Experimental Actinobacillus pleuropneumoniae infection in piglets with different types and levels of specific protection: Immunophenotypic analysis of lymphocyte subsets in the circulation and respiratory mucosal lymphoid tissue

Article (PDF Available)inVeterinary Immunology and Immunopathology 107(1-2):143-52 · September 2005with130 Reads
DOI: 10.1016/j.vetimm.2005.04.007 · Source: PubMed
Abstract
Actinobacillus pleuropneumoniae (APP) infection in piglets results in severe and fatal fibrinous hemorrhagic necrotizing pneumoniae. The aim of our study was to analyze changes in lymphocyte subset distribution in peripheral blood, bronchoalveolar lavage fluid (BALF) and tracheobronchal lymph nodes (TLN) in non-immune piglets upon a challenge with a high dose of APP and to compare the quality of such changes in unprotected piglets with counterparts exhibiting specific immunity mediated by high titers of colostrum-derived APP-specific antibodies and/or a low dose APP infection in the early postnatal period. Challenge with APP resulted in a massive increase in CD8-negative gammadelta T-cells in parallel with a reduction in numbers of CD3-CD8low cells in BALF independent of the type and level of immunity and this seems to be a general phenomenon associated with experimental infection. An increase in B-lymphocyte numbers in TLN was another characteristic feature accompanying APP infection in all experimental groups. In piglets with colostrum-derived APP-specific antibodies, this was associated with higher relative numbers of IgM+CD2+ lymphocytes in TLN, while B-cells with the CD2- surface phenotype apparently expanded in the absence of passive humoral immunity.

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Available from: Miroslav Toman, Dec 03, 2014
Lymphatic organ development in dogs: major lymphocyte
subsets and activity
M. Faldyna
a,
*
, J. Sinkora
b
, P. Knotigova
a
,L.Leva
a
, M. Toman
a
a
Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic
b
DakoCytomation AG, Brno, Czech Republic
Received 2 March 2004; received in revised form 18 November 2004; accepted 2 December 2004
Abstract
In the present study, we have characterized lymphocyte subsets and activity in peripheral blood, spleen, mesenteric and
popliteal lymph nodes in pups from birth till the age of one month and compared the results with the situation in the group of
three adult dogs. In neonatal pups, lower numbers of CD3
+
T-cells were detected in both the spleen and peripheral blood than in
lymph nodes. In contrast to the other compartments, CD21
+
B-cells prevailed in the spleen, which resulted in low values (<1) of
the CD3
+
/CD21
+
ratio. Low numbers of CD8
+
lymphocytes were characteristic in all compartments immediately after birth;
consequently a high CD4
+
/CD8
+
ratio has been calculated. Postnatal development was characterized by an increasing frequency
of CD8
+
lymphocytes in all organs studied. Another typical feature of the early period of life was a relative decrease of B-cell
numbers, which was compensated by an increasing proportion of T-lymphocytes, particularly in the peripheral blood and spleen.
DNA synthesis in newborn pups’ cells as measured by in vitro thymidine incorporation was surprisingly high in non-stimulated
control samples, notably in the spleen. Further development of lymphocyte activity was characterized by the decline in
spontaneous activity in all organs. Stimulation indices upon mitogen-induced proliferation increased proportionally to the
decrease in spontaneous activity. Based on our experimental data, we have concluded that pups are born with a relatively
competent immune system the structure of which, however, markedly develops during a few postnatal weeks.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Canine; Development; Lymphocyte subsets; Flow cytometry; Proliferation
1. Introduction
The dog is a very important species both as a
companion animal and a patient. Dogs may suffer
from various diseases, in which pathogenesis of the
immune system is involved (Felsburg, 1994; Pedersen,
1999) including primary immunodeficiency (Felsburg
et al., 1999) and autoimmunity (Lewis et al., 1965).
The dog has also been used as a biomedical model,
namely in studies on hematopoietic stem cell
transplantation (Ladiges et al., 1990; Wagner and
Storb, 1996).
www.elsevier.com/locate/vetimm
Veterinary Immunology and Immunopathology 104 (2005) 239–247
* Corresponding author. Tel.: +420 5 3333 1301;
fax: +420 5 4121 1229.
E-mail address: faldyna@vri.cz (M. Faldyna).
0165-2427/$ see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2004.12.002
Information on prenatal and early postnatal
development of lymphoid compartments in dogs,
however, is rather scarce (reviewed by Banks, 1981;
Felsburg, 1998, 2002; Krakowka, 1998) and little is
known about lymphocyte subset development and
activity. The dog is a species with the gestational
period of 6063 days. The thymus and lymph nodes
are colonized by lymphoid cells at the 35 and 46 day
of gestation (DG35 and DG46), respectively (Bryant
and Shifrine, 1972). Spleen cells splenocytes are
responsive to the plant lectin phytohemagglutinin
(PHA) at DG45, while rst PHA-induced stimulation
of cells isolated from the thymus has been observed 5
days later (Bryant et al., 1973). Dog fetuses mount
specic antibodies when injected with the bacter-
iophage ØX-174, ovine erythrocytes and Brucella
canis at DG40, DG48 and DG50, respectively (Jacoby
et al., 1969; Shifrine et al., 1971).
Immunological immaturity of newborn pups has
been deduced from a weaker in vitro response of blood
cells to mitogenic stimuli (Gerber and Brown, 1974;
Krakowka and Koestner, 1976), a diminished protec-
tion upon vaccination (Lewis et al., 1973) and a higher
susceptibility to distemper virus infection (Krakowka
and Koestner, 1976).
We have previously described postnatal develop-
ment of peripheral blood lymphocyte subset distribu-
tion (Faldyna and Toman, 1998; Faldyna et al., 2001a)
and their mitogen-induced activity (Toman et al., 2002);
the bone marrow mononuclear cell compartment has
been dened in another paper (Faldyna et al., 2003).
In the present study, we extend our studies on
lymphocyte subsets and activity to lymphatic organs
spleen, mesenteric and popliteal lymph nodes in pups
during the early period of postnatal life and we
compare the data with the situation in adult dogs.
2. Materials and methods
2.1. Animals
Sixteen pups (four per group) aged 1 day, 1 week,
2 weeks and 1 month and three adult dogs were
sacriced under general anaesthesia by complete
exsanguination using cardial puncture. Newborn pups
of different breeds originated from supernumerary
litters from private owners and older Beagle-pups
were from the Veterinary Research Institute, Brno,
Czech Republic. The animals were euthanized under
the agreement of the Branch Commission for Animal
Welfare of the Ministry of Agriculture of the Czech
Republic. Adult dogs (Beagles) were sacriced
because of orthopedic or cardial disorders and were
7-, 8- and 10-year-old. The samples from almost all
dogs examined in this study were used in the work
published by Faldyna et al. (2003), too.
2.2. Sample collection and cell isolation
Heparinized peripheral blood (PB) was obtained by
cardial puncture. Organ samples were collected into
RPMI1640 medium, minced and the cells were teased
as described elsewhere (Sinkora et al., 2000).
Peripheral blood mononuclear cells (PBMC) were
isolated by density gradient centrifugation using a cell
separating medium with a density of 1.077 g/ml
(Verogran, Leciva Prague, Czech Republic). Such
gradient isolation procedure was successfully used
by Strasser et al. (1998) and does not inuence
lymphocyte subset distribution in samples. Our
previous work showed no difference in lymphocyte
subset distribution between samples processed by
whole-blood lysis technique and stained after gradient
centrifugation (Faldyna et al., 2001a).
2.3. Immunostaining
After ammonium chloride-mediated lysis of ery-
throcytes in lymphoid organ suspensions, the cells were
washed twice in washing and staining buffer (WSB)
(PBS with 0.2% gelatine from cold water sh skin,
0.1% sodium azide and 0.05 mM EDTA, all reagents
from Sigma) and resuspended in WSB supplemented
with 10% non-immune heat-inactivated pig serum to
the density of 5 10
6
ml
1
. Peripheral blood leuko-
cytes were stained using the whole-blood lysis
technique as described previously (Faldyna et al.,
2001a).
Cells were stained using the indirect immuno-
uorescence technique. Primary monoclonal antibo-
dies are listed in Table 1. FITC-conjugates swine anti-
mouse immunoglobulin fraction (Sevac, Prague,
Czech Republic) diluted 1:360 was used as the
secondary immunoreagent. Samples stained with the
secondary antisera only were used as controls.
M. Faldyna et al. / Veterinary Immunology and Immunopathology 104 (2005) 239–247240
In 12 mm 75 mm ow cytometry tubes, 50 mlof
blood or cell suspension was incubated with a primary
monoclonal antibody at room temperature for 15 min.
In peripheral blood samples, erythrocytes were lysed
by adding 3 ml of a haemolytic solution (8.26 g
NH
4
Cl, 1 g KHCO
3
and 0.037 g Na
4
EDTA/1 l of
distilled water) and samples were washed twice in
WSB. Secondary antiserum was added after the
second washing and the tubes were incubated at 4 8C
for 20 min. After another washing, the cells were
resuspended in the WSB.
2.4. Flow cytometric analysis
Data were acquired on a standard FACSCalibur
TM
ow cytometer (Becton Dickinson, Mountain View,
CA) operated by the CELLQuest
TM
software. In each
sample, 10 000 to 50 000 cells were measured and the
data were saved in the list mode. Propidium iodide was
used to stain DNA in dead and damaged cells and to
exclude such events from analysis. The WinMDI
TM
or
PC-lysys
TM
software was used for data processing.
Gating was based on forward angle and right angle
scatter signals. The common leukocyte antigen
(CD45) and CD172a expression (Faldyna et al.,
2001a) was used for the ‘‘lymphogate’’ set-up and
lymphocyte purity determination. Results obtained for
the other surface markers were recalculated to 100%
of CD45
+
and CD172a
cells in the ‘‘lymphogate’’.
2.5. Lymphocyte transformation test
The activity of lymphocytes was determined using
the lymphocyte transformation test as described
previously (Toman et al., 1998; Faldyna et al.,
2001b). Briey, the density of the cell suspension
was adjusted to 10
6
ml
1
of RPMI 1640 medium
(Sigma, St Louis) supplemented with 10% precolostral
calf serum, 100 000 U/l penicillin and 0.2 g/l strepto-
mycin. Two hundred microlitres of the suspension was
transferred into the wells in a 96-well at-bottomed
microtitre plate (Gama, Ceske Budejovice, Czech
Republic). Mitogenic lectins were used at the following
concentrations: phytohaemagglutinin (PHA) 40 mg/ml,
concanavalin A (ConA) 10 mg/ml, pokeweed mitogen
(PWM) 10 mg/ml. All samples were run in triplicates.
The microplates were incubated at 37 8C under the 5%
CO
2
atmosphere for 3 days. Twenty hours before
harvesting, 50 ml medium with
3
H-thymidine (5 mCi/
ml) was added. The incorporation of
3
H-thymidine was
measured with a liquid scintillation counter (Packard
Tricarb CA 1600, Canberra-Packard Instruments). The
stimulation index (S.I.) has been calculated as the ratio
of counts per minute (CPM) in stimulated samples
versus CPM in non-stimulated controls.
2.6. Data analysis
Because of relatively small numbers of experi-
mental animals in individual groups, non-parametric
tests were used. Differences between lymphoid
compartments in newborn pups were assessed with
non-parametric ANOVA test for repeated measures
(Friedmans test) with Dunns post-test for compar-
ison of all pairs. Age-dependent changes were tested
with non-parametric KruskalWallis test. The one-tail
MannWhitney non-parametric test was used for
comparison of 1-day-old pups with adult dogs.
Differences with P < 0.05 were considered statisti-
cally signicant. All calculations were performed with
MS-Excel
1
(Microsoft Corp. Inc.) and Prizma
1
(Graph Pad Software Inc.) software.
M. Faldyna et al. / Veterinary Immunology and Immunopathology 104 (2005) 239247 241
Table 1
Monoclonal antibodies used within the study
Antibody Specicity Dilution Cell phenotype Source
CA17.2A12 CD3 1:5 T-lymphocytes Dr. P.F. Moore
a
CA13.1E4 CD4 1:10 T-helper Dr. P.F. Moore
CA9.JD3 CD8 1:10 T-cytotoxic Dr. P.F. Moore
CA2.1D6 CD21-like 1:5 B-lymphocytes Dr. P.F. Moore
CA20.8H1 gd TCR 1:5 gd lymphocytes Dr. P.F. Moore
CA12.10C12 CD45 1:10 All leukocytes Dr. P.F. Moore
DH59B CD172a (SWC3) 1:100 Monocytes + granulocytes VMRD Inc.
a
University of California, Davis, U.S.A.
3. Results
3.1. Lymphocyte subset distribution in newborn
pups
Immediately after birth, the relative numbers of
respective lymphocyte subsets differed between
lymphoid compartments studied (Table 2). When
compared to lymph nodes, lower percentage of CD3
+
T-lymphocytes was detected both in blood circulation
and the spleen. Especially spleen represented the site
with a profoundly low CD3
+
lymphocyte frequency.
On the other hand, CD21
+
B-cells strongly prevailed in
the spleen, which was reected by the CD3/CD21
lymphocyte ratio values below 1 (Fig. 1). Relatively
low numbers of CD8
+
lymphocytes were present in all
newborn compartments, which resulted in unusually
high CD4/CD8 values (Fig. 2). As expected, T-cells
bearing the TCRgd variant of the T-cell receptor (TCR)
were also unequally distributed between circulation
and individual solid lymphoid organs in neonatal
puppies. The highest relative numbers of gd T-cells
were found in the spleen (more than 5 %) and very few
gd T-cells were present in lymph nodes (Table 2).
3.2. Postnatal development of lymphocyte subset
distribution
The most pronounced age-related change in relative
lymphocyte numbers was an increase of the frequency
of CD8
+
lymphocytes in all compartments investigated
(compare Tables 2 and 3), which was accompanied by a
decrease of the CD4/CD8 values with age (Fig. 2). It is
well known that the CD8
+
lymphoid compartment
contains also CD3
NK cells in different species (Yang
and Parkhouse, 1996). Our recent, yet unpublished
results have conrmed an existence of such a subset in
dogs, which, however, always represented a marginal
population in all organs studied. As dogs are similar to
humans and mice in terms of the scarceness of
peripheral double positive (CD4
+
CD8
+
) T-cells, the
CD4/CD8 number can be regarded as an approximate
number reecting the ratio between the helper/
regulatory and cytotoxic lineages of T-cells. In other
words, with increasing age the proportion of cytotoxic
T-cells both in circulation and solid lymphoid organs
increased at the expense of CD4
+
T-cells.
Another profound effect was a signicant increase
of CD3
+
T-cells in blood and spleen, which was
accompanied by decreased relative numbers of CD21
+
B-cells therein (compare Tables 2 and 3, and Fig. 1). In
lymph nodes, on the other hand, no such effect could
be observed. Interestingly enough, the total sum of
CD4
+
+ CD8
+
lymphocytes have regularly been lower
than the proportion of CD3
+
T-cells. The existence of
CD4
CD8
double negative T-cells in dogs cannot
thus be excluded.
A comparison (non-parametric MannWhitney
test) with the situation in newborn pups is illustrated
in Table 3, too.
3.3. Proliferation activity of lymphocytes in
newborn pups
Lymphocytes from all organs and PBMC prolif-
erated upon activation with all mitogenic lectins used
M. Faldyna et al. / Veterinary Immunology and Immunopathology 104 (2005) 239247242
Fig. 1. CD3/CD21 ratio in different compartments: blood (&),
spleen (~), popliteal (^) and mesenterial (*) lymph nodes.
Puppies in the age of 1 day (1), 1 week (2), 2 weeks (3), 1 month
(4) are compared to adult dogs (5).
Fig. 2. CD4/CD8 ratio in different compartments: blood (&),
spleen (~), popliteal (^) and mesenterial (*) lymph nodes.
Puppies in the age of 1 day (1), 1 week (2), 2 weeks (3), 1 month
(4) are compared to adult dogs (5).
in our study (Table 4). Importantly, neonatal S.I. in
both organs and blood is biased to lower values by a
relatively high level of thymidine incorporation in
non-stimulated samples. Such an effect is most
pronounced in the spleen, where unusually high
amounts of spontaneously incorporated thymidine
have been recorded. The non-parametric comparison
for repeated measures (Friedmans test) has revealed
that the lymphocyte capability of being activated with
lectins as determined by the stimulation index
calculation signicantly differed (P < 0.001) in cells
isolated from different organs; stimulation index was
always higher in the lymph nodes than in the spleen
and peripheral blood. Statistical differences are shown
in Table 4.
3.4. Postnatal development of activity of lymphocytes
In the rst week after birth, spontaneous prolifera-
tion activity in the periphery decreased (Kruskal
Wallis, P < 0.05). In both the spleen and popliteal
lymph nodes, a high level of spontaneous incorpora-
tion in vitro persisted until the age of 2 weeks, which
was followed by a sudden decrease of proliferative
activity in control samples (Fig. 3). In contrast,
spontaneous proliferation in cultured mesenteric
lymph node cells and PBMC exhibited a faster
decline during the rst week of life and a steady state
low level of spontaneous incorporation was reached in
the following 3 weeks (Fig. 3). As thymidine
incorporation in control samples decreased, stimula-
tion indices reached higher values. Table 5 shows the
summary of results obtained in three adults and the
comparison with newborns.
4. Discussion
In the present study, we extend our studies on
lymphocyte subsets and activity in peripheral blood
(Faldyna and Toman, 1998; Faldyna et al., 2001a;
Toman et al., 2002) to lymphatic organsspleen,
mesenteric and popliteal lymph nodes in pups during
the early period of postnatal life and we compare the
data with the situation in adult dogs.
Immunophenotyping of cells isolated from blood
and peripheral lymphoid organs in pups and adult dogs
has revealed marked differences between relative
numbers of major lymphocyte subsets. When com-
pared to adults, peripheral blood and spleen contained
M. Faldyna et al. / Veterinary Immunology and Immunopathology 104 (2005) 239247 243
Table 2
Lymphocyte subset distribution in different compartments in 1-day-old pups (n =4)
Subset Peripheral blood Spleen Mesenteric lymph node Popliteal lymph node
CD3 (%) 55.8 8.5 (54.5) 26.3 4.3 (26.3)
*
74.6 3.9 (75.0)
*
69.0 5.9 (68.4)
CD4 (%) 47.5 4.2 (46.0) 10.8 1.1 (10.4)
*
56.2 2.6 (55.8)
*
41.2 1.3 (41.3)
CD8 (%) 5.1 1.1 (5.0) 5.0 1.1 (5.0) 2.4 0.4 (2.4) 2.6 0.3 (2.5)
gd TCR (%) 3.0 1.7 (2.8) 7.7 1.2 (7.6)
*
2.8 0.6 (3.0) 1.0 0.3 (1.0)
*
CD21 (%) 36.2 2.8 (35.8) 52.1 6.9 (54.4)
*,+
20.3 1.6 (20.1)
*
21.2 5.3 (21.4)
+
3/21 (ratio) 1.6 0.3 (1.5) 0.5 0.1 (0.5)
*
3.7 0.3 (3.6)
*
3.5 1.2 (3.3)
4/8 (ratio) 9.5 1.5 (9.2) 2.2 0.4 (2.2)
*
24.6 4.7 (23.4)
*
16.3 2.0 (16.6)
Data are expressed as mean S.D. (median). Parameters with signicant differences (P < 0.05) are marked * and +, respectively.
Table 3
Lymphocyte subset distribution in different compartments in adult dogs (n =3)
Subset Peripheral blood Spleen Mesenteric lymph node Popliteal lymph node
CD3 (%) 83.9 1.7 (84.3)
*
52.1 3.3 (51.9)
*
76.0 5.5 (74.2) 63.4 2.3 (63.9)
CD4 (%) 58.6 3.7 (57.2)
*
32.0 4.0 (32.7)
*
41.1 1.2 (41.1)
*
44.6 2.9 (46.0)
CD8 (%) 15.1 4.4 (13.1)
*
7.9 1.4 (7.5)
*
26.6 2.5 (26.4)
*
16.7 2.6 (15.7)
*
gd TCR (%) 1.6 1.1 (1.2) 7.2 2.3 (6.4) 2.2 0.9 (1.8) 0.9 0.2 (1.0)
CD21 (%) 15.7 1.8 (14.8)
*
24.7 2.3 (25.5)
*
23.6 4.6 (25.1) 32.3 0.4 (32.4)
*
3/21 (ratio) 5.4 0.7 (5.7)
*
2.1 0.2 (2.1)
*
3.3 1.0 (3.0) 2.0 0.0 (2.0)
*
4/8 (ratio) 4.1 1.1 (4.7)
*
4.2 1.2 (4.4)
*
1.6 0.2 (1.5)
*
2.7 0.3 (2.8)
*
Data are expressed as mean S.D. (median).
*
The parameters in which the signicant difference (P = 0.0571) was found when compared with 1-day-old pups (Table 2).
relatively low numbers of CD3
+
T-cells, which was
compensated by higher proportions of CD21
+
B-cells.
In addition, the CD4/CD8 ratio was very high in all
organs studied; MHC class II-restricted T-lympho-
cytes thus prevailed, while class I-restricted T-cells
represented a minor subset in the T-lymphoid
compartment. It must be kept in mind that there are
essentially no activated and memory cells in the
neonatal immune system and lymphocyte circulation
is low due to minimal antigen stimulation. Therefore
we speculate that the great majority of both T- and B-
lymphocytes isolated from neonatal puppies are
immature and naive cells. This is supported also by
previously published ndings (Somberg et al., 1996;
HogenEsch et al., 2004).
Upon exposure to myriads of antigens including
microora colonizing the external and internal
surfaces and penetrating into the body, huge activation
and redistribution of peripheral lymphocytes occur,
which results in the appearance of lymphocytes in
previously empty spaces (e.g. intestinal mucosa),
changes in lymphoid tissue size and structure
(secondary follicles), and re-circulation of newly
formed, activated subsets of lymphocytes. This is at
least partially reected by a transient, two-fold
increase of lymphocyte density in the peripheral
blood in 1-week-old pups (Faldyna and Toman, 1998;
Faldyna et al., 2001a). The most pronounced age-
related effect described in the present work was a
sudden decrease of the CD4/CD8 ratio in all organs
and circulation. Also, expansion of CD3
+
lymphocytes
occurred in the spleen and blood before weaning and
the CD3/CD21 ratio increased accordingly. Such
changes in canine peripheral blood have already been
reported (Somberg et al., 1996; Faldyna and Toman,
1998; Faldyna et al., 2001a; Heaton et al., 2002b).
Similar trends have also been described in cats, the
most related species studied (Sellon et al., 1996;
Bortnik et al., 1999; Heaton et al., 2002a), and in
children (Carver et al., 1991; Denny et al., 1992;
Heldrup et al., 1992; Neubert et al., 1998). Unusually,
high proportions of B-cells in the spleen of 48-week-
old pups have been found by Somberg et al. (1994) in
their study of T-lymphocyte development in dogs with
X-linked severe combined immunodeciency.
Our explanation of the observed phenomena is
based on the fact that, in contrast to lymph nodes,
spleen represents one of the primary B-lymphopoietic
sites, wherein immature B-cells are transported from
the bone marrow via blood circulation. Before birth,
the lymphatic tissues are lled with early stages of
peripheral lymphocyte differentiation and the overall
M. Faldyna et al. / Veterinary Immunology and Immunopathology 104 (2005) 239247244
Table 4
Activity of lymphocytes from different compartments in 1-day-old pups (n =4)
Mitogen Peripheral blood Spleen Mesenteric lymph node Popliteal lymph node
None CPM 758 594 (493)
*
9342 2643 (8990)
*,§,&
1427 491 (1367)
§
1725 619 (1545)
&
ConA CPM 4202 1746 (3585)
*,§
14904 8277 (14299)
&,+
59821 22560 (63771)
*,&
56354 17598 (57026)
§,+
SI 6.5 1.6 (7.1)
*,§
1.5 0.5 (1.6)
&,+
41.4 7.2 (41.7)
*,&
36.1 15.0 (40.4)
§,+
PHA CPM 1915 1627 (1352)
*,§
11886 5649 (11241)
&,+
28984 7737 (30967)
*,&
30747 12074 (30348)
§,+
SI 2.5 0.9 (2.3)
*,§
1.2 0.3 (1.2)
&,+
20.9 3.9 (20.3)
*,&
18.9 8.2 (19.1)
§,+
PWM CPM 1715 516 (1480)
*,§
12502 11118 (8368)
&,+
39195 21760 (43259)
*,&
45624 22519 (37168)
§,+
SI 3.7 1.1 (3.7)
*,§
1.2 0.7 (0.9)
&,+
25.7 10.4 (30.3)
*,&
27.7 13.8 (23.6)
§,+
Data are expressed as mean S.D. (median). CPM, counts per minute; SI, stimulation indexes; ConA, concanavalin A; PHA, phytohae-
magglutinin; PWM, pokeweed mitogen. Parameters with signicant differences (P < 0.05) are marked *, §, & and +, respectively.
Fig. 3. Spontaneous activity of lymphocytes from different com-
partments: blood (&), spleen (~), popliteal (^) and mesenterial
(*) lymph nodes. Puppies in the age of 1 day (1), 1 week (2), 2
weeks (3), 1 month (4) are compared to adult dogs (5).
picture reects this situation: blood and spleen contain
numerous immature B-cells on their way to the full
maturation, while in lymphatic nodes naive T-cells
strongly predominate. Upon immune system activa-
tion, T-cells start to re-circulate and increase their
numbers in blood and blood-lled organs like spleen.
Some immature B-cells, on the other hand, pass the
process of peripheral negative selection in the spleen
and re-circulate to the induction sites including lymph
nodes. Later on, effector and memory lymphocyte
populations appear and the dynamic equilibrium of
lymphocyte subsets is reached in all lymphoid
compartments.
It is not easy to explain the inversion of the CD4/
CD8 ratio in the early postanatal period of life. Bortnik
et al. (1999) concluded from their study in cats that the
maturation of CD8
+
T-cells had been preferred to that
of their CD4
+
counterparts during postnatal thymo-
poiesis. Without any kinetics data, such a conclusion
remains in the eld of speculations. Because huge
expansion and redistribution of peripheral lympho-
cytes occur upon exposure to external antigens
including intracellular bacteria and viruses, it is
probable that not only thymic but also peripheral
regulatory mechanisms are involved in the increased
numbers of MHC class I-restricted CD8
+
T-cells.
Altogether, lymphocyte subset composition in
neonatal dogs resembles the situation known in other
mammalian species studied so far with the exception
of cloven-hoofed animals, which are famous for their
high numbers of gd T-cells in the blood during early
stages of postnatal period of life (Hein and Mackay,
1991; Joling et al., 1994; Yang and Parkhouse, 1996).
Postnatal development of canine blood lymphocyte
capability of proliferating upon lectin stimulation has
been described by several authors. Gerber and Brown
(1974) observed a signicant increase in the respon-
siveness to mitogens in Beagle-pups 612-week-old,
when compared to the situation between 0 and 4 weeks
of age. The intensity of the response reached its
maximum between 6 weeks and 6 months and then
decreased signicantly. Similar results have also been
published by Krakowka and Koestner (1976). Our
previously published data (Toman et al., 2002) have
showed that blood lymphocytes in pups younger than
3 months do not reach the mitogen-induced prolifera-
tion level in adult dogs.
The S.I. values do not reect only the number of
cells synthesizing DNA upon stimulation. Extensive
cell division in the control samples, namely from the
spleen, decreases the S.I. values signicantly and it
may be dangerous to directly compare results from
experimental groups with different levels of sponta-
neous proliferation. Our results clearly show that
neonatal organs and blood are a good source of
proliferating, probably precursor stages of hematopoi-
esis, which is in a good agreement with other studies in
dogs (Bryant et al., 1973). Isolation techniques and
cultivation conditions (e.g. the quality of the plastic,
serum and medium) may also inuence the sponta-
neous proliferation capacity in vitro as individual
laboratories often differ in S.I. values in similar
samples (our and Bryant et al., 1973 results versus
Hartnett et al., 2000). The lymphoid subset composi-
tion in the blood and organ suspensions will also play
an important role in overall mitogen-induced pro-
M. Faldyna et al. / Veterinary Immunology and Immunopathology 104 (2005) 239247 245
Table 5
Activity of lymphocytes from different compartments in adult dogs (n =3)
Mitogen Peripheral blood Spleen Mesenteric lymph node Popliteal lymph node
None CPM 132 11 (133)
*
1168 768 (1208)
*
300 100 (344)
*
512 148 (535)
*
ConA CPM 6523 708 (6907) 15585 8850 (16358) 59785 31055 (67660) 57983 12060 (63098)
SI 49.5 2.5 (48.6)
*
14.3 2.2 (13.5)
*
189.2 47.8 (196.7)
*
123.7 55.0 (124.6)
*
PHA CPM 4442 1766 (3533) 5066 3969 (7068) 10992 7167 (12218)
*
10850 3391 (9776)
*
SI 34.6 16.8 (26.6)
*
3.7 2.3 (4.0) 33.9 16.5 (33.0) 24.1 15.3 (18.3)
PWM CPM 2901 215 (2834)
*
6720 2910 (7209) 24825 14057 (27280) 36161 18350 (44296)
SI 22.2 3.6 (21.3)
*
7.0 2.9 (7.7)
*
78.4 28.6 (73.7)
*
81.6 57.6 (82.8)
Data are expressed as mean S.D. (median). CPM, counts per minute; SI, stimulation indexes; ConA, concanavalin A; PHA, phytohae-
magglutinin; PWM, pokeweed mitogen.
*
The parameters in which the signicant differences (P < 0.05) were found when compared with 1-day-old pups (Table 4).
liferation. It is very well known that B- and T-cells
respond differently to plant lectins and the same can be
said about naive and memory subsets of individual
lymphopoietic lineages. The level of bleeding before
sacrice, anaesthesia and even the number of cell
washes can inuence the data. Taken together, CPM
values in stimulated neonatal lymphocytes from all
organs studied respond pretty well to mitogen
stimulation but the S.I. values are low due partially
to high CPM values in controls. There are differences
between lectins used and more detailed study
combining DNA and surface staining will be required
to dene which lymphocyte subsets proliferate
spontaneously and which neonatal cells are the targets
for lectin-induced DNA synthesis.
Acknowledgement
This study was supported by the Grant Agency of
the Czech Republic (524/00/0474, 524/02/P010).
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    • "One of the most striking findings was the apparent proportional predominance of CD8 À cd T lymphocytes recruited to the compromised uterine tissue. Although cd T lymphocytes represent only a small population in the peripheral blood and lymphoid organs of adult dogs (Faldyna et al., 2001bFaldyna et al., , 2005a ), they can be plausibly recruited to sites of inflammation as described in piglets (Faldyna et al., 2005b), where cd T lymphocytes recruited to the bronchoalveolar space after experimental infection with Actinobacillus pleuropneumoniae are also predominantly CD8 À (Faldyna et al., 2005b). Based on evidence derived from swine, in which CD8 À cd T lymphocytes occur predominantly in blood, while CD8 + cd T lymphocytes are mainly found in tissue (Yang and Parkhouse, 1996), blood can represent a source of CD8 À cd T lymphocytes. "
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