Influence of in vivo interferon treatment on mitogen responsiveness of T- and B-cells is dependent on the mouse genotype

Immunology Letters 5 (1982) 175-180

Elsevier Biomedical Press

INFLUENCE OF IN VIVO INTERFERON

TREATMENT ON MITOGEN RESPONSIVENESS

O F T- A N D B - C E L L S IS D E P E N D E N T O N T H E M O U S E G E N O T Y P E Kris HUYGEN and Kamiel PALFLIET Pasteurinstituut van Brabant, 28 Stoomslepersstraat, 1040 Brussels, Belgium

(Revised version received and accepted 10 July 1982)

1. Summary Intravenous inoculation of moderate doses of viral interferon (up to 104 I.U./mouse) induced subsequent modification of the mitogen responsiveness of T- and B-lymphocytes from the spleen. The modulation, however, was dependent on the mouse genotype. Whereas BALB/c mice demonstrated enhanced T-cell responses (especially to PHA), C57B1/6 mice were generally suppressed in their T-cell response by the same treatment. B-cell proliferation to LPS showed a moderate enhancement in both strains, probably due to activation of accessory cells.

2, Introduction Besides their characteristic property of affecting virus multiplication, interferons (IFN) exert multiple other biological effects, such as inhibition of cell division, modulation of the immune response and antitumor activity in vitro as well as in vivo (for a review see [ 1]). A well known effect is the suppression of cellular proliferative responses to various mitogens, following in vitro IFN treatment. This was first described by Lindahl-Magnusson et al. [2] for the PHA response of C3H splenocytes. Similar suppressive effects were later reported with another T-cell mitogen, Con A [3] and with the B-cell mitogens, PWM

and LPS [4]. Finally, inhibition of mitogen responses was observed after an in vivo IFN pretreatment for 24 h [5,61. De Maeyer et al. have described the genotype dependency of IFN production and subsequent sensitivity in the mouse [7]. C57B1/6 mice produce 10 times as much IFN as BALB/c mice upon induction with Newcastle Disease Virus. On the other hand, 10 times more IFN is needed to inhibit proliferation of committed erythroid precursors (CFU-E) from C57B1/6 than is needed for comparable inhibition of CFU-E from BALB/c. BALB/c are also more readily protected by IFN against viral EMC challenge than are C57B1/6 mice. In this paper we have shown the effects of in vivo IFN administration on in vitro mitogen responses of BALB/c and C57B1/6 spleen cells. T-cell responses, especially to PHA, were enhanced in BALB/c mice and suppressed in C57B1/6 mice. B-cell responses to LPS could be enhanced irrespective of the mouse strain.

3. Materials and methods 3.1. Animals BALB/c/Cnb and C57B1/6 Pfd mice were used at the age of about 12 weeks, weight about 20 g. All the animals were supplied by the Mouse Breeding of the laboratory.

Key words: interferon - mitogen responsiveness - geno-

type - immunomodulation Abbreviations: IFN, interferon; Con A, concanavalin A; PHA,

pbytohemagglutinin; LPS, lipopolysaceharide; SRBC, sheep red blood cells; PWM,poke weed mitogen.

3.2. Interferon Partially purified murine type I IFN (kindly given to us by Dr. H. Heremans, Rega Institute, Leuven) was prepared in mouse L929 cells, grown to near con-

0165-2478/82/0000-0000/$2.75 © 1982 Elsevier Biomedical Press

175

fluency in roller bottles with Newcastle Disease Virus as the inducer. Culture supernatant was harvested after 18 h of incubation and purified by precipitation with ammonium sulfate between 40% and 65% saturation. The specific activity of the IFN (a mixture of IFN-a and IFN-/3) was 1.0-2.0 × 10 6 I.U./mg of protein. Electrophoretically pure IFN (a generous gift from Dr. E. De Maeyer-Orsay) with a specific activity of 1.5 X 10 9 I.U./mg of protein, was prepared by sequential affinity chromatography of C-243 cell IFN on poly(U)- and antibody-agarose columns [8]. 3.3. Interferon assay Interferon was assayed on mouse L929 cells grown as monolayers in MEM-5% heat-inactivated fetal calf serum (FCS), in fiat-bottomed microplates. IFN-containing material was serially diluted and left on confluent monolayers for overnight incubation in a humidified incubator with 10% CO2 atmosphere. Cell

ethanol. Cells were plated in fiat-bottomed microplates (Sterilin) in 200/21 volumes. To activate the lymphocytes, 20/al of freshly prepared mitogen solutions were added per well. Final concentrations of the mitogens were, unless otherwise state d, concanavalin A (Con A; Pharmacia) 2/ag/ml, phytohemagglutinin (PHA; Wellcome) 15 ~ug/ml, and lipopolysaccharide E. coli 055B5 (LPS; Instituut Pasteur Brabant) 5/ag] ml. Cells were cultured for 72 h in a humidified COz incubator, pulsed with 0.4/aCi ]3HI thymidine (Amersham; 8.3 mCi/mg)/well and harvested 5 h later on a Titertek Cell Harvester (Skatron). The radioactivity recovered from the filters was measured in a Beckman LS.230 liquid scintillation counter. All experiments were done in quadruplicate. Results are expressed as the mean + S.D. in counts per minute (cpm) and are representative of several experiments. Percent inhibition or stimulation was calculated as follows:

cpm (mitogen, IFN) - cpm (no mitogen, IFN) . . . . . \ c p m (mitogen, no IFN) - cpm (no mitogen, no IFN) cultures were then challenged with vesicular stomatitis virus (VSV) at multiplicity of infection of 1. Protection against the cytopathic effect (CPE) was determined 24 h later. The IFN titer is given as the reciprocal of the highest dilution of the sample resulting in a 50% protection against CPE. One laboratory unit equals 2 reference units G-002-904-511 (NIH, Bethesda). 3.4. Interferon treatment Various dilutions of IFN were made in phosphatebuffered saline (PBS) and 0.2 ml/mouse was administered by intravenous inoculation in the tail. Control mice received PBS only. 24 h later, mice were sacrificed (at least 3 mice]group), spleens removed aseptically and cells were prepared in a loosely fitting Dounce Homogenizer. Spleen cells were washed in RPMI-1640 medium and counted in a Coulter Counter (Type Dn). 3.5. Mitogen stimulation Unfractionated spleen cells were suspended at 5 X l0 s cells/ml in RPMI-1640 medium, supplemented with 5% FCS, antibiotics and 5 X 10 -s M 2-mercapto176

\

1) X 100

4. Results 4.1. Effect o f lFN pretreatment on the proliferation of unstT"mulated cells As shown in Table 1, intravenous inoculation of IFN resulted in increased thymidine uptake (up to 70%) by spleen cells in the absence of mitogen. Whereas BALB/c demonstrated maximal enhancement at the dose of 100 I.U./mouse, with a gradually decreasing effect at higher doses, C57B116 showed maximal increases at slightly more elevated doses. 4.2. Effect of lFN pretreatment on the proliferative response to concanavalin A As shown in Table 2, the effect of IFN on Con A response was dependent on the mouse genotype. Treatment of BALB/c mice with 100 I.U./animal resulted in a 50% enhancement of the response, the intermediate dose of 103 I.U./mouse had almost no effect, whereas 104 I.U./mouse induced a 30% suppression of the response. C57B1/6 mice on the other hand showed a 30% suppression of their Con A response when treated with 100 I.U. Moreover, we

Table 1 Effect of in vivo IFN treatment on the proliferation of BALB/c and C57B1/6 spleen cells Expt. no.

IFN/mouse

BALB/c thymidine incorporation

C57B1/6 thymidine incorporation

cpm a

% modulation b

cpm

0 10 I.U. l0 s

3050 ± 195 3375 ± 615 NS 5455± 825

+11 +79

0 103 104

4010 ± 560 5195 ± 1190 NS 4715 ± 300 NS

+30 +18

% modulation

3355 ± 140 3515 ± 145 NS 4435± 535

+5 +32

8455 ± 580 14,980 ± 845 14,510 ± 1235

+77 +72

aMean ± S.D. of quadruplicate cultures; all data were significantly different from controls (P < 0.05) unless NS indicated. bpercent modulation, calculated as described in Section 3.5.

,s[

were unable to induce more suppression with higher doses up to 104 I.U. This could indicate that only a subpopulation o f cells responsive to Con A is affected, As can be learned from Fig. 1, the I F N modulation could be observed over a wide range of Con A concentrations and was most significant at suboptimal doses.

E o ~o

QC

4.3. Effect of lFN pretreatment on the proliferative

response to phytohemagglutinin PHA responses were modulated in a similar way as Con A responses, but the differences between the two strains were much more pronounced (Table 3). An increase of about 70% o f the PHA response was observed in BALB/c mice for the 4 IFN doses used, whereas C57B1/6 mice showed a 50% suppression, again irrespective o f the dose of IFN used. As in the response to Con A, the I F N effect was observed over

'ID I--

ConA

o

50

25

-I(0

0.2

0.4

,

,

,

1

2

4

10

Fig. 1. Incorporation of tritiated thymidine in the presence of increasing doses of Con A (~g/ml) by BALB/c spleen cells untreated (e e) or pretteatedby 103 I.U. of IFN ( o - - - o ) , C57B1/6 spleen cells, untreated ( , - - m ) or pretreated with 103 I.U. of IFN ( o - - - n ) .

Table 2 Effect of IFN pretreatment on responsiveness to concanavalin A Expt. no.

IFN/mouse

BALB/c thymidine incorporation

C57B1/6 thymidine incorporation

cpm

% modulation

cpm

% modulation

0 10 102

80,080 ± 12,660 88,185 ± 19,745 NS 120,480 ± 13,135

+10 +49

39,265 + 2960 36,468 + 6005 NS 27,585 + 4260

-8 -36

0 10 3 104

90,685 ± 10,345 96,300 ± 2940 NS 67,155 ± 3140

+6 -29

53,135 + 3610 49,420 + 2970 NS 48,765 + 2605

-23 -23

177

Table 3 Effect of IFN pretreatment on responsiveness to phytohemagglutinin Expt. no.

IFN/mouse

BALB/c thymidine incorporation

C57B1/6 thymidine incorpgration

cpm

% modulation

cpm

% modulation

0 10 10 2

25,025 ± 3590 37,760 ± 3255 43,760 ± 4335

+56 +75

16,785 ± 4090 10,245 ± 2185 NS 10,130± 410

-50 -58

0 103 10'

16,480 -+ 1020 24,575 -+ 2765 28,345 ± 2990

+55 +89

34,610 +- 4065 24,930 ± 2490 24,690 _+ 1340

-62 -61

Table 4 Effect of IFN treatment on responsiveness of B-cells to LPS Expt. no.

IFN/mouse

C57B116 thymidine uptake

BALB/c thymidine uptake cpm

% modulation

cpm

% modulation

0 10 10 2

18,390 ± 1730 17,970 ± 1170 NS 24,680 ± 3205

0 +31

38,935 ± 4150 31,755 ± 1710 41,015 ± 4775 NS

-21 +2

0 103 10'

26,095 ± 1735 31,201 ± 2155 31,825 ± 4290

+15 +22

66,575 ± 2745 89,030 x 1910 75,865 ± 4180

+26 +6

Table 5 Mitogen responsiveness of spleen cells pretreated with electrophoretically pure interferon Control a BALB/c Control Con A PHA LPS

2744 104,088 26,422 13,134

C57B1/6 Control Con A PHA LPS

3066 45,104 10,020 21,872

± 304 ± 5616 ± 2893 ± 1964

± ± ± ±

336 2686 1364 1794

Interferonb

% modulation

6163 116,499 42,437 30,582

± 730 ± 8629 -+ 3696 -+ 2502

+125 +9 +53 +135

8224 52,496 8522 47,478

+ 754 -+ 2658 ± 693 -+ 2141

+168 +5 -96 +108

a0.01 M citrate buffer, containing 0.1 mg]ml bovine serum albumin 0.2 ml intravenously/mouse. bElectrophoretically pure interferon in control buffer - 103 I.U. intravenously/ mouse. 178

a wide range of PHA concentrations and expressed especially at suboptimal doses. 4.4. Effect o f lFN pretreatment on the proliferative response to LPS Enhancement of LPS responsiveness was observed in both mouse strains, although BALB/c mice seemed to be more susceptible to enhancement than C57B1/6 mice and also to a higher extent (Table 4). The B-cell response closely parallelled the response of non-stimulated total cells shown in Table 1.

4.5. Electrophoretically pure interferon has the same effect on mitogen responsiveness Mice were injected intravenously with 0.2 ml buffer (0.01 M citrate, BSA 0.1 mg/ml) or 103 I.U. of electrophoretically pure interferon. Spleens were removed 20 h later (5 mice/group) and tested for their mitogen responsiveness. As can be learned from Table 5, our previous observations were confirmed with pure IFN. T-cell responses of BALB/c mice were enhanced whereas T-cell responses of C57B1/6 mice were suppressed. The discrepancy was very clear for the PHA proliferation, whereas no clear-cut differences were observed at the optimal Con A concentration. (It was observed at other Con A doses; data not shown.) Proliferation of control cells and LPS-activated cells was enhanced in the two mouse strains and even to a greater extent than with IFN of lower specific activity. 30 1

PHA iI

20

~,

,' /I I

b.. .... •

F-

0

.........

L.~,"" 5

I 10 15 20

40

100

Fig. 2. Incorporation of tritiated thymidine in the presence of increasing doses of PHA (#g/ml) by BALB/c spleen cells untreated ( . - - o ) or pretreated with 103 I.U. of IFN ( o - - - o ) and C57B1/6 spleen cells, untreated ( . - - . ) or pretreated with 103 I.U. of IFN ( a - - - a ) .

5. Discussion In vitro and in vivo treatment with interferon is known to inhibit cellular proliferation of murine Tand B-lymphocytes stimulated by mitogens [2,3]. Interferon administered within 24 h of sacrifice of the mice, results in suppression of DNA synthesis of splenic lymphocytes to PHA, Con A and to a lesser extent to LPS [4]. The inhibition is, however, dependent on the dose of interferon employed, with a minimum of 6 × 104 units needed to observe significant suppression [5]. In this paper, we report that a 24 h pretreatment with lower doses of IFN (up to 104 units/mouse) significantly enhances PHA responses (and Con A responses to a lower extent) of BALB/c spleen cells whereas C57B1/6 mice are refractory to this enhancement. Indeed, after interferon treatment, BALB/c mice can respond over a wider range of Con A and PHA concentrations whereas C57B1/6 mice seem to be more restricted in their responsiveness. B-lymphocyte responses to LPS can be enhanced both in BALB/c and C57B1/6 mice. The effect can be attributed to IFN since both semi-purified IFN (spec. act. 106 I.U./ mg protein) and electrophoretically pure IFN (spec. act. -+109 I.U./mg protein) were capable of incuding it. T-lymphocytes have been divided in different subsets. Non-recirculating Trcells from thymus and spleen correspond more or less to the Lyt 123 + subset, whereas circulating T2-cells from lymph nodes and peripheral blood express preferentially the Lyt 1+23or Lyt 1-23 + phenotype [9]. Recent studies indicate that PHA is predominantly a T2-cell mitogen, whereas Con A can activate both T r and T2-cells [10,11]. We believe that in our experiments, intravenous interferon inoculation may increase the 'homing' of circulating T2-cells to the spleen, which then in its turn could result in enhanced proliferation to PHA and Con A (the effects of Con A proliferation being smaller as only part of the responsive cells are T2). Interferon might also promote maturation of T-cells directly, since in vitro IFN treatment at low doses, enhances BALB/c spleen cell responses to PHA and Con A as well (data not shown). In our experiments, C57B1/6 mice were refractory to enhancement but were on the contrary more prone to suppression, especially in their PHA response. These data are in agreement with Sonnenfeld et al. [ 12] who have shown that the antibody response to SRBC of C57B1/6 mice 179

can be more easily inhibited by IFN than that of BALB/c mice. Moreover, injection of low doses of IFN ( 1 0 - 1 0 2 I.U.) in DBA/1 × C57B1/6 F1 animals has a significant enhancing effect on IgG PFC cells [13]. Activation of helper T-cells, as reflected by the increased proliferation to T-cell mitogens we have observed could explain these enhancing effects of low doses of IFN. Recently, interferon treatment of mice has been described to induce peripheral lymphadenopathy and a decrease in the number of peripheral blood lymphocytes [14]. The effect was observed in different strains of mice but only with doses of at least 4 × 104 units/mouse. The effect o f interferon on T-cells can therefore be hypothesized as follows: low doses (<104 units/mouse) would enhance T-cell proliferation but only of certain genotypes whereas high doses (>5 × 104 units/mouse) would suppress T-cell responses, irrespective of the genotype. The observed enhancement of B-cell responses to LPS and of the proliferation of unstimulated cells, independent of the genotype, can probably be attributed to the activation of macrophages. Indeed, recent findings on PWM-induced differentiation of human PBL indicate that doses of IFN up to 1000 I.U./ml enhance immunoglobulin release and this enhancement is mediated by monocytes [15]. Blomgren et al. have recently suggested that substances other than IFN in impure IFN preparation can eventually lead to suppression. We confirm their observation, since pretreatment with pure interferon resulted in more pronounced enhancement of unstimulated spleen cells and LPS-activated B-cells, than pretreatment with semi-purified IFN. To conclude, intravenous inoculation of small doses of IFN (less than 104 units/mouse) can result in enhanced proliferation of T-cells (of certain genotypes) and B-cells. Since current doses given in experimental trials on man are of the order of magnitude of 20/lg/kg/day [ 17] corresponding to about 103 units/ mouse, we believe our observation may have a certain clinical relevance.

180

Acknowledgements We thank Drs. E. De Maeyer and H. Heremans for the generous gifts of interferon, Drs. L. Thiry and J. Content for reviewing the manuscript, Mrs. J. Herinckx for typing the manuscript and Mr. M. Beier for skillful mouse breeding.

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