J.
gen. Virol. (1984), 65, 1487-1495. Printed in Great Britain
1487
Key words: interferon ( BolFN-cq )/alveolar macrophages/BHV- 1
Effect of Recombinant DNA-produced Bovine Interferon Alpha (BolFN-al)
on the Interaction between Bovine Alveolar Macrophages and Bovine
Herpesvirus Type 1
By H. B I E L E F E L D T O H M A N N , * J. E. G I L C H R I S T AND L. A. B A B I U K
Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of
Saskatchewan, and Veterinary Infectious Disease Organization, Saskatoon, Saskatchewan,
Canada, S 7 N 0 WO
(Accepted 4 June 1984)
SUMMARY
Treatment of bovine alveolar macrophages (BAM) with bovine leukocyte interferon
(BoIFN-cq) resulted in reduced bovine herpesvirus type 1 replication and spread. This
was demonstrated by reduced virus yields and number of cells infected. BoIFN-ctm
treatment of BAM also induced enhanced Fc receptor expression and/or avidity by
the cells, increased their activity in antibody-dependent cellular cytotoxicity, and
augmented their extrinsic antiviral activity as measured by a reduction in the
development of plaques in a susceptible cell line. These results are discussed in the
context of the possible role of interferon in activation of AM during the early phases of
a virus infection.
INTRODUCTION
The mononuclear phagocyte system constitutes a central component of the antimicrobial
defence system of an animal, expressing both non-specific and immune-specific functions (Van
Furth, 1980). In the lung, the resident alveolar macrophages (AM) and monocytes, migrating
into the lungs from the blood, offer the first line of cellular defence against viral and bacterial
invasion. Moreover, by interacting with other components of the immune system, the AM can
regulate the antigen-specific immune response, such as antibody production and induction of
cytotoxic T lymphocytes (Lyons & Lipscomb, 1983). The immediate tissue reaction to virus
invasion is the production of interferon (IFN), which has been shown to modulate various
macrophage activities both in vivo and in vitro (for review, see deMaeyer & DeMaeyer-Guignard,
1982). In this context the enhanced cytotoxic (Stanwick et al., 1980, 1982) and antibacterial
(Bielefetdt Ohmann & Babiuk, 1984) activities of macrophages are of interest, as lung infections
by viruses are often complicated by secondary bacterial invasion (Loosli, 1973), and it has been
suspected that this is due to an impairment of AM functions (for review, see Forman et al., 1982 a).
The interaction between bovine AM (BAM) and bovine herpesvirus type 1 (BHV-I) presents
an interesting model for studying AM-virus interaction and determining why discrepancies
occur between observations in vivo and in vitro (Nugent & Pesanti, 1979; Warshauer et al., 1977).
BAM are susceptible to infection with BHV-1 in vitro, resulting in cytopathology and
impairment of various effector functions (Forman & Babiuk, 1982; Forman et al., 1982b), but
less than 0.1 ~ of lavaged cells from experimentally infected calves are productively infected
with BHV-1 and BAM activities are unaltered (Forman et al., 1982a). These results indicate that
factors in the lung microenvironment may render the BAM resistant to BHV-1 infection. One
such factor could be IFN(s) (Rodgers & Mims, 1982). To date, it has not been possible to
determine unequivocally whether IFN or some other factor is responsible for these effects. With
the advent of recombinant DNA technology for production of IFNs, it has now become possible
to use homogeneous preparations of bovine IFN to demonstrate unambiguously the influence of
IFN on virus susceptibility of BAM. In the present communication we report on the in vitro
effects of recombinant bovine alpha interferon (BoIFN-al) on intrinsic and extrinsic antiviral
activities (Morahan & Morse, 1979) of BAM as expressed against BHV-1.
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0022-1317/84/0000-6138 $02.00© 1984 SGM
1488
H. B I E L E F E L D T O H M A N N , J. E. G I L C H R I S T AND L. A. B A B I U K
METHODS
Calves. Holstein and Holstein Friesian calves were obtained by Caesarian sections and artifically reared in
isolation. Two age groups were employed: calves l0 days to 2 months old and calves 6 to 12 months old. All calves
remained negative for BHV-1 throughout the investigations as judged by periodic serological surveys.
Cells. Georgia bovine kidney (GBK) cells were cultured in Eagle's base M E M supplemented with 10~o heatinactivated foetal bovine serum (FBS), non-essential amino acids, gentamicin and HEPES buffer as described
previously (Rouse & Babiuk, 1974).
Alveolar macrophages were obtained by fibre optic bronchoscopic lavage, as described elsewhere (McGuire &
Babiuk, 1982). After two washings in Hanks' balanced salt solution (HBSS), cells were suspended in RPMI 1640
medium with 10~ horse serum (HS), polymyxin B, penicillin and streptocillin, and either seeded in tissue culture
plates as detailed below or incubated in suspension in polyethylene tubes with snap caps (no. 14-956-IF, Fisher
Scientific, Edmonton, Alberta, Canada), under rotation at 2 to 4 r.p.m, at 37 °C. Plated cultures were incubated
for 2 to 3 h at 37 °C in a humidified 5 ~ CO2 atmosphere, washed twice with M E M and re-fed with RPMI 1640
containing 10~o HS or FBS. Some cultures were treated with BolFN-cq as detailed below.
Viruses and antiserum. The origin and growth of BHV-1 strain P8-2 has been described previously (Rouse &
Babiuk, 1974). Anti-BHV-1 serum was obtained as described by Babiuk & Rouse (1978).
Interferon. BolFN-cq, produced in Escherichia coil and purified to homogeneity (lot no. 1572-22a), was
generously provided by Genentech Incorporated (South San Francisco, Ca., U.S.A.). The IFN preparation, which
was greater than 9 9 ~ pure, had a specific activity of 1.7 x 107 units/mg protein when titrated on Madin-Darby
bovine kidney (MDBK) cells challenged with vesicular stomatitis virus (VSV) in a virus c.p.e, inhibition assay.
ViralreplicationinBAM. Alveolar macrophages were seeded in 24-well plates (Corning), at 1.5 x 106to2 x 106
cells/well. After 3 h of adhesion, cultures were rinsed and reincubated for 24 h with M E M / 1 0 ~ FBS with or
without BolFN-cq in doses varying from 10 to 105 units/ml. Cultures were then rinsed thoroughly with MEM,
inoculated with BHV-1 at an m.o.i, of 0-01 and incubated for 1 h at 37 °C. Following two washings in M E M to
remove unadsorbed virus, the cultures were reincubated with M E M / 5 ~ FBS. Subsequent procedures for
harvesting of cultures and titration of infectivity by plaque assay on GBK cells were performed as described
elsewhere (Forman et al., 1982b).
Immunofluorescence assay. Alveolar macrophages were seeded in eight-chamber slides (Lab-Tek Products,
Westmont, II1., U.S.A.), treated with BolFN-ct 1 as above, at doses ranging from 102 to l0 s units/ml, followed by
infection with BHV-1 in the usual manner. After 48 h of infection, the cultures were fixed and stained as described
by Forman et al. (1982b).
InJectious centre assay. The experimental procedure followed a protocol previously described (Forman et al.,
1982b). Briefly, BAM were cultured in 16ram diam. wells of cluster plates (Costar no. 3524), treated with BolFNcq for 24 h and infected with BHV-I at an m.o.i, of 0-01. After adsorption for 1 h and removal of unadsorbed virus,
the cells were reincubated with MEM/10)/o FBS containing 4 units anti-BHV-1 antibody. At various times postinfection the cells were removed from the wells by detachment with 9 mM-lidocaine solution (Astra Chemicals
Ltd., Mississauga, Ont., Canada) in HBSS. The cell samples were washed, counted and diluted in M E M
containing anti-BHV-1 serum. Triplicate fivefold dilutions of these cells were added in 1 ml volumes to confluent
GBK cell monolayers in 24-well plates. After 48 h of incubation, the monolayers were fixed, stained and virus
plaques were enumerated microscopically.
AssayJbr interJeron production by AM. AM were seeded in 24-well plates, treated with BolFN-cq and infected
with BHV-1 as described above. Culture fluids were harvested 24, 48 and 72 h after infection and centrifuged
(1000 g) to remove cellular debris. After neutralization of BHV-1 in the supernatants by incubation with antiBHV-1 serum for 1 h, levels o f l F N activity were determined by the ability to inhibit VSV replication in GBK cells
as described previously (Babiuk & Rouse, 1976).
Virus plaque inhibition assay. Virus plaque inhibition by BolFN-cq-treated A M was performed as described by
Rouse & Babiuk (1975). AM were pretreated with BolFN-cq for 8 or 24 to 36 h prior to use as effector cells.
Assay for spontaneous cytotoxicity (SCT). AM (effector cells) were pretreated with B o l F N - ~ for 24 to 36 h. GBK
cells were grown in 25 cm 2 culture flasks (Corning) in M E M with 10 H FBS. When confluent, the monolayers were
inoculated with BHV-1 at an m.o.i, of 1. After adsorption and removal of unadsorbed virus, the cultures were
reincubated for 14 to 16 h with MEM/5 ~ FBS. Cells were then dispersed by mild trypsinization, washed once and
suspended at a concentration of 5 x 106 cells/ml in M E M / 2 ~ FBS and labelled for 1 h with 100 pCi Na: 5~CrO4
per ml (New England Nuclear). Labelled target cells were washed three times with MEM/5 ~ FBS and suspended
at a final concentration of 5 x 10~ ceUs/ml in the same medium. Mock-infected GBK cells were similarly
prepared. The microcytotoxicity assay was performed according to Stanwick et al. (1980), with final effector to
target cell ratios of 25 : 1, 50 : 1 and 100 : 1. Chromium release was calculated according to the formula: ~ sl Cr
release for SCT = 100 (2A/(A + B)), where A is the c.p.m, in 100 p.l supernatant and B is the remaining radioactivity in the well (supernatant plus cell pellet), determined after lysis of the cells. Specific release was defined as
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I F N effect on virus-macrophage interactions
1489
the ~o s 1Cr released from target cells in the presence of BAM minus the percentage released from target cells alone.
The average 51Cr leakage from uninfected and infected cells was less than 30~ in 24 h.
Antibody-dependentcellular cytotoxicity (ADCCOassay. The assay was performed as described previously (Rouse
et al., 1976) with minor modifications. Target cells were prepared as described above for the SCT assay. BAM
effector cells were incubated in the presence or absence of BoIFN-ct t in polyethylene tubes (Fisher Scientific) in
R P M I / 1 0 ~ HS at a cell concentration of 1-5 x 106 to 2.0 × 106 cells/ml under continuous rotation at 2 to 4 r.p.m.,
at 37 °C for 3 to 4 h. Only cell isolates composed of more than 95 ~o AM and monocytes as monitored by staining for
non-specific esterase and/or differential counts on cytosmears stained with Wright's stain were used. Cells were
washed three times in HBSS and suspended at various concentrations in MEM/5 ~ FBS. The cytotoxicity assay
was performed in round-bottom well microtitre plates at effector cell: target cell ratios of 50:1 and 25:1, and a
final concentration of anti-BHV-1 serum of 1:50. The incubation was terminated after 6 or 15 h, 5 0 ~ of the
culture fluids aspirated and the radioactivity measured. Total releasable radioactivity was estimated as the amount
released from target cells lysed in 5 ~ Triton X-100. Controls included cultures containing either antibodysensitized target cells alone or non-sensitized target cells plus BAM. Background release in all experiments was less
than 10~. The specific release was estimated as: ~ SR = (c.p.m. test c.p.m, control)/(total releasable c.p.m. c.p.m, control) x 100.
Fc receptoractivities. Washed bovine red blood cells (BRBC), in a 5 ~ suspension, were sensitized for 30 min at
37 °C with rabbit anti-BRBC serum, washed and resuspended to a final concentration of 0.2~ in MEM. BAM,
pretreated with BolFN-~ as described for the ADCC assay, were dispensed in 200 ~tl volumes in Eppendorf
tubes, pelleted, the medium was discarded and the cells were resuspended in 200 ~tl of sensitized BRBC. The
mixture was then centrifuged at 40 g for 3 min and incubated for 30 rain at 37 °C. After incubation, the pellet was
gently disrupted, a drop diluted in 0-1 ~o trypan blue and examined by light microscopy. BAM binding three or
more BRBC were considered to be Fc receptor (FcR)-positive. For estimation of FcR-mediated phagocytosis the
BAM-BRBC mixture was again pelleted, resuspended in HBSS, diluted 1:4 with water to lyse attached BRBC,
and reconstituted with double-strength MEM. A drop was again diluted in trypan blue and the number of cells
containing one or more BRBC were scored. On a few occasions, the FcR activities were quantified by the use of
chromium-labelled sensitized BRBC, as described elsewhere (Grewal et al., 1978).
RESULTS
Effect o f BolFN-~l on BHV-1 replieation in B A M and endogenous I F N production
A l t h o u g h v i r u s r e p l i c a t i o n is r e s t r i c t e d in B A M as c o m p a r e d to b o v i n e k i d n e y cells, t h e s e cells
d o s u p p o r t v i r u s g r o w t h ( F o r m a n et al., 1982b). P r e t r e a t m e n t o f t h e B A M w i t h B o I F N - c q for
24 h b e f o r e v i r u s i n f e c t i o n c o n f e r r e d p a r t i a l p r o t e c t i o n , as r e v e a l e d b y b o t h a r e d u c t i o n in t h e
n u m b e r o f p r o d u c t i v e l y i n f e c t e d cells ( T a b l e l), in v i r u s yield a n d a d e l a y in d e t e c t a b l e virus
p r o d u c t i o n ( T a b l e 2). I n a c c o r d a n c e w i t h t h e results o f t h e v i r u s t i t r a t i o n s , a d e c r e a s e in t h e size
of t h e i n f e c t e d foci was n o t e d b o t h in t h e i n f e c t i o u s c e n t r e a s s a y a n d i m m u n o f l u o r e s c e n c e assay.
A n a l y s i s by P A G E o f B o I F N - ~ l - p r e t r e a t e d a n d n o n - t r e a t e d , B H V - l - i n f e c t e d B A M d i d n o t
r e v e a l a n y selective r e d u c t i o n in a n y o f t h e v i r u s - i n d u c e d p o l y p e p t i d e s ( d a t a n o t s h o w n ) . I n all
a s s a y s d e s i g n e d to look at v i r u s r e p l i c a t i o n a n d s p r e a d , it was c o n s i s t e n t l y o b s e r v e d t h a t t h e
o p t i m u m dose o f B o I F N - ~ x r a n g e d b e t w e e n 102 a n d 103 u n i t s / m l . H i g h e r q u a n t i t i e s o f B o I F N ~ , a l t h o u g h still c a p a b l e o f r e d u c i n g v i r u s r e p l i c a t i o n a n d s p r e a d , w e r e less efficient. T h u s ,
n u m b e r s o f i n f e c t e d cells ( T a b l e 1) as well as v i r u s yields ( T a b l e 2) were all l a r g e r t h a n at l o w e r
T a b l e 1. Effect o f BoIFN-cq * pretreatment on BHV-1 replication in bovine alveolar rnacrophages
Interferon treatment level (units/ml)
A
Assay
0
102
103
104
105
No. of fluorescing foci/wellt
Infectious centres§
97:~
53125
42
3750
40
5125
50
13125
81
46875
• Treatment was for 24 h prior to infection with BHV- 1, strain P8-2. The results shown are representative of four
or five separate experiments with each assay, performed with different animals.
Infection dose was 100 p.f.u./well.
:1:Average of two wells per IFN dose.
§ Number of infectious centres 56 h after infection of BAM.
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H. BIELEFELDT OHMANN, J. E. GILCHRIST AND L. A. BABIUK
1490
Table 2. Effect of BolFN-~l on BHV-1 replication in bovine alveolar macrophages*
Interferon treatment level (units/ml)
Time of
harvest (h)
24
48
72
96
A
0
101
102
103
70I
1.7 × 103
6"5 × 104
1"2 x 104
10
4 x 102
2"5 × 103
2.7 × 103
0
1.4 × 102
6-5 x 10z
0
55
4"5 × 102
104
0
10
1 x 102
105
0
45
1"2 × 103
5"5 × 102
5"5 × 102
1"0 × 103
5"0 X 103
* Treatment was for 24 h prior to infection with an m.o.i, of 0-01 of BHV-1 strain P8-2.
"~Virus titre (p.f.u./ml) in culture supernatants, titrated on GBK cell monolayers.
Table 3. Effect of BolFN-~l pretreatment of bov&e alveolar macrophages on their production of
interferon upon infection with BHV-1
Interferon pretreatment dose (units/ml)*
A
r
Calf no.
83-51
83-52
0
2048~"
512
102
1024
128
103
512
32
104
512
32
* Alveolar macrophages were pretreated for 24 h, washed twice and then infected with BHV-1 at an m.o.i, of
0-01.
t Interferon titre/ml in culture fluids determined at 48 h by inhibition of VSV replication on GBK cells after
neutralization of BHV-1 with hyperimmune serum.
I F N concentrations. I n addition to the antiviral effect of BolFN-~I on BAM, an alteration of
cellular metabolism was also detected. Thus, pretreatment of BAM with BoIFN-~I reduced the
endogenous I F N production in BAM cultures, following BHV-1 infection, in a dose-dependent
m a n n e r (Table 3).
Effects of BoIFN-~l on antibody-independent extrinsic antiviral activities of B A M
One of the modes by which some macrophage populations can express antiviral activity is
revealed in vitro as a non-cytotoxic viral plaque inhibition (VPI) (Hayashi et al., 1980; M o r a h a n
et al., 1980; Rouse & Babiuk, 1975). As shown in Table 4, BAM also possess the capacity to
inhibit viral plaque development in vitro, and this activity can be enhanced in a dose-dependent
m a n n e r by pretreatment of the BAM with BoIFN-~I for 8 h, with maximal inhibition reached at
the highest dose tested. The increase in VPI activity was reflected in an effect on plaque
numbers as well as on size, without a general trend for either one or the other being dominant.
The possibility that the observed effect was due to carry-over of I F N seems remote, as the
procedure to procure the A M (non-treated and IFN-treated) by lidocaine detachment involved
at least five or six washing sequences. Furthermore, if carry-over had been a problem, there
should have been higher levels of I F N in the BAM treated with 104 units/ml in Table 3, rather
than lower levels as observed.
W h e n BAM were pretreated for longer periods of time, i.e. 24 to 36 h, prior to employment in
the VPI assay, the activity was either unchanged or even suppressed as compared to non-treated
cells. The latter event was manifested as increased cytopathology in the BHV-l-infected G B K
monolayer (data not shown). A n attempt was therefore made to determine whether this
apparent decrease in VPI activity represented a true inhibition of the BAM function or whether
the prolonged I F N treatment had rendered the BAM cytotoxic to infected G B K cells, thus
generating plaques that were not virus-induced. BAM, treated with BoIFN-cq for various times,
were used as effector cells in a 51Cr release assay. I n no case could we demonstrate spontaneous
cell-mediated lysis of either infected or uninfected target cells (data not shown).
One of the mechanisms whereby BAM might exert the non-cytotoxic antiviral activity is by
altering target cell metabolism (Hayashi et al., 1980; Keller, 1976; Morse & Morahan, 1981).
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Table 4. Effect of BolFN-cq pretreatment of bovine alveolar macrophages on their intrinsic
antiviral activity as revealed by virus plaque inhibition
Virus*
(p.f.u./well)
100
50
25
Virus
control'~
N × A§
499.5
358.6
184
Level of IFN pretreatment~ (units/ml)
1.5 × 102
1"5 x 104
c
~'
~ c - - h- BAM added per well (x 10-5)
5-0
1-25
2-5
5-0
1.25
2-5
5.0
79.6
50-7
87.4
88.0
97.4
97.5
93.6
85.8
70.2
86.7
84.8
86.0
86.9
93.5
74.4
62.7
79.6
80.0
64.9
81-5
94.9
0
r - - ; ' - - ~
1-25
23.311
45.8
37.2
2-5
69-0
52.7
62-5
* Initial input virus/culture.
t Area of plaque formation in cultures without BAM added.
:~BAM were treated for 8 h with various levels of BolFN-cq, washed and added to GBK cell cultures
immediately following virus adsorption.
§ Number of plaques (N) x area of plaque (A).
II Percentage of plaque inhibition exhibited by BAM.
Table 5. Cytostasis mediated by bovine alveolar macrophages against GBK cells*
Incorporation of [3H]thymidinet
t
Sample
Targets~
Non-treated, 6 h
3.0 x 102 units/ml,
3-0 x l0 s units/ml,
Non-treated, 28 h
3.0 × 102 units/ml,
3.0 × 105 units/ml,
C.p.m. + s.~.~,i.
2879 + 214
6h
6h
28 h
28 h
A
No. of BAM/well
1.2 × 104
2-5 × 104
.
.
.
84.5
75.8
57-2
52-3
46.4
41-7
49.5
42.3
44.3
34.9
41.8
37.8
5 × 104
.
59.7
37-2
38.6
31.3
26.8
29.7
1 x l0 s
47.5
30-8
29.7
25.5
15.3
14.9
* Target cells were cultivated for 12 h with or without BAM, either non-treated or pretreated with BolFN-ctI as
indicated in MEM with 5~o FBS. Labelling was for 1 h with 0-5 IxCi/well of [3H]TdR.
t Percentage of incorporation as compared to GBK cells alone, was calculated after subtraction of [3H]TdR
incorporation into BAM without target cells (300 c.p.m.), or BAM cultivated in the presence of mitomycin-treated
GBK cells.
.I:GBK cells seeded at 1 x 104 celts/welt 12 h prior to addition of BAM.
Analysis of D N A synthesis in non-infected G B K cells co-cultivated with non-treated BAM
revealed that BAM can reduce [3H]thymidine incorporation in the G B K cells in a dosedependent manner. Similar results were obtained when [3H]/cytidine was used (data not shown).
BAM, cultivated alone or co-cultured with mitomycin-treated G B K cells, incorporate only
negligible amounts of thymidine under the culture conditions employed [low serum content in
medium (McGuire & Babiuk, 1982)]. Pretreatment of BAM with BolFN-~I for 6 h substantially
enhanced their cytostatic activity (Table 5). In contrast, the effect was only marginally apparent
after 28 h of pretreatment when compared to the normally increased activity of control BAM
caused by in vitro maturation and/or activation of the ceils (Table 5). The arguments against
possible carry-over of I F N , mentioned above, also apply to this experimental series.
Effect of BolFN-~ 1 on B A M ADCC and Fc receptor activities
Alveolar macrophages from young calves (2 months) express A D C C of BHV-l-infected cells
rather slowly. However, pretreatment of the cells with BolFN-cq boosted this activity. The
effect was dose-dependent with maximal effect seen after 3 h at an I F N level of 1 x 102 units/ml
(Fig. la). Incubation for longer periods (i.e. 6 h) did not result in a quantitative or qualitative
change in activity. Results of experiments in which the expression of F c R activities were studied
by rosette formation and phagocytosis of Ig-sensitized erythrocytes suggested that the enhanced
A D C C activity was not only due to increased F c R expression and/or avidity (Fig. lb, c, d), but
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1492
H.
BIELEFELDT
40
OHMANN,
J.
E.
(a)
30
T
GILCHRIST
AND
L.
A.
BABIUK
14o
I
::.),i ':!.:
..~ . .
20 !
?_
0
•~> 70
0
101 10 2 10 3 10 4
(c)
0
10 t 10 z 10 3 10 4 10 5
16
[14
(d)
60
12~
50
40
_~ 30
: :ii[i 6
:!?'4
2O
0
10 ~ 10 2 10 3 10 4 10 5
0
101 10 2 10 3 l 0 4 l 0 5
BolFN % concn. (units/ml)
Fig. 1. Effect of pretreatment with BolFN-~ on expression of ADCC and Fc receptor activities by
bovine alveolar macrophages. Plastic adherent (ADCC) or suspended (Fc receptor assays) AM were
treated with various concentrations of BolFN-c~ for 3 h, washed thoroughly and employed in the
assays. (a) ADCC against BHV-l-infected cells. The results represent average activity of six
independent experiments conducted with AM from different animals, expressed as percent of nontreated cells. [Specific release in 6 h assay was 13.2 (+ 4-3)~. Background release for target cells alone,
for target cells and BAM without serum and for target cells and serum without BAM were less than 5 ~.]
Bars indicate S.E.M. (b) Fc receptor-mediated rosette formation with sensitized (serum dilutions 1/100)
erythrocytes. The results represent average of five separate experiments. Cells binding three or more
erythrocytes represented 53-7 (_+ 8-3)% of the non-treated AM population. (e) Fc receptor-mediated
phagocytosis. Cells binding one or more sensitized erythrocytes were counted and amounted to an
average of 39.2 (___15-8)~ in five independent experiments. The results are given as percent of the
activity in non-treated cells. (d) Quantification of FcR-mediated phagocytosis. BRBC were sensitized
with 1/50 dilution of rabbit-anti-BRBC serum and labelled with 5~Cr. The results are representative of
three separate experiments done in triplicate on three different animals.
also the subsequent cytolytic mechanism(s) was increased. However, t r e a t m e n t with B o l F N - ~
did not increase the m a x i m a l A D C C activity, reached at 14 to 16 h of i n c u b a t i o n above the
n o r m a l plateau activity of control cells (results not shown), i n d i c a t i n g that the effect of B o I F N ~1 is on the m e c h a n i s m of A D C C rather t h a n on the cytotoxicity.
DISCUSSION
In the study reported here it was d e m o n s t r a t e d that p r e t r e a t m e n t of B A M with BoIFN-a~ in
vitro resulted in increased resistance to infection with BHV-1 a n d increased extrinsic antiviral
activity of the cells as expressed by i n h i b i t i o n of spread of BHV-1 in b o v i n e k i d n e y cell cultures,
a n d by A D C C . Thus, the results would a p p e a r to support the i n t r i g u i n g suggestion by B l a n d e n et
al. (1976), that the p r i m a r y function of I F N s in recovery from viral infection m i g h t be to protect
m a c r o p h a g e s from those viruses capable of infecting them, a n d the cells t h e n in turn can act as
the principal mediators of specific and non-specific recovery processes.
Quite unexpectedly, it was found that the antiviral effect of B o I F N - a l exerted on BHV-1
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I F N effect on virus-macrophage interactions
1493
replication in BAM reached an optimum and then declined, resulting in a suboptimal effect at
high levels of BolFN-~ 1 (Table 2). It is conceivable that the protective effect seen is the result of
the combined activities of BolFN-cq and endogenous I F N produced by the BAM, the latter
having a greater effect on the BAM than what is recognized from titration with VSV and G B K
cells (Bell et al., 1983; Hailer et al., 1980; Maehara et al., 1977; Sen & Herz, 1983). Therefore, by
abolishing the production of endogenous I F N in the BAM cultures, the absolute protective
effect exerted on the BAM declines, allowing viral replication and spread to resume. Thus, the
protection seen at high levels of BolFN-~I may be (almost) exclusively due to exogenous IFN.
Such an effect should be taken into account when I F N is being used clinically (Babiuk &
Bielefeldt Ohmann, 1984).
As found in other species (Oud Alblas & van Furth, 1979; Zwilling et al., 1982) only a
subpopulation of freshly isolated resident BAM from normal calves express Fc receptor activity,
as revealed by rosette formation with Ig-sensitized erythrocytes. However, exposure to BolFN~1 for as short a time as 3 h enhances the percentage of erythrocyte-binding and -phagocytosing
cells substantially, and increases the number of sensitized erythrocytes binding to each BAM. By
assaying the cells in suspension, it was excluded that the increase was due merely to a steric
effect following increased cell spreading (Babiuk & Rouse, 1978). Thus, the results indicate that
the eft'ect is due both to expression of Fc receptors on previously negative cells and to an
increased number and/or avidity of the receptors on a particular cell. This is in agreement with
recently published reports indicating that the enhancing effect of IFNs is due to an increase both
in number and surface density of Fc receptors on cell membranes (Vogel et al., 1983; Yoshie et
al., 1982).
Increased Fc receptor activity probably also contributed to the enhancement of ADCC ~
expression of BAM, but cannot account for all of the increase, as judged by their different dose
optimum and percentage increase (Fig. 1). The ADCC process is composed of an initial immune
specific recognition and binding (Fc-mediated), followed by triggering of the macrophage and
expression of cytotoxicity. Either or both of the latter steps may also be enhanced. However,
exposure to BolFN-~I did not induce spontaneous cytotoxicity of the BAM as expressed against
BHV-l-infected cells, such as has been reported in other species (Pace et al., 1983 ; Sen & Herz,
1983; Stanwick et al., 1980), but the treatment did enhance their cytostatic activity, especially
after a relatively short exposure. The absence of spontaneous cytotoxicity may be a quality
related to the target cell system rather than to the BAM. Thus, BAM can spontaneously kill cells
infected with bovine parainfluenza 3 virus (Probert et al., 1977), suggesting that the virusspecific glycoproteins expressed on BHV-l-infected cells (Misra et al., 1981, 1982) do not have
sufficient 'foreignness' to trigger BAM cytotoxicity, even though they had been primed by
exposure to BolFN-cq (Pace et al., 1983).
Expression of extrinsic antiviral activity by macrophages is not limited to cytolytic functions,
but can be conveyed also by a mechanism involving the host cell metabolism. This activity is
expressed in vitro as an inhibition of viral plaque formation (Morse & Morahan, 1981 ; Rouse &
Babiuk, 1975), probably via a cytostatic mechanism(s) (Keller, 1976; Morse & Morahan, 1981).
Since BHV-1 replicates better in rapidly dividing cells, a cytostatic effect exerted on bronchiolar
and alveolar epithelial cells by the BAM might be anticipated to serve to limit virus spread,
especially early in infection, until specific immune mechanisms can come into effect. Thus, as
IFN is produced early in an infection, the interactions between the resident AM and the I F N
system may play a major role in the development of AM resistance in vivo (Forman et al., 1982a;
Rodgers & Mims, 1982), and in the subsequent protection of the lung tissue proper. An effect on
the AM may also be one of the mechanisms whereby exogenously applied B o l F N - ~ exerts its
protective effect against a BHV-1 infection in calves, and increases their resistance to secondary
bacterial infection (Babiuk & Bielefeldt Ohmann, 1984).
The authors thank Genentech, Inc. for providing the recombinant bovine alpha interferon, Greg Krakowski for
assistance with the lavages and Bonnie Neufeld for typing the manuscript. Financial support was provided by
grants from the Medical Research Council of Canada and Farming for the Future. H.B.O. was a post-doctoral
fellow of the NATO Science Fellowship Programme (grants no. 23-03 51/81 and no. 23-03 38/82).
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1494
H. B I E L E F E L D T O H M A N N , J. E. G I L C H R I S T A N D L. A. B A B I U K
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(Received 21 March 1984)
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