Abstract Book of EAVLD2012 - eavld congress 2012

S1 - P - 39
BOVINE BLOOD DENDRITIC CELLS IN CATTLE INFECTED WITH BOVINE LEUKAEMIA VIRUS (BLV)
Maria Szczotka, Jacek Kuźmak
National Veterinary Research Institute, Biochemistry Department, Pulawy, Poland
Bovine leukemia, dendritic cells, immunophenotype
Introduction
Dendritic cells (DCs) play an important role in the immune
system. These cells are specialized in the presentation of
antigens. DCs are leukocytes derived from bone marrow and
they are the only cells able to present antigen to naïve T cells.
They initiate primary immune response, exist in all lymphoid and
most non-lymphoid tissues and express both MHC class I and
class II antigens. They not only have an enhancing effect on the
acquired immunity development, but can induce tolerance and
can be manipulated to prevent and treat allergic and autoimmune
diseases. These cells are present in most tissues and comprise a
small fraction of the leukocytes through the body, representing
only about 1% of peripheral blood mononuclear cells. DCs are
heterogenous cells that can be divided into the few functionalldistinct
subsets on the basis of their phenotype, possibility of
migration and ability for cytokine production. They modulate both
innate and adaptive immune response.
The aim of the study was the isolation of monocytes from bovine
blood with the use of immunomagnetic beads coated with
monoclonal antibody, generation of DCs and determination of
immunophenotype and cytokine expression.
Materials & methods
Isolation of monocytes.
Investigations were performed on a group of 9 cows naturally
infected with BLV and 4 healthy, BLV-negative animals in ELISA
and PCR that served as negative control. Peripheral blood
mononuclear cells were isolated from whole blood treated with
EDTA-K 2, by standard density centrifugation in Histopaque
(density 1.077). The cells from interphase were collected,
washed twice by centrifugation and incubated with
immunomagnetic Microbeads (Miltenyi Biotec, Germany), coated
with monoclonal mouse anti-human CD14 antibody. After
incubation, cells were centrifuged, cell pellet was resuspended in
AutoMacs Rinsing Solution and placed on a magnetic column
MACS LS (Miltenyi Biotec). The magnetically labelled CD14+
cells were retained on the column and eluted after removal of the
magnetic column from the magnet. The viability of the cells was
assessed by trypan blue exclusion. The purity of the monocyte
fraction was higher than 90%, determined by FACS analysis.
Monocyte culture.
Isolated monocytes were cultured in RPMI 1640 medium
(containing FCS, glutamax and antibiotics) at 37 0 C in humidified
5%CO 2 atmosphere, in the presence of granulocyte-macrophage
colony stimulating factor (GM-CSF) and recombinant bovine
interleukin- 4. The cells were fed every 3 day and cultivated for 7-
10 days.during this period, the monocytes transformed into DCs.
Cytology of DCs
After 7-10 days, the cultured cells were dislodged by gentle
pipetting, washed, cytocentrifuged onto slides, air-dried at room
temperature and stained with Giemsa’s stain. Morphological
observations were performed under the light microscope
Olympus, equipped with computer system Lucia (Nikon).
Ultrastructure of DCs was examined using scanning electron
microscopy (SEM). Briefly, the cells were fixed in 2,5%
glutaralaldehyde in 0.1M cacodylate buffer, dehydrated in serially
graded alcohols (70%-100%), and embedded. Ultrathin sections
were cut, counterstained, and then observed under SEM.
Immunophenotype of DCs in both groups of cows was
determined in flow cytometer with the use of specific monoclonal
antibodies for CD markers and fluorescent conjugates.
Results
In normal bovine blood, the number of monocytes was very low:
1%-4%. After magnetic separation we obtained a fraction of
positively enriched (95%-97%) labelled cells. The CD14 positive
cells had typical monocyte morphology with great nucleus and
small cytoplasm. These cells cultured 24 h in the presence of
GM-CSF and IL-4 in a growth medium transformed to the
dendritic cells. Some of them were elongated and had dendritelike
processes or edges. After 72 h, almost all cells had typical
dendrites: some of them were divided on the end. DCs of infected
cows cultured in vitro had very strong expression of CD11a,
CD11b, CD11c, MHC-I and MHC-II. Expression of gp51
glycoprotein was determined in BLV infected cows.by flow
cytometry
Tab.1. The immunophenotype of dendritic cells in leukaemic and
control cattle (% cells – mean values)
BLV(+)
CD
14
CD
11a
CD
11b
CD
11c
MHC
I
MHC
II
gp5
1
1 week 83,6 42,5 89,5 58,4 84,6 17,0 5,6
2 weeks 87,9 53,6 84,9 30,0 79,5 16,4 4,1
3 weeks 92,7 63,2 95,7 31,6 77,5 20,5 5,4
BLV (-)
CD
14
CD
11a
CD
11b
CD
11c
MHC
I
MHC
II
1 week 49,4 38,8 67,3 26,5 80,4 20,5 -
2 weeks 41,6 27,3 75,7 26,5 51,3 9,2 -
3 weeks 88,4 23,4 72,2 11,2 28,9 10,5 -
4 weeks 12,7 10,4 41,3 2,1 1,4 30,7 -
gp5
1
Discussion & conclusions
The great amount of work performed in the recent years has led
to the definition of DCs as cells identified by peculiar morphologic
characteristics and specific functions, such as the ability to
activate naive T cells. Very little is known about their origin and
pathways of differentiation. Developing the immunogenic
potential of DCs for vaccines – and what is very important for
cancer therapy in humans – requires better access to this cell
type. This report demonstrates that DCs can be derived from
positively-selected monocytes in cattle. Large amounts of DCs
can be obtained after the in vitro exposure of peripheral blood
monocytes to the effects of cytokine combinations, mainly
interleukin IL-4 and GM-CSF, but the ability of monocytes to
convert to DCs in vivo has not been so far established. Although
their origins are still controversial or remain unknown, myeloidderived
and lymphoid-derived DCs lineages have been
described. Through their ability to determine the differentiation of
either Th1 or Th2 cells, distinct DCs subsets may present
antigens in vivo in an immunogenic or tolerogenic fashion. It is
important that the immunostimulatory properties of DCs are
dependent on their maturity state: immature DCs have been
shown to be capable of inducing antigen-specific inhibition of in
vivo T-cell function in humans. Moreover, DCs are potent to
improve vaccine efficacy and are still being investigated as an
important tool in cancer therapy. The availability of sufficient
numbers of efficient antigen-presenting DCs facilitates effective
vaccine development and the study of T-cell-mediated responses
to tumour-associated antigens and cancer therapy.
References
1.Banchereau, J, Steiman, R.1998, Dendritic cells and the control of
immunity. Nature, 392, 245-252
2. Dubois, B, Fayette, J.1999. Dendritic cells directly modulate B cell
growth and differentiation. J Leukoc Biol, 66, 224-230.
3. Pinchuk LM, Boyd BL, Kruger EF. 2003. Bovine dendritic cells
generated from monocytes and bone marrow progenitors regulate
immunoglobulin production in peripheral blood B cells. Comp Immunol
Microbiol Infect Dis, 26, 233-249.