CELLULAR IMMUNOLOGY 119,382-392 (1989)

Prostaglandin E2 Production Is Enhanced in Mice Genetically Selected to Produce High Affinity Antibody Responses

CATHERINE PHILLIPS

Immunology Unit. Department ofMedical Microbiology, London SchoolofHygiene and Tropical Medicine, London WCJE 7HT, United Kingdom

Received Juiy 21, 1988

Spleen cells from mice selectively bred to produce high affinity antibody responses to protein antigens (HI) had reduced responses to both T and B cell mitogens when compared to those from mice selectively bred to produce low affinity (LO) responses. The reduced response by spleen cells from HI mice was partially reversed by the addition of indomethacin in vitro. Spleen adherent cells from HI mice had increased production of prostaglandin E2 when compared to those from LO mice. In addition, spleen adherent cells from mice which fail to show affinity maturation not only produced lower amounts of PGEz than those from HI mice but also a decreased proportion of spleen cells adhered to plastic in these mice. To test the possibility that the increased PGEP production in HI mice was responsible for the production of high affinity antibodies, indomethacin was administered in vivo and resulted in a significant reduction in antibody affinity. The possibility that PGE2 production may control the balance between the TH 1 and TH2 cells of Mosmann and Coffman is discussed. o 1989 Academic press, inc.

INTRODUCTION

High affinity antibodies have been shown to be more effective in a number of bio- logical reactions including virus neutralisation, protection against bacterial infec- tions, and complement fixation (reviewed Ref. (1)). In addition, the extensive produc- tion of low affinity antibodies has been shown to play an important part in determin- ing susceptibility to chronic immune complex disease (2-4). Despite the biological importance of antibody affinity, the mechanisms controlling affinity maturation have yet to be fully elucidated.

The clonal selection hypothesis of Siskind and Benacerraf (5) suggests that the se- lection of high affinity clones by limiting doses of antigen is the main driving force in affinity maturation. Work from Milstein’s laboratory (6-8) has demonstrated the crucial role of somatic mutation in this process. However, the production of high affinity antibodies has also been shown to be under the genetic control (9, 10). Early workers have shown that adult thymectomy brings about an increase in antibody affinity in rabbits and mice and that transfer of activated T cells resulted in a reduc- tion in antibody affinity. In addition, macrophage function has been shown to corre- late with antibody affinity in certain instances (reviewed Ref. (1)). However, the un- derlying basis of the control of antibody affinity is yet to be fully elucidated.

An originally outbred strain of mouse (TO) has been separated into two lines of mice on the basis of the affinity of antibody produced in response to protein antigens

382

0008-8749189 $3.00 Copyright 0 1989 by Academic Press, Inc. All ri&ts ofreproduction in any form reserved.

PGEz IS RAISED IN HIGH AFFINITY BRED MICE 383

in saline (10). As well as confirming that antibody affinity is under genetic control, the availability of these lines of mice provides a unique opportunity to study the cellular basis of the control of antibody affinity. During the breeding programme brother-sister matings were avoided in order to preserve the outbred nature of the lines and to maximise the possibility of selecting solely for factors controlling anti- body affinity. Thus any consistent differences between the lines are very likely to be relevant to the control of antibody affinity maturation.

In vivo studies have shown that the low affinity line is more susceptible to tolerance induction than the HI affinity line (1 I), and more recently, elimination of suppressor cell precursors by administration of low doses of cyclophosphamide has been shown to enhance antibody affinity in mice unable to show affinity maturation ( 12). In vivo clearance studies have revealed no differences between these lines in their ability to clear polyvinylpyrrolidone, aggregated IgG, or preformed immune complexes. How- ever, macrophages play a pivotal role in the immune response which depends not only on their ability to clear antigen but also on their ability to present it in conjunc- tion with class II MHC. In addition, through the release of cytokines such as IL-l and PGE2 , they can control the balance between help, suppression, tolerance induction, and potentiation ( 13- 15).

Despite a number of in vivo studies of the differences between the HI and the LO antibody affinity producing lines, this report represents the first study of the differ- ences in cellular function in vitro followed by an attempt to relate these findings to the production of high affinity antibody responses in vivo.

MATERIALS AND METHODS

Mice

High (HI) and low (LO) affinity mice were obtained from an initially randomly bred population of TO mice by selective breeding for 23 generations on the basis of the affinity of the antibody produced after four injections of a protein antigen in saline (10). Affinity nonmaturing mice (NM) were derived from the original low affinity line by interbreeding of those mice which failed to produce high affinity antibody after injection of antigen with Freund’s complete adjuvant (FCA) (4). Male and fe- male mice were used at 8 to 12 weeks of age.

Spleen Cell Cultures

Spleens cells were cultured in 96well plates at 2 X lo5 cells per well in medium consisting of RPM1 1640 (GIBCO) containing 10% foetal calf serum (GIBCO), 20 mM Hepes (Sigma), and antibiotics. Concanavalin A (Con A) (Sigma), phytohae- maglutinin (purified) (PHA) (Wellcome Diagnostics), or lipopolysaccharide from Salmonella minnesota (LPS) (Sigma) was added to triplicate cultures at final concen- trations of 2,0.4, and 12.5 pg/ml, respectively. Indomethacin (Sigma) was dissolved in 0.1 M sodium bicarbonate and added to cultures in 1 O-p1 aliquots to give a final concentration of 1 pg/ml. Cellular proliferation was measured by the addition of 1 MCi of tritiated thymidine (5 Ci/mmol) (Amersham International) per well 18 hr be- fore cell harvest using an Ilcon cell harvester. Tritium incorporation was measured using a Beckman LS 9800 B counter.

384 CATHERINE PHILLIPS

Prostaglandin E2 Measurement

Spleen adherent cells were obtained by incubation of 2 X 1 O6 spleen cells per well in 96-well plates for 90 min at 37°C followed by removal of nonadherent cells by washing. Two hundred microlitres of fresh medium was added to each well and the plates were incubated for a further 6 hr, when supernatants were harvested and stored at -20°C until they were assayed. PGEz was measured using a New England Nu- clear Kit.

Protein Measurement

Protein concentration was measured by naphthol yellow S binding (16). Briefly, adherent cells were fixed in 4% formol saline and incubated with 0.1% naphthol yel- low S in 1% acetic acid at room temperature for 1 hr. Plates were washed in 1% acetic acid, the bound dye was extracted in 4% decon 90, and the optical density at 4 10 nm was read on a Dynatech MR 60 microplate reader.

Inoculation of Mice

One milligram human serum albumin (HSA) (Miles Scientific) was injected in FCA (Difco Laboratories, Detroit, MI) by the intraperitoneal route (ip) in 0.2 ml phosphate-buffered saline (PBS). A stock solution of indomethacin (Sigma) (5 mg/ ml in ethanol) was prepared and stored at -70°C. Just before use, the stock solution was diluted l/25 in PBS and 0.1 ml (1 mg/kg) was injected into mice ip each day of the treatment.

Antibody Level and Ajinity Measurement

The level (Abt, picamoles of binding sites per 10 ~1 serum) and relative affinity (K,, litres/mole) of antibody to HSA was measured by a double isotope ammonium sulphate globulin precipitation method which incorporated a 22Na volume marker ( 17). Free and antibody-bound antigen was determined at equilibrium over a range of antigen concentrations in antigen excess. Abt was obtained by extrapolation of a Langmuir plot of the reciprocal of the bound antigen versus the reciprocal of free antigen:

1 fb = l/K. l/c. l/Abt + l/Abt,

where b = bound antigen; c = free antigen, and K = affinity. A curvilinear plot was obtained and antibody affinity calculated by linear regres-

sion analysis using data points where less than 50% of the total antigen was bound. Affinity was expressed as the reciprocal of the free antigen concentration when 50% of the total antibody binding sites was bound to antigen.

Statistical Analysis

Statistical significance was assessed using Student’s t test.

RESULTS

Mitogen Stimulability of Spleen Cells An initial kinetic study of tritiated thymidine incorporation by spleen cells from

the different mouse lines in response to the T cell mitogens, Con A and PHA, and

cpm x lo-3t

300 -

200 -

100 -

PGE2 IS RAISED IN HIGH AFFINITY BRED MICE

A B C

* + +* 3

385

I I I 1 I I

2 4 2 4 2 4 day

FIG. 1. The proliferative response to T and B cell mitogens by spleen cells from HI (squares), LO (circles), and NM (triangles) mice: (A) with Con A; (B) with PHA; (C)with LPS. Tritiated thymidine incorporation (cpm) on Days 2 and 4 of cultures (10 mice per group + SD). *Pi 0.01; ‘P < 0.01; OP i 0.05.

the I.3 cell mitogen LPS (data not shown) suggested that the spleen cells from NM mice might reach peak proliferation earlier than those from LO or HI mice. Accordingly, in 10 further experiments comparing mitogen stimulability between the different mouse lines, tritiated thymidine incorporation was measured on both Day 2 and Day 4 of in vitro culture (Fig. I). Spleen cells from HI mice showed a reduced proliferative ability to all three mitogens as compared to those from both LO and NM mice, the differences being significant on Day 2 in response to PHA and between LO and HI mice on Day 2 in response to LPS. There were no significant differences in the sponta- neous incorporation of tritiated thymidine between spleen cells from HI, LO, and NM mice (data not shown).

The Efect of Indomethacin in Vitro

It has been shown in some instances that mitogen stimulability can be reduced in the presence of PGEz (18). Thus, in order to assess whether the reduced mitogen stimulability seen in HI mice may be due to an increased production of prostaglan- dins by HI spleen cells, the effect of indomethacin on mitogen stimulability was as- sessed. The in vitro addition of indomethacin (1 rg/ml) significantly enhanced the proliferative response of HI spleen cells compared to untreated HI controls both to Con A on Day 2 and to PHA on Day 4 (Fig. 2). Indomethacin had no significant effect on the mitogen stimulability of either LO or NM spleen cells (data not shown).

PGE, Production by Spleen Adherent Cells from HI, LO, and NMMice

Spleen adherent cells from HI mice were found to produce significantly more PGEz than those from LO and NM mice at both the basal and the LPS-stimulated rate (Table 1). A significantly lower amount of protein adhered to plates after incubation

386 CATHERINE PHILLIPS

cpm * 10-3 t Con A PHA

200 -

lSO! :i--::

+

100

* 50

A f

+

*

I 1 , ,

2 4 2 4 day

FIG. 2. The effect of indomethacin in vitro on the proliferative response of spleen cells from HI mice to Con A and PHA: tritiated thymidine incorporation (cpm) on Days 2 and 4 of cultures by spleen cells incubated either with (open circles) or without (solid circles) 1 &ml indomethacin (five mice per group + SD). 'P < 0.05; +P < 0.05.

with NM spleen cells compared to either HI or LO spleen cells (Fig. 3). Despite this, the difference between NM and HI mice was still significant when the value for PGE2 production by NM mice was corrected for the smaller amount of adherent material found in these mice (Table 1).

The Eflect of Dlflerent Weekly in Vivo Regimes oflndomethacin on Antibody Afinity and Antibody Levels in HI Mice

In order to assess whether the increased production of PGE2 by spleen cells from HI mice may contribute to the higher affinity antibody responses seen in these mice, indomethacin in vivo was administered at different weekly intervals before and after

TABLE 1

PGE2 (pp/ 100 ~1) Produced in 6 Hours at 37’C by the Spleen Adherent Cells Present in 2 X 1 O6 Spleen Cells from Untreated HI, LO, and NM Mice with or without 12.5 &ml LPS (5 Mice per Group + SD)

Treatment

Mouse line Unstimulated” LPS stimulatedb

HI 9.9 + 1.7&t,* 29.3 +0.35&T,* LO 2.5 *4.3t 6.2 f 4.6t NM 0.61 _+ 0.98* 11.4-+2.0* NM (corrected) 1.01 + 1.69 18.7 -r- 3.49

- Note. As spleen cells from NM mice contain proportionately less protein that will adhere to plastic than

do those from LO or HI mice, NM (corrected) shows PGE2 production corrected by this proportion. "*P<0.001;~P<0.005;~P<0.001. b*P<0.001;tP<0.001;~P<o.005.

PGE2 IS RAISED IN HIGH AFFINITY BRED MICE 387

.024 - *

t *

016 -

.008 -

HI LO NM

FIG. 3. Spleen adherent cells: total protein adhering to plastic tissue culture plates after incubation with spleen cells from HI, LO, and NM mice for 1 hr at 37°C (optical density (O.D.) of naphthol yellow S bound and extracted with decon 90; five mice per group f SD). *P < 0.0 1; 'P < 0.0 1.

challenge with 1 mg HSA in FCA. Antibody affinity was measured using the double isotope method of Gaze et al. (17). This assay has been shown to rank the affinities of monoclonal antibodies in the same order as the more classical method of affinity measurement, equilibrium dialysis, although it does tend to overestimate the affinity of low affinity antibodies and underestimate that of high affinity antibodies (19). In addition, it has been successfully applied to the measurement of affinity of antihapten antibodies and antibodies to protein antigens such as HSA, transferin, HCG, and myelin basic protein (20-22).

In viva indomethacin treatment during the first week after antigen challenge sig- nificantly reduced antibody affinity in HI mice compared to that of the control (P < 0.05) (Fig. 4) as well as compared to HI mice receiving indomethacin at any other time tested (P < 0.05) (Fig. 4). The open circles in the control group in Fig. 4 represent another group of HI mice whose antibody affinities to HSA in FCA were measured a year earlier than those represented by the solid circles. The mean and standard devia- tion of both groups are almost identical, demonstrating the reproducibility of the double isotope method of affinity measurement and that the wide deviation in anti- body affinities in HI control mice is due to the outbred nature of the mice rather than due to irregularities in the affinity assay used here. The regime of indomethacin which led to a decrease in antibody affinity in these mice led to a concomitant decrease in antibody levels (Fig. 5).

DISCUSSION

Spleen adherent cells from mice selectively bred to produce high affinity antibody responses (HI mice) were found to produce significantly higher levels of PGEp than those from mice producing low affinity antibody responses (LO and NM mice) and,

388 CATHERINE PHILLIPS

Kr L/M t

x 10-6 0

1 control pretreat 1st week 2nd week

FIG. 4. The effect of different regimes of indomethacin on the anti-HSA antibody affinity (K, in litres/ mole) present in the sera of HI mice 21 days after 1 mg HSA in FCA ip. Daily injections of indomethacin were given for 7 days before antigen challenge (pretreatment), for 7 days immediately after antigen chal- lenge (first week), or for 7 to 14 days after antigen challenge (second week). (Each circle represents one mouse + SD; open circles represent an experiment done at another time from that represented by the solid circles.) ‘P < 0.05; *Pi 0.02; +P < 0.05.

in addition, HI mice produced substantial amounts of PGEP upon adherence to plas- tic alone. Administration of indomethacin in vivo to HI mice during the first week after antigen challenge resulted in a significant reduction in antibody affinity, suggest- ing that PGE2 may have a significant role in the production of the high affinity anti- body responses seen in these mice.

PGE2 is one of the major products of arachidonic acid metabolism of murine mac- rophages (23-25) and has an effect on most of the immunological cells involved in the humoral immune response. Most effects of PGE2 can be replaced by CAMP or

PGEz IS RAISED IN HIGH AFFINITY BRED MICE 389

600

400

200

FIG. 5. The effect of different regimes of indomethacin treatment on the anti-HSA antibody amount (picamoIes/ 10 ~1) present in the sera of HI mice 2 1 days after 1 mg HSA in FCA. Weekly indomethacin regimes were given as per Fig. 4 (five mice per group ?z SD). *P-c 0.05; +P i 0.025.

its analogues (26-33) and although the dominant in vitro effect of PGEz on isolated cells of the murine immune system appears to be suppression (34-37) enhancement has also been reported (38).

However, the overall effect of PGE2 in vivo will be more complex. It will depend upon the relative sensitivities of subsets of cells to PGE2 (39) as well as the specific interactions under consideration. For instance, large amounts of PGE2 in the rheu- matoid joint has been suggested to lead to inhibition of suppressor function with a consequent facilitation of helper activity (40). Thus it could be envisaged that indo- methacin may bring into operation cells, hitherto suppressed by PGE2, which would be counterproductive to affinity maturation. As suppression and ease of tolerance induction have been implicated in the lack of affinity maturation in NM and LO mice, respectively ( 12, 1 l), the cells released from PGE2-induced suppression would presumably be suppressor cells. In addition, indomethacin can enhance the metabo- lism of arachidonic acid via the lipoxygenase pathway (4 1) and leukotriene B4, one of the metabolites of this pathway found in mice (42), has been shown to be able to induce suppressor cells in human lymphocyte cell cultures (43) and to increase cyto-

390 CATHERINE PHILLIPS

toxic cell effectiveness by enhancing target cell recognition (44). However, the ex- treme reproducibility of the high PGE;! levels in HI mice despite the outbred nature of the lines is remarkable. In addition there is a graded decrease in the amount of PGE2 produced by LO and NM mice, respectively, which reflects a graded impair- ment in the ability to produce high affinity antibody responses. Thus the high levels of PGE2 produced by these mice are likely to have an important role in the produc- tion of high affinity antibody responses seen in these mice.

There is evidence that PGE2 may directly enhance early stages of B cell activation but inhibit the proliferation and differentiation of larger, cycling B cells (45,46). Thus it may be possible that by preserving B cells in an earlier state of activation while preventing terminal differentiation into immunoglobulin-secreting cells, PGE2 may function to prolong the period during which somatic mutation can occur, while at the same time providing more strenuous conditions under which high affinity clones compete for available antigen.

Macrophage-induced suppression of low affinity clones in vitro has been shown to be responsible for an increase in affinity of plaque-forming cells in rabbits (47). Thus it must be considered that the high PGE2 production in HI mice may simply suppress low affinity clones, thus increasing average affinity. This mechanism, however, can be ruled out here, as it would predict an increase in antibody levels in conjunction with a decrease in antibody affinity. In contrast, antibody levels were found to de- crease concomitantly with antibody affinity in indomethacin-treated HI mice (com- pare Figs. 4 and 5).

However, another regulatory role of PGE2 should be considered. Koga et al. have shown that administration of indomethacin, or a fall in PGEz levels induced by im- munisation, has been shown to induce an influx of cells from the thymus (48, 49). Preventing this influx, by adult thymectomy before antigen administration, has been shown to enhance antibody affinity in rabbits (50) and mice (5 1,52). Thus it is possi- ble that high levels of PGE2 present in HI mice mimic the effect of thymectomy by preventing influx of cells from the thymus and that the administration of indometha- tin abrogates this.

In a recent review, Swain et al. (53) have attempted to relate earlier work with T cell subsets with the more recent description of subsets of T helper cells on the basis of the lymphokines they produce. They report evidence that the anti-thymocyte serum (ATS) resistant, thymectomy-sensitive T cells of Raff and Cantor (54) and Kappler et al. (55) are the interleukin 4 (IL-4) producing Tu2 of Mosmann and Coffman (56). In addition, it has been shown that there is a decrease in IL-4 producing cells after adult thymectomy in the rat (57). Koga et al. (48) reported that the cells which in- fluxed from the thymus upon indomethacin administration were able to help anti- body responses. Thus it is possible that the reduction in antibody affinity seen in HI mice after indomethacin administration is due to a change in the balance of Tn 1 to Tn2 in favour of Tn2 due to an influx of Tu2’s from the thymus. This favouring of Tn2’s could be further exacerbated by an increased production of IL-l induced by indomethacin (58) as the proliferation of Tu2’s is enhanced by IL-1 while that of Tul’s is not (59, 60). The greater ability of T,2’s to help specific B cell responses compared with Tnl’s (6 1) may not be relevant here as there is no direct relationship between antibody amount and antibody affinity (62). In addition, as well as its many other effects, IL-4 has also been shown to induce CD8 expression on human CD4+ T cell clones (63).

PGE2 IS RAISED IN HIGH AFFINITY BRED MICE 391

With regard to this hypothesis, I have examined the subclass of antibody produced by HI mice with or without indomethacin elsewhere (manuscript in preparation) and have found a decrease in IgG2a concomitant with a decrease in antibody affinity in these mice. This supports the hypothesis that Tn l’s may be more conducive to high antibody affinity responses, as Tn l’s are the subset of T helper cells which have been reported to produce interferon-y which has been shown to be responsible for the production of this subclass of antibody (64). In addition, other workers subsequently have found HI mice particularly resistant to malaria infection (Playfair and Devey, personal communication), once again indicative of Tnl activity as this subgroup is reported to be more effective against intracellular parasites than Tn2’s (6 1).

Thus, although the mechanism by which PGEz exerts its effect has not yet been elucidated, this report raises the possibility that PGE2 may play a significant role in the production of high affinity antibody responses.

ACKNOWLEDGMENT

I thank Madeleine Devey for her helpful comments in the preparation of this manuscript.

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