Surface IgM on lymphocytes from pregnant women

tested immediately after separation and some were incubated overnight in autologous serum at 4” C. bef’orc in testing. Equal volumes of trashed I~mphocytcs PBS/BSA (approximately 1 x 10” concentration) I\er‘c incubated at 4” C. for 30 minutes with Huoresceinlabeled sheep antihuman IgM (Wellcome) diluted 1 :-L. After being washed three times, the lymphocytes WVC’I-c reconstituted to their original concentration with L’ drops mounted on a slide with a coverslip added. ‘l‘hr preparations Lvere examined ~\ith a Lcitz Diavcrt Orthomat microscope cquipprd with phase contrast and incident light fluorescence, each being used alternat& during the counting procedure to exclude the dc-ad lymphocytes. Thr fluores~ein-labeled antisera \I rre ultrawntrituged tin- 30 minutes at 150.000 g before use. The spwificit> ~vas checked as follows: (1) Bloc king expel-imerits with nonconjugated anti-IgM, hut tlot anti-l+ I-ctlucd the count ;,f fluorcwent IgM-bearing ccllb. (2) lmmL~nc)dift‘Llsion of the fluor~scein-lal,eled anti-Igh2 w-urn produced a positive arc onlv with purified human IgM and not Is<;. IgA, or IgD. (3) Immunoelec.troptlor~sis of thr fluoresc~,in-labeled antiIgM serum against whole human serum revealed onl\~ one arc in the IgM region.

THE LYMPHO(:Y~‘k~ is an important cell in immunologic reactions. A study of lymphocyte physi$og) during pregnancy is theref’ore relevant to the problem ot fetomaternal immunologic relationship. During an investigation of the peripheral lymphocyte population in pregnant cvomen. w found signihcantlv more IgM-bearing lymphocytes than in nonpregnant control subjects.’ Kecenr experiments shou that this is due to IgM antibodies in maternal sera. probably in the form of complexes. becoming attached to the surf’acc of‘ a wrtain population of autologous Ipmphocytes. Antibody. alone or in complrx \\-ith antigen. may play an important role in regulating the immune I-csponse by modulating Iymphocytc surface receptors or activating suppressor c ells.” Our observations tnay be indicalivc of a feedback mechanism controlling maternal immunologic I‘C’;IC tivity in the tace of increasing antigenii load during prcgnanc \.

Materials and methods I,ynlphwvtes I\ crc obtained from delibrinattd yeblood 1)). treatment rvith dextran-carbonyl iron mixture, passed owr a strong magnet, and f’urthet purified by the Ficoll-Hypaque gradient technique with Lymphoprcp (Nyegaard. Oslo). as previously dcscribed.’ Detection of surface IgM. Some lymphocytes were I~OLIS

Simultaneous

detection

of SRBC-RFC

and

surface

Following incubation with autologous serum ant1 staining fi)r surf-ace IgM, lymphocytes were spun with an q~1a1 volume of 0.4 per cent sheep I-cd blood ~~11s (SKBC) at 250 g for IO minutes and placctl immediately on ice for 60 minutes. The mixture was thcl1 resuspended gently and exarnined f’or Ivmphot vtc> with simultaneous SKBC rowttt’s and surlk IgR/I I)! phase-contrast and fluorescence microscopy. IgM.

846

Volume Number

Surface IgM on lymphocytes

127 8

Simultaneous detection of EA-RFC and surface IgM. A 2 per cent suspension of Ox RBC was sensitized with an equal volume of a subagglutinating dose of rabbit IgG anti-Ox RBC serum for 45 minutes at room temperature. After washing, these sensitized RBC’s lvcrc diluted to a concentration of 1 per cent, added to an equal volume of maternal lymphocyte suspension which had been incubated with autoloeous serum and stained for sui-face IgM, and centrifuged at 250 g for 3 minutes at room temperature. This was then allowed to stand on ice for 30 minutes. after lvhich the centrifuged pellet tvas resuspended gently and examined mider phase contrast and fluorescence microscopy for lymphocytes with simultaneous rosettes and surface IgM. Immunodiffusion. IgM levels of .50 midterm pregnant and .50 nonpregnant sera were tested by radial immunodiff.usion on commercial plates (Hyland, United States). Ultracentrifugation. Maternal sera were spun in an MSF. Superspeed 65 at 4” C. and at 37” C. for varying periods oi time. The supernate fractions and the ultracentrifuged deposits were then incubated with autologous cells. Separation of T and B lymphocytes. Lymphocytes \verc incubated with an equal volume of washed 0.4 per (wit SRB(Z for 10 minutes at 37” C. The mixture was then spun at 250 g f’or 10 minutes and left for 60 minutes on ice. After resuspending the pellet gently, 3 ml. of the mixture were layered on to 2 ml. of Lymphoprep (dtnsits. 1.077) in “Ll” bottom 1 % by 10 cm. tubes and spun with an interface speed of 400 g for 40 minutes. ‘l%e interface layer nmv contained cells depleted of T cells and were therefore mainly B cells. The SKBC rosetted pellet at the bottom of the tube consisted mainly of T cells. The rosettes were dissociated by agitation at 37” (:. and a T-cell suspension free of’ SRBC obtained by layering 2 ml. of the cell mixture on to 2 ml. of Lymphoprep and spun for 15 minutes at an interface force of 400 g. ”

Results In pregnant women, there was a marked increase in the number of lymphocytes with detectable IgM on their surface after incubation overnight with autologous swum at 4” C. This also occurred in nonpregnant women but to a much lesser extent (see Table I). Heating at 37” (1. resulted in a rapid disappearance of the surface IgM within a f&v minutes, but this did not occur in the presence of sodium azide (3 x IO-” molar). In a group of’ women monitored throughout preg-

847

Table I. Showing increase in pcwentage ot‘ IgM-bearing cells after incubation overnight with autologous sera at -4” (Z.

Pregnant Nonpregnant

Table II. Showing percentage of IgM-bearing cells after incubation with autologous sera at different stages of pregnancy

O-12 wk. 12.3

20-40

wk.

133

nancy, it was found that the number of’ l~mphocytex with surface IgM after incubation 1, ith autologons ._ serum increased immediately during the first trimcster, remained high throughout pregnant).. and tell after parturition (see Table II). When lymphocytes from the postpartum peGot1 were incubated with autologous serum collected tluring midpregnancy. it was found that the tl~rmtm01’ IgM-bearing cells rose to the level normally swn in midpregnancy (see Table III). The IgM antibodies could be spun do\vn at 3ti.000 r.p.m. for 6 hours and 2 1 minutes-at -1‘ (:.. or for 2 hours and 48 minutes at 37” (I., \z hich c-orwspo~~tlcd IO a sedimentation coef’hcient of‘ 25s. No rheumatoid activit!, was tletcctcd in pwgmnt sera by the Rose Waaler or Latex tests (\l’dlcc~m~). The IgM antibody was not cytotosic. to autologous lymphocytes when incubated ltith added rabbit complement. Immunodiffusion estimation ot lg.\1 shol\vd a ~nc’an value of 12X.9 mg. per deciliter in pregnant st‘ra ;~nd 125.7 mg. per deciliter in nonpregnant scra. I‘hc dill ference was not statistically significant. When examined simultaneously with phasw ontmst and fluorescence microscopy, a large number. (up to 90 per cent) of EA-RFC was found to passively acquire serum IgM after incubation \\ ith autologous SCT~II~. but only a very small number (up to 3 per cent) ot‘ SKBC-KFC did so. Separation of maternal l~inphoc~tc populatio~~~

848

Loke, Brook, and Allen

Table III. Showing percentage of IgM-bearing collected at the same time or previously during

cells rqhen lymphocytes were incubated another stage of pregnancv

Table IV. Showing percentage of IgM-bearing cells in T-cell enriched and T-cell depleted fractions after incubation overnight with autologous sera at 4” C. IgM-bmring

lyrrzphocytr

~

T-cell depleted fraction T-cell enriched f’raction

8.0 None

3 I .o 8.25

showed that cells mainly from the T-cell depleted fi-action passively acquired serum IgM whereas only a small percentage of cells from the T-cell enriched fraction did so (see Table IV). Prior treatment of maternal lymphocytes with 0.2.?1 per cent trypsin for 30 minutes at 37” C. did not affect the cells’ abilities to passively acquire serum IgM.

Comment Our experiments have shown that the surface IgM detected on peripheral lymphocytes from pregnant women I\ ere passively acquired from autologous serum. These antibodies were not autolymphocytotoxins. On ultracentrifugation, these cytophilic IgM antibodies were found to occur as relatively large molecules with a sedimentation coefficient of 27s. suggesting they tvere in the form of aggregates or complexed with antigen. That they could be deposited by ultracentrifugation at 4” C. as well as at 37” C., suggests that these large IgM molecules were probably present in vivo and not merely aggregated in thr cold in vitro. Immunodiffusion estimation revealed no significant difference in the level of free IgM antibodies between pregnant and nonpregnant sera. The number of IgM-bearing lymphocytes I-ose within the first trimester, remained at a high level throughout pregnancy, and fell after parturition. The results of incubation of lymphocytes from the postpartum period with autologous sera collected previously

with autologous

sera

during pregnancy indicate that this observed variation in the number of IgM-bearing lymphocytes can be largely attributed to changes in the amount of maternal serum IgM aggregates or complexes. Only certain maternal peripheral lymphocytes appear to be involved, as repeated incubation with flesh aliquots of autologous serum did not increase tht number of cells showing surface IgM to beyond that found after the first incubation. Separation of maternal peripheral lymphocytes has shown that cells mainly from the T-cell-depleted fraction passively acquired serum IgM 11hcreas only a fthn from the T-cell-enriched fraction did so. Furthermore. a large number of cells with 1gG Fc-receptors (up to 90 per cent) was found to have passively acquired surface IgM, but hardly any of the cells lvhich rosetted \+ith SKBC (up to 3 per cent) were found to do so. Thcsc observations seem to indicate that maternal serum Ighl hinds mainly to the surface of autologous B cells. Kewptora for IgM ha\,e been demonstrated on B cells” and T cells.‘. ’ Recently. receptors for IgM complexes were found on murine splenic B and T cells.” These receptors were trypsin insensitive. appeared to bind the Fc portion of IgM, and 1, ere distinct from IgG Fc-receptors. In our experimrnts, receptors for maternal serum IgM were not affected by trypsin, and also appeared to be distinct from IgG Fc-receptors since IgG F(--rosettes and surface IgM could still be SCVII OII the same cell e\‘en after saturating IgM receptor sitc.s by repeated incubation 1, ith f’resh alicluots of. autologous sera. We could detect surface IgM on maternal lymphocytes after incubation with autologous serum only at ‘1” C. When warmed to 37” C., the surface IgM rapidly disappeared. However, in the presence of sodium azide. the surface IgM did not disappear when warmed. A possible interpretation of these findings is that IgM aggregates or complexes in pregnant srra are constantly fixing on to a population of autologous lymphocytes via surface receptors in vivo. When mcmbrane activity is not artificially suppressed by lowering

Volume Number

the

127 8

Surface

temperature

with

or by sodium

its surface

a mechanism phocyte

receptors for

surface

the

azide.

may continuous

receptors

this

then by

IgM

be shed, modulation

serum

factors

together providing of

We Hospital

thank for

the their

consultants

IgM on lymphocytes

and

staff

of the

Mill

849

Road

help.

lym-

during

pregnancv.

REFERENCES

I. Lake,

Y. W., Brook,

GYNECOL.

122:

561,

S. S., and Allen,

G. E.: AM. J. OBSTET.

1975.

2. Immunological Tolerance: British Medical Bulletin, (British Council, London,) 32: No. 2, 1976. 3. Basten, A., Warner. H. L., and Mandel, T.: J. Exp. Med. 135: 627. 1972.

4. Moretta. L.. Ferrarini, M., Immunol. 5: 565, 1975. 5. McConnell, I.. and Hurd. 1976. 6. Lamon. E. W., Andersson, Immunol. 116: 1199, 1976.

Durante. C. M.:

M. L., et al.: Eur. Immunology

B.. Whitten,

J.

30: 835,

H. D., et al.: J.