invasive lung AHR
measurement technique correspond to those for the invasive
lung function measurements ( Figure 2g and data not shown).
As Sema4A deficiency obviously increases most measured
features of an acute allergic response, we therefore concluded
that Sema4A functions as a downregulatory molecule in the
experimental model of allergic asthma.
Both lung and BM Sema4A expression are critical for the
downregulation of allergic asthmatic response
We previously reported that in contrast to the lymphoid tis-
sues, mouse lung tissue and cell Sema4A expression is generally
low and is only slightly upregulated by allergen challenge. 20,21
Therefore, our next set of experiments was aimed to determine if
Sema4A expression by the lung-resident or BM-derived inflam-
matory cells is more critical for the regulation of the severity of
allergic airway response.
We generated BM chimeras where irradiated WT or
Sema4A − / − mice received BM from either WT or Sema4A − / −
mice. These chimeric mice were then subjected to allergen treat-
ment ( Figure 1a ). As expected and as shown in Figure 3a,b ,
OVA-challenged Sema4A − / − mice that received Sema4A − / −
BM cells, namely Sema4A − / − (donor (d)) − Sema4A − / − (recipi-
ent (r)), showed a robust allergic eosinophilic response in their
BAL and lung tissues. Concurrently, we observed significantly
lower levels of BAL eosinophilia in WT (d) − Sema4A − / − (r)
mice and Sema4A − / − (d) − WT (r) mice as compared with
Sema4A − / − (d) − Sema4A − / − (r) mice (120,900 ± 1,900 compared
with 150,800 ± 500 and 352,500 ± 1,400 cells, respectively). WT
(d) − WT (r) group demonstrated an increased number of BAL
macrophages as compared with all other chimeric mice treated
with allergen. Importantly, the level of eosinophilic infiltra-
tion into BAL in WT (d) − WT (r) chimeras was significantly
lower than that observed in a positive control of this study,
Sema4A − / − (d) − Sema4A − / − (r) mice (101,200 ± 21,200 cells
vs. 352,500 ± 1,400 cells, respectively). In addition, eosinophilia
in WT chimeras was lower as compared with the chimeric mice
involving the absence of Sema4A expression either on struc-
tural or hematopoietic cells, which additionally supports the
important regulatory role of Sema4A in the outcome of aller-
gic lung inflammatory response. The lower density and size of
allergen-induced infiltrates was also noted in the lung tissues of
corresponding chimeric mice when compared with a positive
control for this study, Sema4A − / − (d) − Sema 4A − / − (r) mice
( Figure 3b ). Acute inflammatory infiltrates in BM chimeric
mice consisted predominantly of eosinophils, neutrophils, and
lymphocytes associated mostly with perivascular and peri-
bronchial areas. The periodic acid-Schiff (PAS) staining was
the most prominent in Sema4A − / − (d) − Sema4A − / − (r) mice
where > 50 % of bronchioles were PAS positive, whereas for
other chimeric mice this number was not that remarkable, with
only 2 – 3 bronchial profiles per tissue displaying PAS positivity
( Figure 3c ). It is noteworthy that no such inflammation was
found in the BAL (data not shown) and lungs ( Figure 3b ) of
any chimeric mouse challenged with PBS. Therefore, both lung
and BM Sema4A expression is equally critical for the allergic
inflammatory response regulation. However, it is not equally
efficient for the local lung cytokine response.
Cytokine content in 5 × BALF was measured using CBA
inflammatory cytokine (data not shown) and Th1 / Th2 / Th17 kits
( Figure 3d ). Consistent lower levels of IL-6 and tumor necrosis
factor- ? were detected in BALF of WT(d) − Sema4A − / − (r) mice
as compared with other chimeric counterparts. Interestingly, we
observed the increased local IL-17A levels ( Figure 3d ) in both
chimeric mice lacking Sema4A lung tissue expression.
Sema4A − / − CD4 + T cells are more effective in transferring
allergic response than WT CD4 + T cells
In the lymphoid tissues, Sema4A is expressed on APCs, mainly
on DCs, 9,15,20,21 and function as a costimulator of T-cell acti-
vation. 10,11 We found that lung tissue DCs did not show any
measurable Sema4A cell surface expression in either steady-state
or inflammatory condition. 20,21 On the other hand, it is also
known that Sema4A expression is induced on activated CD4 +
T cells mainly of Th1 phenotype 15 that might either downregu-
late 22 or potentiate 23 the allergic inflammatory response. As
reported above ( Figure 2e,f ), we did not find any significant
differences in the BALF interferon- ? contents between WT
and Sema4A − / − mice, suggesting that Sema4A deficiency does
not affect the overall negligible Th1 response to allergen in this
model. Based on all of the above, we next asked the question
whether Sema4A expression by CD4 + T cells or DCs is more
critical for the regulation of the allergic response severity. For
that purpose, we isolated DCs and CD4 + T cells from the lungs
and spleens of allergen-challenged Sema4A − / − mice and trans-
ferred them into naïve mice intranasally followed by three con-
secutive OVA nebulizations over a period of 72 h ( Figure 1b ).
MucosalImmunology | VOLUME XX NUMBER X | MONTH 2012
PBSOVA PBSOVAPBS OVA
Absolute number of BAL
cells, × ×103
WT into Sema4A–/–
Sema4A–/– into WTSema4A–/– into Sema4A–/–
WT into Sema4A–/–
Sema4A–/– into WT Sema4A–/– into Sema4A–/–
WT into Sema4A–/–
Sema4A–/– into WTSema4A–/– into Sema4A–/–
Figure 3 Semaphorin 4A (Sema4A) expression on bone marrow (BM)-derived cells and lung-resident cells is critical for the downregulation of
allergic airway response. The indicated BM chimeras were generated as described in the Method. Nonirradiated wild-type (WT) mice and the chimeras
were immunized with Alum or ovalbumin (OVA) / Alum and challenged as indicated. ( a ) Lungs were lavaged and the cells in the bronchoalveolar lavage
(BAL) were analyzed by the differential counting. The average numbers of BAL total cells (white bars) macrophages (black bars), eosinophils (hatched
bars), lymphocytes (dotted bars) and neutrophils (grid bars) ± s.e.m. are shown ( n = 3 – 4 mice per group). * , † , # P < 0.05, differences in absolute numbers
of total cells, macrophages and eosinophils, respectively, in BAL of Sema4A − / − (donor (d)) − Sema4A − / − (recipient (r)) vs. WT (d) − Sema4A − / − (r)
or Sema4A − / − (d) − WT (r). WT (d) − WT (r) group was assessed in a separate experiment. ( b ) Representative lung histology (hematoxylin and eosin
(H & E), × 40; inserts, × 100) from phosphate-buffered saline (PBS)- and OVA-challenged mice is shown. ( c ) Representative periodic acid-Schiff stain
(PAS) for mucous cell hyperplasia. Magnification used for pictures is × 40. ( d ) Cytokine contents in concentrated BAL measured using a Cytometric
Bead Array (CBA) Th1 / Th2 / Th17 kit. Data are shown as FlowJo-generated dot plots and CBA software-calculated cytokine concentrations for polled
samples from two mice in one of two representative experiments. IL, interleukin; TNF- ? , tumor necrosis factor- ? .
VOLUME XX NUMBER X | MONTH 2012 | www.nature.com/mi
We have found that Sema4A − / − CD4 + T cells but not DCs were
able to induce a substantial allergic eosinophilic response in the
cell-recipient WT and Sema4A − / − mice ( Figure 4 ) upon in vivo
Ag challenges. Moreover, Sema4A − / − CD4 + T cells were as
effective in WT hosts as in Sema4A − / − hosts for the induction
of eosinophilia after allergen challenge, as the relative numbers
of BAL eosinophils were not different between the cell-recipi-
ent groups. At the same time, WT CD4 + T cells were not as
efficient to transfer the allergic response as Sema4A − / − CD4 +
T cells. Examination of histology slides demonstrated the pres-
ence of multifocal perivascular and peribronchial mono-lympho-
eosinophilic infiltrates in the lungs of WT and Sema4A − / −
mice that received adoptively transferred Sema4A − / − CD4 +
The downregulatory effect of Sema4A on allergic airway
inflammation is associated with increased Treg numbers
Considering an emerging critical role of Tregs in the Th2
response suppression, 22,23 we analyzed the numbers of Tregs
in the lungs of WT and Sema4A − / − mice subjected to the
allergen priming and challenges. For this flow cytometry
analysis we employed corresponding Abs for the Treg detec-
tion ( Figure 5a ). We found significant differences in the rela-
tive and absolute numbers of lung Tregs between WT and
Sema4A − / − mice (46.9 – 51.7 % and 31.1 – 38.0 % Foxp3 + cells
among CD25 + CD4 + T cells, respectively). At the same time,
the numbers of both CD8 + and CD4 + T cells in the OVA-
challenged Sema4A − / − mice as compared with similarly treated
WT mice were increased in the lungs (5.4 ± 0.4 and 8.1 ± 0.5 %
compared with 2.4 ± 0.5 % and 6.1 ± 0.2 % , respectively, P < 0.003
and P < 0.006) and spleens ( Figure 5a,b ), whereas spleen B-cell
numbers were downregulated ( Figure 5b ). This suggests that
a defined increased inflammatory response in Sema4A − / −
mice is associated with increases of T-cell lung infiltration but a
downregulation of Tregs.
Sema4A deficiency upregulates the in vitro recall T-cell
response to OVA 323 – 339 peptide but not to OVA protein
As Sema4A expression on APCs is reported to be a critical
costimulator for Ag-specific T-cell activation, 10 we analyzed
the in vitro spleen mononuclear cell (MNC) response to OVA
restimulation ( Figure 6 ). Employing two distinct methods
for T-cell proliferation measurement, we have found that a
proliferative response to a whole OVA protein was not affected
by Sema4A deficiency, whereas the response to OVA 323 – 339 pep-
tide was significantly upregulated ( Figure 6a,b ). As Tim-2 is a
reported functional Sema4A receptor on T cells, 10 we concluded
that Sema4A / Tim-2 interaction may play an important but not
Sema4A–/– DCSema4A–/– CD4+ T cells
0.7 ± 0.3 % 0.7 ± 0.3 %20.0 ± 5.6 %
1.0 ± 1.0 % 1.5 ± 1.3 % 24.8 ± 10.4 %
WT DCWT CD4+ T cells
1.4 ± 0.7 %8.1 ± 2.9 %
Figure 4 CD4 + T cells but not dendritic cells (DCs) from ovalbumin (OVA)-challenged Sema4A − / − mice transfer allergic eosinophilic response to
naive mice followed by allergen challenges. Mice that received either lung DCs (CD11c + CD11b + cells) or spleen CD4 + T cells obtained from either
OVA-challenged wild-type (WT) or Sema4A − / − mice were subjected to three consecutive allergen nebulizations and assessed for lung inflammatory
response 24 h later ( n = 3 – 5). Allergen-challenged mice that did not receive cell transfer served as controls. Note the inflammatory cell accumulations in
the lung tissues of WT and Sema4A − / − mice after Sema4A − / − CD4 + T cell transfer followed by allergen nebulizations. Sema4A, semaphorin 4A.
MucosalImmunology | VOLUME XX NUMBER X | MONTH 2012
exclusive role in the downregulation of the in vivo response to
allergen. This conclusion is based on the reported above obser-
vation that the in vitro T-cell recalled response to a whole protein
was not compromised by Sema4A deficiency, and on the results
of our previous study showing that the specifics of the lung tis-
sue Sema4A and its receptors peptide was affected by Sema4A
deficiency. More prominent effect was found on Th2 cytokine
(IL-5 and especially IL-13) production by OVA 323 – 339 -restimu-
lated spleen MNCs ( Figure 6c ). No significant differences in the
cell culture supernatant levels of IL-4, interferon- ? , IL-6, and
IL-10 were found between WT and Sema4A − / − OVA 323 – 339 -
restimulated spleen MNCs. The noted higher levels of IL-17A
in Sema4A − / − cultures (7.8 ± 1.8vs. 4.1 ± 2.0 pg ml – 1 in WT mice)
were not statistically significant.
In this study we demonstrate that Sema4A, which initially
was reported to act as a critical positive regulator of T-cell acti-
vation and function, plays a downregulatory role in allergic
airway inflammatory response. Its absence in vivo in Sema4A − / −
mice upregulated many features of allergic response such as
eosinophilic BAL and lung tissue infiltration, mucous cell
hyperplasia, AHR to methacholine challenges, sera Ag-specific
IgG1 / IgG2b / IgE contents, and IL-13 levels in BAL, sera, and
The latter is especially interesting as it is well known that IL-
13 is a critical Th2 cytokine for the induction and regulation of
allergic response. 24 – 28 When IL-13 was blocked systemically,
it resulted in a complete reversal of the allergen-induced AHR
and mucus overproduction in the mouse experimental model of
asthma. 24 However, this blockade did not affect significantly air-
way eosinophilia and sera OVA-specific IgE levels. Nevertheless,
when IL-13 was blocked locally, allergen-induced pulmonary
eosinophilia was significantly attenuated. 25 Furthermore, the
importance of IL-13 in the induction and regulation of airway
eosinophilia was defined in the studies employing a recom-
binant IL-13 administration to the mice 24,25 that alone, in
IL-4R ? – dependent manner, was able to induce eosinophilic
0 380 2.91
21.8 61.3 0.4843.8
Figure 5 Increased T-cell and decreased regulatory T cell (Treg) numbers are associated with a heightened allergic response in Sema4A − / −
mice. ( a, b ) Wild-type (WT) and Sema4A − / − mice were immunized and challenged with ovalbumin (OVA). Lungs were digested with collagenase-
DNAse and spleens were sterilely processed omitting a digestion step. Single-cell suspensions were analyzed by fluorescence-activated cell
sorting (FACS) for the expression of CD4 and CD8 T-cell markers. ( a ) Gated CD4 + T cells were further analyzed for the expression of CD25 and
Foxp3 and the relative number of CD4 + Foxp3 + cells was ascertained in comparison with a control rat IgG protein stain. ( b ) Spleen T- and B-cell
relative numbers in one out of three representative independent experiments are shown. Sema4A, semaphorin 4A.
VOLUME XX NUMBER X | MONTH 2012 | www.nature.com/mi
infiltration and time-dependent increases in total sera IgE lev-
els. 26 Therefore, IL-13 alone is able to control a full asthmatic
phenotype. Indeed, studies in IL-13 transgenic mice have dem-
onstrated the IL-13-dependent MNC / eosinophil inflammation,
AHR, and mucous cell metaplasia. 27 We found a clear associa-
tion of the increased lung local IL-13 levels and increased mucus
secretions observed in OVA-challenged Sema4A − / − mice as
compared with WT mice ( Figure 2c,d ). The importance of
IL-13 in the mucous cell hyperplasia was demonstrated in sev-
eral previous studies that have shown that the mice lacking IL-13
[ref. 28] or IL-4R ? (a common receptor subunit for IL-4 and
IL-13[ref. 29]) or STAT6 (the IL-4R ? signaling component 30 )
lack goblet cell hyperplasia in response to Ag challenges.
Needless to say, the mechanism of IL-13 regulation by Sema4A
remains to be determined.
Following OVA treatment, the Sema4A deficiency resulted in
increased AHR in response to increasing doses of methacholine
as compared with the similarly treated WT mice ( Figure 2g ).
Interestingly, PBS-treated Sema4A − / − mice show higher airway
reactivity when compared with PBS-treated WT mice. We have
shown previously that OVA treatment leads to the induction
of Sema4A expression in the airway smooth muscle cells. 20,21
Therefore, it might play some currently undefined role in the
smooth muscle cell function. The lack of Sema4A may increase
the sensitivity of the response of smooth muscle cells to an
external pharmacological contractile stimulant such as metha-
choline, resulting in a higher airway reactivity that we observed
in PBS-treated Sema4A − / − mice. 31 The increased AHR in OVA-
treated Sema4A − / − mice might also be directly associated with
increased local IL-13 levels. 26,27
Sema4A deficiency is also associated with a systemic Th2
dysregulation ( Figure 6c ). Previous observation has shown
the induction of Sema4A expression on activated Th1 cells
and its necessity for the optimal in vitro Th1 generation and
T-bet expression that significantly affected Th1 / Th2 differen-
tiation. 15 At the same time, the in vivo Th1 cell response (inter-
feron- ? and IgG2b production) to Ag was downregulated in
Sema4A − / − mice, whereas Th2 response (IL-4 and IgG1 pro-
duction) was not affected. It is noteworthy that we did not find
any significant differences in interferon- ? contents in BALF
( Figure 2e,f ) and Ag-restimulated spleen cell culture super-
natants (data not shown) between Sema4A − / − and WT
mice. Interestingly, we find increases in the sera OVA-specific
Th2-dependent IgG1 and IgG2b 32 levels in allergen-treated
Sema4A − / − as compared with WT mice ( Figure 2e ). The seem-
ing discrepancy in the results could be explained by the different
immunization protocols and different adjuvants used. Indeed,
Sema4A − / − mice immunized with OVA in complete Freund ’ s
adjuvant CFA showed a substantially lower recall response
in the delayed-type hypersensitivity model as compared with
WT mice. 15
Our study of the in vivo response of chimeric mice to aller-
gen has shown that Sema4A on BM-derived inflammatory cells
and on lung-resident cells is critically important for the allergic
response regulation ( Figure 3a ). However, the AHR examination
of chimeric mice demonstrated no differences between groups
(data not shown), suggesting that the intensity of inflammatory
response might dissociate with AHR, 33 and that lung-resident
cell Sema4A expression, airway smooth muscle cells in particu-
lar, 20,21 most probably plays a protective role.
The especially interesting observation includes a low but
significant increase of IL-17A in Sema4A − / − mice in allergen-
induced inflammation model ( Figure 2f ) and in chimeric mice
lacking Sema4A on lung-resident cells ( Figure 3d ). The role of
Th17 cytokines IL-17A and IL-17F in asthma has been a subject
of numerous recent basic research and clinical studies. 34 – 38 Recent
studies in animal models have shown that the allergic sensitiza-
tion through the airways but not through the peritoneum induced
a strong Th17 response with AHR and lung neutrophilic but not
0 0.0010.011 100
OVA, mg ml–1
OD (450 nm)
Cytokine, pg ml–1
Figure 6 Semaphorin 4A (Sema4A) deficiency does not affect
the in vitro recall T-cell response to a whole allergen but upregulates
the response to the OVA 323 – 339 peptide challenge. ( a – c ) Spleen
mononuclear cells (MNCs) were obtained from allergen-treated wild-
type (WT; open circles or bars) and Sema4A − / − (black circles or
bars) mice as described in the Methods. Cells were stimulated with
either increasing doses of ( a ) ovalbumin (OVA) or ( b , c ) 200 ? g ml – 1 of
OVA 323 – 339 peptide for 72 h. ( a , b ) Proliferation was measured in triplicate
wells. [H] 3 was added to the cultures for the last 24 h before cell harvest.
Data are presented as mean c.p.m. ± s.e.m. of [H] 3 incorporation in 72 h
cultures. ( b ) Means and s.e.m. are shown for the measured optical
density (OD) of cells incubated with WST-1 solution for the colorimetric
proliferation visualization. Means and s.e.m. of [H] 3 incorporation in
72 h cultures. ( c ) Supernatants were taken at 72 h from the in vitro cell
cultures restimulated with OVA 323 – 339 peptide. Interleukin (IL)-5 and
IL-13 contents are expressed as the average of corresponding cytokine
concentration for two representative samples for each point.
MucosalImmunology | VOLUME XX NUMBER X | MONTH 2012
eosinophilic inflammation. 36 IL-17R deficiency downregulated
the numbers of tissue-infiltrating neutrophils but upregulated
eosinophil numbers in this model. The other study employing
intraperitoneal OVA sensitization has shown that CD11b + F4 /
80 + macrophages but not T cells in the airways of challenged
mice were the major cellular source of IL-17. 35 Interestingly, stud-
ies in human asthmatic patients have shown the increased num-
bers of IL-17 + cells in sputum and BAL in subjects with asthma
as compared with control nonasthmatic subjects. 34 Moreover,
eosinophils were active producers of this cytokine that was also
detected in a subset of sputum and BAL T cells. We did not find a
significant difference in IL-17A levels in supernatants harvested
from spleen MNC cultures with OVA peptide, although there was
a trend for a higher concentration of this cytokine in Sema4A − / −
cell cultures. Based on all of the above, it is tempting to conclude
that Sema4A regulates, either directly or indirectly, Th17 response
to allergen systemically and in the lung.
We show that allergic response was equally efficient in both
WT and Sema4A − / − mice after Sema4A − / − CD4 + T cell
transfer, whereas the in vivo Ag-primed lung Sema4A − / − DCs
were not able to transfer such response ( Figure 4 ). It is cur-
rently well established that T cells of Th2 phenotype are criti-
cal for asthma initiation and progression. 18,19 DCs are critical
for Th2 priming and necessary for the development of both
acute and chronic disease phenotypes. 39 We were interested to
determine whether allergen-primed Sema4A − / − DCs or CD4 +
T cells are able to transfer the allergic inflammatory phenotype
to naive mice followed by allergen nebulizations. It is known
that OVA treatment through the airways without any adju-
vant does not lead to the inflammatory response 40,41 that we
observed in the control allergen-nebulized WT and Sema4A − / −
animals in this study ( Figure 4 ). The effective transfer of the
allergic disease by allergen-primed WT CD4 + T cells has been
described previously 40,42 as well as the ability of the in-vivo-
transferred allergen-primed BM-derived WT DCs to induce
asthmatic responses. 43,44 However, lung DCs, most probably
because of their low number and difficulties in isolation, were
used only in one cell transfer study. 45 This particular study has
shown that two distinct subpopulations of lung DCs, namely
CD11c high CD11b low and CD11c low D11b high when adoptively
transferred to OVA-sensitized and challenged mice before last
allergen nebulization, inhibited AHR and lung inflammation.
It is noteworthy that sorted lung DCs used for our study were
CD11c + CD11b + and, therefore, consisted of these two differ-
ent lung DC subpopulations. Again, as noted above, sorted lung
DC subpopulations were transferred to the allergen-primed and
-challenged mice followed by a single challenge, 45 whereas we
used naive DC recipient mice.
As expected, based on the observed increased severity of
allergic response in Sema4A − / − mice, we found increased num-
bers of T cells in the lung tissue single-cell suspensions using
a flow cytometry assay with corresponding Abs ( Figure 5 ).
However, Treg numbers were downregulated in OVA-
challenged Sema4A − / − mouse lungs. It is well established
that CD4 + CD25 + Foxp3 + Tregs downregulate Th2 response
by multiple mechanisms and, vise versa, that Th2 cytokines
display a downregulatory effect on Tregs. 46 Therefore, the increased
allergic response in Sema4A − / − mice compared with WT mice
as reported here can be explained, in part, by the increased
local IL-13 levels ( Figure 2e ) and decreased Treg numbers
( Figure 5 ). The decreased Treg numbers in Sema4A − / − CD4 +
T-cell preparations used in the cell transfer experiments can also
explain the superior efficiency of Sema4A − / − CD4 + T cells as
compared with WT CD4 + T cells in transferring the allergic
response to naive mice ( Figure 4 ).
In conclusion, our previous observations on Sema4A receptor
expression 20,21 and our new functional data reported here sug-
gest that a downregulatory effect of Sema4A in allergic asthma
is a complex venue that cannot be simplified by its role in DC /
T-cell interaction and involves other cells, lung-resident cells,
and inflammatory cells, many of which express functional recep-
tors for Sema4A such as Plexin B1 and Plexin D1. 20,21 These
Plexins play important roles in cell migration 47 and angiogen-
esis, 14 both of which are critical components of asthma patho-
genesis. If expressed on immune cells, 20,21 these Plexins may
also play a role in their activation and function.
Mice . The generation and characterization of Sema4A − / − mice
has been described in detail previously. 15 C57BL / 6 mice (WT) were
purchased from Taconic (Hudson, NY). Mice were bred and main-
tained under specific pathogen-free conditions within the animal
facility at University of Maryland School of Medicine. All procedures
on mice were performed according to the animal protocol approved
by the animal care and use committee of the University of Maryland
School of Medicine. Age- and sex-matched mice were used in all
Anesthetic . Avertin in dose of 0.3 or 2 mg kg – 1 by intraperitoneal injec-
tion was used as previously described 32 to anesthetize or kill the mice,
Experimental model of allergic airway response . Mice were treated
with chicken OVA (Sigma, St Louis, MO) as described previously 20
( Figure 1a ). Briefly, 100 ? g OVA per 2 mg Alum per 200 ? l was deliv-
ered intraperitoneally to WT and Sema4A − / − mice on days 0 and 5.
Control mice were injected with sterile endotoxin-free PBS / Alum. On
days 12 and 14, mice received a 40-min aerosol challenge of either PBS
(control animals) or 1 % (w / v) OVA using Invacare Envoy aerosol com-
pressor (Elyria, OH). At 24 h after last nebulization, the AHR in response
to the increasing doses of methacholine was measured as an indicator of
changes in the airway resistance. Mice were killed 48 h after the last OVA
nebulization for other analyses.
Generation of BM chimeras . Bone marrows were collected and single-
cell suspensions were prepared as detailed previously. 30 BM recipient
mice were irradiated with a single dose of 500 Rad using Sheppard Mark
I model 68 irradiator (San Fernando, CA). Within 6 h after irradiation,
8 × 10 6 BM cells were injected intraperitoneally into the recipient mouse.
Allergen treatment according to the defined above protocol ( Figure 1a )
started 6 weeks after transfer.
In vivo adoptive cell transfer . For the adoptive cell transfer experi-
ments, the cell-donor mice were treated with OVA as described above
and shown in Figure 1a . Lungs and spleens were harvested on days 16
and 19, respectively, and processed sterilely to obtain single-cell suspen-
sions. 48,49 Lung DCs were isolated using anti-CD11c (N418) plus anti-
CD11b (120-000-300) beads and LS-positive selection columns (all from
VOLUME XX NUMBER X | MONTH 2012 | www.nature.com/mi
Miltenyi Biotechnology, Auburn, CA). Spleen CD4 + T cells were isolated
using EasySep Negative Selection Mouse CD4 + T-cell Enrichment Kit
and an EasySep column-free magnet, both from Stem Cell Technologies
(Vancouver, Canada), according to the manufacturer ’ s instructions. The
purity of isolated DCs (90 % ) and CD4 + T cells (95 % ) was ascertained
by flow cytometry analyses. Enriched cell populations were resuspended
in sterile endotoxin-free PBS and 2 × 10 6 DCs per 50 ? l per mouse or
3 × 10 6 CD4 + T cells per 50 ? l per mouse were transferred intranasally to
naive mice. The cell-recipient mice were then nebulized with 1 % OVA in
PBS (or PBS alone for control animals) on days 1, 2, and 3 after transfer
( Figure 1b ). On day 4 after transfer, BAL and serum were retrieved and
lung tissues were collected.
AHR measurements . AHR measurements to methacholine challenges
were performed 24 h after last Ag nebulization using either noninvasive
(BUXCO Electronics, Wilmington, NC) or invasive (FlexiVent, SCIREQ,
Montreal, Canada) techniques as previously described. 50,51
Histochemistry . Core Facility at the Center for Vascular and
Inflammatory Diseases was used for histochemistry (hematoxylin and
eosin and PAS stains) of deparaffinized lung tissues. 30
Cellular composition and cytokine / chemokine content in BALs and
lung tissue lysates . BALs were performed 48 h after last Ag nebulization,
cells and BALF collected, cytospin made, and cell counts performed as
described earlier. 51 BAL and lung lysate cytokine and chemokine levels
were determined using Searchlight Proteome Array (Aushon, Billerica,
MA), enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems,
Minneapolis, MN), and CBA kits (552364, BD Biosciences). 30,32 Array and
CBA data were analyzed using the ArrayVision software (Piscataway, NJ)
and FlowJo plus BD CBA softwares (Ashland, OR), respectively. All ELISA
plates were read on the Emax Precision Microplate reader (Molecular
Devices, Sunnyvale, CA) using the manufacturer-specified wavelengths
for each assay. Intact BALFs were used in the Proteome Array and ELISA.
In the CBA assays, whole-lung tissue lysates 51 and 5 times concentrated
(Amicon Ultra 3K membranes, Millipore) BALFs were used.
Sera Ab measurements . OVA-specific IgG Ab levels in sera were
determined using corresponding IgG subclass standards and
AP-labeled anti-IgG Ab (Southern Biotechnology, Birmingham,
AL) as previously described. 32 Plates were covered with an optimal
concentration of 10 ? g ml – 1 of OVA (Sigma) defined in the previous
dose – response ELISA assays. OVA-specific IgE levels were measured by
ELISA (MD Bioproduct, St Paul, MN) according to the manufacturer ’ s
instruction. Total serum IgE was measured by ELISA using matching
antibody pairs (R35-72 and R35-92) obtained from BD Pharmingen
(San Jose, CA) as previously described. 32
Proliferation assays . Cell proliferation was measured using either
[H] 3 incorporation assay or Quick cell proliferation assay kit (ab65473,
Abcam, Cambridge, MA). Briefly, single-cell suspensions were
prepared from spleens of either PBS- or OVA-challenged mice on day
5 after challenge. 32 Spleen MNCs were plated to a density of 1 × 10 6 cells
per 200 ? l in 96-well tissue culture plates (Cellstar, Greiner, Monroe,
NC) and stimulated with either ConA (10 ? g ml – 1 ), lipopolysaccharide
(100 ? g ml – 1 ), OVA (from 0.001 to 100 mg ml – 1 ), or OVA 323 – 339 peptide
(200 ? g ml – 1 ) as previously described. 30,32,49 After 72 h of incubation,
[H] 3 thymidine (Perkinson-Maer, Waltham, MA) was added to the
wells and plates were harvested on Packard Filtermate harvester
(Packard Instruments, Meriden, CT) 24 h later. In a colorimetric pro-
liferation assay, 10 ? l of tetrazolium salt WST-1 solution was added
to each well followed by 4 h of further incubation. The plates were
read in the ELISA plate reader at 450 nm with a reference wavelength
of 650 nm.
Flow cytometry . Flow cytometry of lung and spleen single-cell
suspensions was performed as previously described 30,48 using the
BD Biosciences (San Jose, CA) Abs for the following cell markers:
I-A b (PE), CD4 (FITC), CD8 (PerCP), CD25 (PE), and B220 (FITC).
Intracellular staining for Foxp3 was done using anti-Foxp3 (APC) Ab
or rat IgG2a (APC) isotype control Ab (both from eBioscience, San
Diego, CA). 30 Cells gated by forward- and side-scatter parameters were
analyzed using either the CELLQuest or FlowJo software (Ashland, OR)
at the Flow Cytometry Facility, Center for Vascular and Inflammatory
Statistics . Data were summarized as mean ± s.e.m. To calculate the
significance levels between the experimental groups, Student ’ s t -test
(Microsoft Excel) and Mann – Whitney test (Prizm-4, San Diego, CA)
We thank Dr Stephen Liggett (Department of Medicine) at the University
of Maryland School of Medicine for the use of the FlexiVent machine in his
laboratory. We thank Dr Sun Min Lee, a Scientist at the BD Biosciences,
for the calculations of CBA data using the company ’ s CBA software. This
work was supported by NIH grant R21AI076736 (to S.P.C.).
The authors declared no conflict of interest.
© 2012 Society for Mucosal Immunology
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