ISRAEL JOURNAL OF
VETERINARY MEDICINE
VOLUME
54 (3) 1999
DETECTION OF IL-12 ON PERIPHERAL BLOOD
MONONUCLEAR CELLS OF BOVINE LEUKEMIA VIRUSINFECTED CALVES BY FACS ANALYSIS
H. Ungar-Waron, R. Paz, J. Brenner and Z. Trainin
Department of Immunology, Kimron Veterinary Institute, 50250 Beit Dagan, Israel
Abstract
An experimental model of bovine leukemia virus (BLV) infection was established
in young calves. All animals used in the experiment became infected but the patterns
of cellular response varied among individual animals during infection. At early stages
of infection a short-termed IL-12p40 gene expression in peripheral blood
mononuclear cells (PBMC) was observed which seemingly directed the subsequent
stages of infection. We were able to demonstrate that in addition to IL-12p40 cytokine
gene expression, the presence of the cytokine on PBMC of infected calves could be
detected by means of flow cytometry studies (FACS analysis). The transient nature of
its appearance made the kinetics of its manifestation difficult to follow and not in all
the calves was it possible to assess the participation of this key cytokine of cellmediated immunity in the immunopathology of BLV retroviral infection.
Introduction
In a previous paper, we reported on short-termed expression of interleukin12 (IL-12) during experimental BLV infection (1). This cytokine plays a central
role in the regulation of Th1 and Th2 cellular responses. Whereas Th1 cells
are involved in cellular immunity, Th2 cells address functions of the humoral
response. Optimal generation of Th1 CD4+ T cells and CD8+ cytotoxic T
lymphocytes (CTL) requires IL-12 expression (2). Because of the critical role
of IL-12 during different infections, the immune response in Th1-mediated
pathogen resistance can be distinguished by the kinetics of IL-12 production
(3).
We have established an experimental model of BLV infection (4). This
retrovirus induces a chronic infection in cattle leading in some cases to
persistent lymphocytosis (PL+) due to intensive polyclonal B-cell expansion
(5). In this model the early stages of infection could now be investigated and,
in particular, levels of cytokine expression. However, no uniform pattern of cell
proliferation nor of cellular immune response was observed in infected calves
throughout experimental infection (1,4).
A short-termed IL-12p40 gene expression was observed in PBMC of two out
of 4 infected animals, one to 3 weeks after infection. It was of interest to
examine whether expression of the IL-12p40 protein molecule on the cell
surface of BLV-infected PBMC could possibly be detected while it was being
secreted.
Materials and Methods
Experimental animals
Six Friesian-Holstein, 7 months old, steers were employed. They were
clinically healthy and BLV-free at the beginning of the experiment. Four calves
(numbers, 056, 077, 714, 715) were infected by intravenous injection (jugular)
with 106 bovine embryo kidney cells (BEK) persistently infected with BLV
(BEK-BLV). This cell line was obtained as described (1). Two other calves
were mock-infected each with 106 BEK cells co-cultivated with normal PBMC.
Monoclonal antibody to IL-p40
This is a product of PharMingen (20711D) developed for specifically
measuring human IL-12p40 subunit in an ELISA capture assay and was used
in this study for flow cytometry.
Enumeration of blood lymphocytes
Peripheral blood was collected by venipuncture into tubes containing EDTA
as anticoagulant, and total leukocyte counts were determined on an
automated cell counter (Technicon H. 1ETM System, Miles Tarrytown, NY)
using the manufacturer’s instructions.
Detection of antibodies to BLV
A standard diagnostic test, AGID, as recommended by the Office
International des Epizooties, (1992), was used (6). Serum samples were
tested for precipitating antibodies to commercially available BLV glycoproteins
(Pittman-Moore, Atlanta, GA) which contained both the Mr 51000 BLV
envelope glycoprotein (BLV-gp51) and a small amount of Mr 24000 viral core
protein.
Flow cytometry (FACS analysis)
PBMC were obtained from each blood sample by Ficoll-Hypaque
centrifugation, and 106 cells were incubated with
the anti IL-12p40 specific mAb for 30 min at 40C. After washing, FITCconjugated (Fab’)2 anti-mouse IgG (Fc portion) was added at an appropriate
dilution and incubated as before. After additional washings, cytofluorographic
analysis was performed in a fluorescence-activated cell analyzer (FACScan,
Becton-Dickinson, CA).
Results and Discussion
Establishment of an experimental model of BLV infection in calves
Four calves were experimentally infected with BLV as described. Infection
with BLV was assessed by detection of antibodies to BLVgp51 antigen in the
sera of all the experimentally infected animals by AGID from the fourth to fifth
week after infection. These persisted to the end of the experiment. Infection
was likewise assessed either by BLVgp51 mRNA expression or by DNA
amplification to detect provirus-infected cells as reported elsewhere (1). Mockinfected calves remained BLV-PL- negative to the end of the experiment, 6 to
12 months later.
In two calves (056 and 715), lymphocytosis developed gradually (Table 1)
and PL+ positive was established permanently after 4 weeks. Transient
lymphocytosis between the third and fifth week after infection was observed in
calf 714. PBMC counts stayed low and the state of PL- negative persisted for
at least 3 months but it eventually became PL+ by the end of the experiment.
Calf 077 stayed PL negative for 11 weeks and became PL+ after 4.5 months.
IL-12p40
We have previously reported (1) that two BLV experimentally infected
calves, 056 and 077, exhibited strong IL-12p40 mRNA expression one to
three weeks after infection which decreased to undetectable levels by 12
weeks and was not detected further before the end of the experiment 18
months later. However when PBMC of these animals were stimulated with
conA, IL-12p40 gene expression was detected throughout the entire
experimental period. Six months after infection, PBMC of calves 056 and 077,
were labeled with anti IL-p40 monoclonal antibody and examined by FACS
analysis. As seen in Figure 1, 17 to 19 percent of PBMC were IL-12p40
positive.
In PBMC of the two other calves, 714 and 715, IL-12p40 gene expression
was not detected at any stage of the infection. However by FACS analysis it
was possible to detect a noticeable amount of the IL-12p40 on PBMC of calf
714 two weeks after infection which gradually decreased during the following
weeks (Table 2). No such reactivity was detected in PBMC of calf 715.
We conclude that the kinetics of expression of IL-12p40 cytokine gene in
PBMC or its detection on their surface have no direct connection with
lymphocyte counts or the state of infection of the infected calves.
IL12-p40 mRNA expression and protein secretion have been demonstrated
in human monocytes that were activated by Brucella abortus (7). Similarly,
IFN-gamma protein was detected in supernatants of rat peritoneal mast cells
following IL-12 treatment (8). Thus both cytokine gene expression and protein
secretion can be studied in various systems. Detection of IL-12p40 on PBMC
of experimentally BLV-infected calves is an example of such a system in
which its transient presence is identified possibly as a step in its secretion.
Acknowledgement
This project was supported by US-Israeli Binational Agriculture Research
and Development Fund. (BARD) US-2367-94.
References
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