IFN - Ehrlichia
IFN - Ehrlichia
IFN - Ehrlichia
a r t i c l e i n f o a b s t r a c t
Article history: Ehrlichia canis is an obligate intracellular bacterium that infects the macrophage–monocyte
Received 27 December 2012 cells of dogs, causing canine monocytic ehrlichiosis. Interferon-␥ (IFN-␥), along with other
Received in revised form 2 September 2013
cytokines, mediates the immune response to such intracellular bacterial invasions. To deter-
Accepted 23 September 2013
mine the role of IFN-␥ in the immunity of dogs to E. canis infection, peripheral blood
mononuclear cells (PBMC) and white blood cells (WBC) were collected from E. canis-infected
Keywords:
dogs and added to a culture of E. canis in DH82 cells. The number of E. canis inclusion-positive
Cellular resistance
Dog cells was significantly reduced in cultures containing PBMC and WBC from E. canis-infected
Ehrlichia canis dogs compared to uninfected dogs. However, this resistance was inhibited by the addition
Interferon gamma of an anti-dog IFN-␥ antibody. Resistance was also observed when PBMC were added to
the Cell Culture Inserts, which prohibited contact of PBMC to DH82 cells, while allowed
the diffusion of soluble cell products. The results of this study indicate that resistance was
not dependent on cell to cell contact, but was associated with soluble cell products, such
as IFN-␥. The addition of recombinant canine IFN-␥ to the E. canis culture also reduced the
number of infected cells. A commercial recombinant canine IFN-␥, which is sold in Japan,
was also effective at reducing E. canis-infected cell number. These results indicate that IFN-
␥ has an inhibitory effect on the frequency of E. canis-infected cells in vitro and that contact
between effector and target cells is not necessary for the resistance.
© 2013 Elsevier B.V. All rights reserved.
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http://dx.doi.org/10.1016/j.vetimm.2013.09.014
T. Tajima, M. Wada / Veterinary Immunology and Immunopathology 156 (2013) 200–204 201
us to hypothesize that canine IFN-␥ mediates resistance to a 24-well culture plate with 5 × 105 WBC or PBMC cells, and
E. canis infection at the cellular level. To test the hypoth- were placed in a total volume of a 1.0 ml/well. On another
esis, we examined the inhibitory effect of IFN-␥ on the plate, 1 g/well of anti-canine IFN-␥ antibody, which was
frequency of E. canis-infected cells in vitro. produced by goats (R & D Systems, Inc., Minneapolis, MN,
USA), was added to the culture. The cells were collected
2. Materials and methods from 4 separate wells on days 2, 3, and 4 of culture, and
Cytospin smears were prepared as described previously
2.1. Animals and design (Barnewall and Rikihisa, 1994). The smears were stained
with Diff-Quik (Sysmex Co., Kobe, Japan), and examined for
E. canis Oklahoma strain was kindly supplied by Dr. Y. the presence of inclusions. At least 120 cells per well were
Rikihisa, The Ohio State University, Columbus, OH, USA. examined, with the mean and standard deviation of the
The agent was cultured in DH82 cells, which is a dog percentage of inclusion-positive cells in the 4 wells being
macrophage–monocyte cell line established from canine calculated.
malignant histiocytosis (Wellman et al., 1988), using Dul- Cell Culture Inserts (BD Falcon, Franklin Lakes, NJ, USA)
becco’s modified Eagle’s MEM (D-MEM), containing 5% were placed into the wells of a 24-well culture plate con-
fetal calf serum. taining E. canis-inoculated DH82 cells. PBMC from infected
Two female beagles (of 2–3 years in age) were intra- or uninfected dogs, 1 × 105 cells was added to each Cell Cul-
venously inoculated with 5 × 106 E canis-infected DH82 ture Insert, and cultured for 3 days. Smears of each DH82
cells (of which 93% of the cells were infected) in 5 ml culture were prepared, stained, and examined for the pres-
of D-MEM. Before inoculation, both dogs tested nega- ence of inclusions.
tive for E. canis using indirect fluorescent-antibody (IFA)
testing and Polymerase Chain Reaction (PCR), as previ-
2.4. Effect of recombinant canine IFN- (r-IFN-) and
ously described (Wen et al., 1997). Although slight fever
commercial recombinant canine IFN- (com-r-IFN-) on
(39–40 ◦ C, 8–10 days post-inoculation [DPI]) was observed
the frequency of E. canis-positive cells
in both dogs, they had good appetites, with no clinical
abnormalities being observed during the experiment. E.
To examine the effect of r-IFN-␥ (R & D Systems, Inc.),
canis infection was confirmed by PCR using DNA extracted
r-IFN-␥ was added to E. canis-inoculated DH82 cells at a
from the buffy coat cells obtained from the peripheral blood
concentration of 0.625, 1.25, 2.5, and 5.0 ng/ml. A com-r-
of each dog at 14 DPI. At 16 DPI, blood was drawn from
IFN-␥, which is named INTERDOGTM (Kyoritsu Seiyaku Co.,
both dogs. Two additional female beagles (of 4–6 years of
Tokyo, Japan), has been manufactured and sold in Japan
age) were used as uninfected controls. The animal exper-
since 2005 to treat atopic dermatitis (eczema) in dogs.
imentation protocols of this study were approved by the
As r-IFN-␥ (R & D Systems, Inc.) is only used for research
Osaka Prefecture University’s Animal Research Commit-
purposes, com-r-IFN-␥ was used to examine the utility of
tee.
r-IFN- for clinical application. Commercial recombinant
canine IFN-␥ was added to the culture at a concentration
2.2. Preparation of white blood cells (WBC) and
of 0.05, 0.5, 5, and 50 ng/ml. The experiments were carried
peripheral blood mononuclear cells (PBMC)
out twice for each recombinant canine IFN-␥.
Table 1
Effect of white blood cells (WBC) collected from E. canis-infected and uninfected dogs on the frequency of E. canis-positive cells. A dog macrophage–monocyte
cell line, DH82 cells, were infected with E. canis and cultured with WBC. The cells were collected from 4 separate wells on days 2, 3 and 4 of culture. Smears
of each culture were prepared, stained, and examined for the presence of E-canis inclusions.
Dog code WBC of E. canis-infected dogs (n = 2) Dog code WBC of uninfected dogs (n = 2)
E. canis E. canis + WBC E. canis + WBC + Abc E. canis E. canis + WBC E. canis + WBC + Ab
2 I-1 16.70 ± 4.26 4.71 ± 0.49 14.88 ± 4.87 N-1 10.24 ± 0.91 13.00 ± 2.81 13.95 ± 1.16
I-2 9.85 ± 0.70 4.70 ± 2.55 8.55 ± 1.44 N-2 14.58 ± 6.13 13.61 ± 1.93 14.97 ± 2.20
Mean 13.27 ± 4.32 4.71 ± 1.59d 11.72 ± 4.43 Mean 12.41 ± 4.88 13.31 ± 2.11d 14.46 ± 1.61
3 I-1 18.94 ± 2.56 3.59 ± 1.43 16.29 ± 4.07 N-1 14.10 ± 1.32 14.83 ± 1.27 12.86 ± 3.03
I-2 12.37 ± 2.25 5.28 ± 0.94 10.51 ± 2.54 N-2 17.99 ± 1.37 16.41 ± 2.93 16.54 ± 1.61
Mean 15.65 ± 3.89 4.44 ± 1.34e 13.40 ± 4.12 Mean 16.05 ± 2.27 15.62 ± 2.11e 14.70 ± 2.80
4 I-1 34.36 ± 2.80 14.37 ± 3.68 30.74 ± 3.50 N-1 25.43 ± 3.90 29.27 ± 6.25 28.35 ± 8.79
I-2 24.17 ± 2.89 12.60 ± 1.76 21.79 ± 3.04 N-2 32.49 ± 1.66 28.59 ± 4.11 32.81 ± 2.48
Mean 29.26 ± 5.66 13.48 ± 2.65f 26.27 ± 5.30 Mean 28.96 ± 4.38 28.93 ± 4.59f 30.58 ± 6.02
a
Percentage of E. canis-positive DH82 cells; mean ± standard deviation of the 4 cultures for each dog, and 8 cultures (total of 2 dogs) for Mean.
b
Days post-inoculation.
c
Anti-dog interferon gamma antibody.
d
There was a statistically significant difference between the infected and uninfected groups (p ≤ 0.001) when using the t-test.
e
There was a statistically significant difference between the infected and uninfected groups (p ≤ 0.001) when using the t-test.
f
There was a statistically significant difference between the infected and uninfected groups (p ≤ 0.001) when using the t-test.
the culture, which indicated that IFN-␥ contributes to the 3.2. Effect of PBMC in the Cell Culture Inserts
reduction of E. canis-positive cells. However, this inhibi-
tion effect was limited to 4 days. At 5 and 6 days after Peripheral blood mononuclear cells from E. canis-
cultivation, the frequency of inclusion-positive cells was infected dogs inhibited the frequency of E. canis-positive
more than 30% in all cultures. On the basis of these results, cells on the culture plate. The Cell Culture Inserts pre-
all subsequent cultures were conducted for a maximum of vented PBMC from forming contact with DH82 cells, while
4 days. allowing the diffusion of soluble cell products. The present
The number of E-canis inclusion-positive cells was also results indicate that inhibition is not caused by cell–cell
significantly reduced in cultures containing PBMC from contact, which is the mechanism of cytotoxic T-cells, but is
the 2 E. canis-infected dogs compared to the 2 uninfected caused by soluble cell products, such as IFN- (Table 3).
dogs (Table 2). Differences were not observed between
WBC and PBMC cultures, indicating that granulocytes 3.3. Effect of r-IFN-
are not essential for inhibiting frequency of E. canis-
positive cells. Hence, PBMC were used in all subsequent A significant reduction in the incidence of inclusion-
experiments. positive cells was observed in E. canis-infected DH82 cells
Table 2
Effect of peripheral blood mononuclear cells (PBMC) of E. canis-infected and uninfected dogs on the frequency of E. canis-positive cells. A dog macrophage-
monocyte cell line, DH82 cells, were infected with E. canis and cultured with PBMC. The cells were collected from 4 separate wells on days 2, 3 and 4 of
culture. Smears of each culture were prepared, stained, and examined for the presence of E-canis inclusions.
Dog code PBMC of E. canis-infected dogs (n = 2) Dog code PBMC of uninfected dogs (n = 2)
c
E. canis E. canis + PBMC E. canis + PBMC + Ab E. canis E. canis + PBMC E. canis + PBMC + Ab
2 I-1 16.38 ± 3.03 4.66 ± 2.43 14.36 ± 3.22 N-1 16.97 ± 4.86 11.52 ± 2.23 11.05 ± 2.34
I-2 20.89 ± 3.17 5.13 ± 2.73 18.77 ± 2.28 N-2 13.37 ± 3.81 13.79 ± 0.22 14.88 ± 0.80
Mean 18.64 ± 3.51 4.90 ± 2.25d 16.56 ± 3.27 Mean 15.17 ± 4.19 12.65 ± 1.78d 12.79 ± 2.44
3 I-1 19.27 ± 1.36 7.19 ± 1.07 14.79 ± 2.84 N-1 16.76 ± 5.34 11.83 ± 1.14 12.83 ± 1.28
I-2 23.58 ± 2.92 7.13 ± 0.92 19.22 ± 1.29 N-2 18.26 ± 2.40 19.34 ± 1.45 18.79 ± 0.41
Mean 21.42 ± 2.92 7.16 ± 0.86e 17.01 ± 2.93 Mean 17.51 ± 3.67 15.58 ± 3.92e 15.81 ± 3.09
4 I-1 20.94 ± 2.59 12.60 ± 1.34 17.81 ± 1.30 N-1 23.96 ± 5.31 23.78 ± 5.80 19.52 ± 8.99
I-2 27.49 ± 4.00 12.85 ± 3.07 25.87 ± 3.95 N-2 29.32 ± 1.75 31.37 ± 0.92 29.57 ± 2.12
Mean 24.21 ± 4.38 12.73 ± 2.06f 21.84 ± 4.77 Mean 26.64 ± 4.35 27.57 ± 5.23f 24.55 ± 7.56
a
Percentage of E. canis-positive DH82 cells; mean ± standard deviation of the 4 cultures for each dog, and 8 cultures (total of 2 dogs) for Mean.
b
Days post-inoculation.
c
Anti-dog interferon gamma antibody.
d
There was a statistically significant difference between the infected and uninfected groups (p ≤ 0.001) using the t-test.
e
There was a statistically significant difference between the infected and uninfected groups (p ≤ 0.001) using the Mann–Whitney rank sum test.
f
There was a statistically significant difference between the infected and uninfected groups (p ≤ 0.001) using the Mann–Whitney rank sum test.
T. Tajima, M. Wada / Veterinary Immunology and Immunopathology 156 (2013) 200–204 203
Table 3 Table 5
Effect of peripheral blood mononuclear cells (PBMC) of E. canis-infected Effect of commercial canine recombinant IFN-␥ (com-r-IFN-␥) on the
and uninfected dogs in the Cell Culture Inserts on the frequency of E. canis- frequency of E. canis-positive cells. Each concentration of com-r-
positive cells. Cell Culture Inserts were placed into the wells of a 24-well IFN-␥ was added to E. canis-inoculated DH82 cells, which is a dog
culture plate containing E. canis-inoculated DH82 cells, which is a dog macrophage–monocyte cell line, and cultured for 3 days. Smears of each
macrophage-monocyte cell line. Peripheral blood mononuclear cells were culture were prepared, stained, and examined for the presence of E-canis
added to each Cell Culture Insert and cultured for 3 days. Smears of each inclusions.
culture were prepared, stained, and examined for the presence of E-canis
inclusions. Concentration Experiment Frequency of E. p value
of com-r-IFN-␥ canis-positive
Origin of PBMC Frequency of E. canis-positive cellsa (ng/ml) cellsa