EFFECT OF SPLENECTOMY AND THE FUNCTIONAL HYPOSPLENISM OF COELIAC DISEASE ON AUTO-ANTIBODY FORMATION

J. G. O'Grady, F. M. Stevens, B. Harding, J. Flynn, M. P. G. Little and C. F. McCarthy

Medical Research Council of Ireland, Coeliac Unit, Regional Hospital, Galway.

Summary AUTO-ANTIBODIES or rheumatoid fac-

tor were detected in 10.3% of 78 normal subjects, 16.8% of 89 splenec- tomised subjects, 15.4% of 39 normo- splenic coeliacs and 18.3% of 49 hyposplenic coeliacs. Auto-antibodies tended to occur in older subjects but the presence of hyposplenism did not result in their formation at a younger age. The presence of the HLA-B8 antigen did not significantly effect auto-antibody forma- tion in any of the 3 study populations. We conclude that auto-antibody forma- tion is not influenced by splenic function or the HLA-B8 antigen.

Introduction The role played by the spleen in the

control of autoimmunity is controversial. As a major source of suppressor T-lym- phocytes, it has been suggested that the spleen has a significant regulatory role in auto-antibody formation (Bullen et al, 1981). On the other hand cell-mediated immunity has recently been shown to be nomal in hyposplenic states (Nielsen and Haahr, 1982). Auto-antibodies we,re found with greater frequency in splenec- tomised subjects than in normal subjects (Spirer et al, 1980; Robertson et al, 1983). Coeliac disease is associated with hyposplenism (McCarthy et al, 1966; Marsh and Stewart, 1970; Ferguson et, a/, 1970; Robinson et al, 1980; Corazza et al, 1981) and increased auto-antibody for- mation has been reported both in coeliac patients (Lancaster, Smith et al, 1974; Ratnaike and Wangel, 1977) and in coeliacs wih hyposplenism (Wardrop et al, 1975; Bullen et al, 1981). The major histocompatibility antigen B8 (HLA-B8) is associated both with coeliac disease (Falchuk et al, 1972) and some auto- immune diseases (Vladutiu and Rose,

1974). In this study we examine auto- antibody formation in large groups of normal subjects, splenectomised sub- jects and coeliac patients, including both normosplenic and hyposplenic coeliacs, with particular reference to the HLA-B8 antigen.

Patients and Methods Sera from 78 normal subjects, 89

splenectomised subjects and 88 coeliac patients were examined for the presence of antibodies to nuclear material (ANF), mitochondria, smooth muscle, gastric parietal cells and reticulin in addition to rheumatoid factor (RF). The RF was detected using latex-RF reagent (Beh- ring). All the other antibodies were detected with an indirect immunofluores- cent technique, using rat stomach, liver and kidney as tissue substrate and fluor- escent antisera against human IgG, IgA and IgM.

The normal controls consisted of healthy volunteers or patients attending a surgical out-patient clinic with minor complaints. There were 41 males and 37 females with a mean age of 37.4 years. The splenectomised subjects had their operation between 1 and 10 years prior to the, time of study for a variety of indications including trauma, haemato- logical disorders and incidental to another surgical procedure e.g. gastrec- tomy. This group consisted of 58 males and 31 females, with a mean age of 39.5 years. The. diagnosis of coeliac disease was made, on the. basis of severe, small bowel mucosal damage while ingesting gluten followed by a histological and/or appropriate, clinical response to with- drawal of gluten from the diet. There were 35 male and 53 female coeliacs with a mean age of 41.8 years and at the time of study 69 were on gluten-free diets

351

352 Ob'Grady et at.

and 19 were untreated. No patient was included in any of the 3 groups who had a coexisting overt disease associated with the presence of autoant ibodies or RF.

'Pitted' erythrocyte counts were per- formed on all subjects by the method of Pearson et at (1978). Three drops of fresh venous blood were fixed in 1 ml of 3% glutaraldehyde pH 7.4. One, thousand consecutive e rythrocytes were examined for the, presence of 'pis' using a Nikon interference-phase microscope and the results were expressed as percentages of the total with one or more 'pit' present. Both the 'pitted' erythrocyte counts and the antibody screens were performed without knowledge of the clinical status of the patient. The HLA typing was car- ried out using the microlymphocytotox- ~ technique. Statistical analysis was performed using the Chi-square test.

Results 'Pitted' erythrocyte counts in the nor-

mal subjects ranged from 0.4-7.5%. The counts in the splenectomised sub- jects ranged from 1.1-44.2% and were greater than 7.5% in 60 cases (67.4%). Of the 29 splenectomised subjects with counts less than 7.5%, 13 had their operation for congenital spherocytosis, 8 for trauma and 8 for other various

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Figure 1 - 'Pitted" erythr(~cyte counts in: normal subjects, coel iac pat ients and' splenectomJsed,

subjects.

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elective, indications. The. 'pitted' erythro- cyte counts in the cceliac patients ranged from 0.6-40.5%. The coeliacs were subdivided into 39 with counts below 7.5% who were defined as having normal splenic function and 49 with counts greater than 7.5% who were said to have hyposplenism (Figure 1).

The HLA-B8 antigen was present in 40.7% of 64 normal controls, 37.3% of 67 splenectomised controls and 85% of 87 patients with coeliac disease.

Ten auto-antibodies (in 8 subjects) were found in the normal subjects, 17 auto-antibodies (in 15 subjects) in the splenectomised group and 18 auto anti- bodies (in 16 patients) in the cceliac population. There were no significant differences between the 3 groups. The type, of auto-antibodies detected are shown in Table. I. Of the 15 coeliacs with auto-antibodies, 6 had normal splenic function (15.4%) and 9 had hyposplen- ism (18.3%).

TABLE I

Inc idence of auto-ant ibodies in normal subjects, splenectomised subjects and coel iac patients.

No,rmal Splenectomised Ccel iac subjects subjects pat ients

Number 78 89 8S Ant i -nuclear factor 1 3 3 An.t imitochondrial ant ibodies 2 1 0 Smooth muscle ant ibodies 0 1 1 Parietal cel l ant ibodies 1 3 3

Rheumato id factor 2 6 3

The details of the age, sex and HLA-B8 status of the subjects with detectable auto-antibodies are given in Table I1. The male/female ratios in each group reflect their respective overall ratios with the exception of the exagge,rated female predominance in the coeliacs, but this is not statistically significant. The mean ages are. very similar and somewhat higher than the mean age,s of the com- plete populations. The incidence of the HLA-B8 antigen parallels that found in the, overall respective groups.

Volume 153 Number 10

TABLE II

Age, sex and HLA-B8 status of subjects with detectable auto-ant ibodies.

Normal Sp lene: tomise : l Coel iac subjects subjects patients

Number 8 15 16

Mean age 46.0 46.1 45.0

Male 4 9 4 Female 4 6 12

HLA-B8 + 3 3 13 HLA-B8 -- 4 8 2

Discussion 'Pi t ted' erythrocytes contain auto-

phagic vacuoles that are normally re- moved from the cell as it passes through the spleen. Their presence in increased numbers in peripheral blood indicates impaired splenic function or hyposplen- ism and 'pitted' erythrocyte counts have been used to assess splenic function in a wide variety of diseases including coeliac disease (Corazza et al, 1981; O'Grady et al, 1983), dermatitis herpeti- formis (Corazza et al, 1981), neonates (Holroyd et al, 1969; Kim et al, 1981), splenic irradiation in Hodgkin's disease. (Coleman et al, 1982) and splenosis (Pearson et al, 1978; Neilan and Perry, 1980; Cohen and Ferrante, 1982). In general 'pitted' erythrocyte counts sensi- tive,ly differentiate normal from splenec- tomised subjects, but low counts may be found post-splenectomy in association with congenital spherocytcsis (O'Grady et al, 1984) and trauma (Pearson et al, 1978; Cohen and Ferrante, 1982). 'Pitted' erythrocyte counts were used in this study to quantitatively assess splenic function in coeliac patients and repre- sents a more sensitive indicator of hypo- splenism than peripheral blood smears markers like Howell-Jolly bodies.

Auto-antibodies have been reported with greater frequency in splenectomised subjects (Spirer et al, 1980; Robertson et al, 1983), coeliac patients (Lancaster- Smith et al, 1974; Ra'tnaike and Wangel, 1977) and coeliacs with hypcsplenism (Wardrop et al, 1975; Bullen et al, 1981 ) than in normal subjects. These findings are not confirmed in this study which

Auto-antibody formation 353

represents the first large series in which all 3 groups are concomitantly studied. Auto-antibodies were detected in just over 10% of the normal subjects in com- parison to an incidence of 21.6% in a large community study in Australia where a wider range of auto-antibodies were investigated (Hooper et al, 1972). Auto- antibodies were present in 16.8% of splenectomised subjects which was not significantly higher than normal controls. Robertson et al (1983) found auto-anti- bodies in 13 of 35 splenectomised sub- jects compared to 7 out of 35 normal subjects. It is noteworthy that in the report of Spirer et a l (1980), the incid- ence of non-tissue specific auto-antibod- ies was not significantly higher than in normal subjects and the reported differ- ence only became apparent when antibodies against heart muscle were included. No difference was found in this study in auto-antibody formation in the coeliac patients compared to either normal or' splenectomised subjects, and within the coeliac population itself, splenic function did not influence the level of auto-antibodies found. This contrasts with the findings of Bullen et a i (1981) and Lancaster-Smith et al (1974), both of whom however included some coeliac patients in their series who had co-existing diseases also associated with the presence of auto-antibodies. Anti-reticular antibodies were detected in 5% of normal subjects in this series which is in keeping with the figure of 6.3% described by Stevens et al (1975). The finding of anti-reticular ~ntibodies in only 9% of ceeliac patient~ was sur- prising, but it probably I reflects the predominance of treated (~oeliacs in this study population as these have a much lower incidence of antibodies to reticulin than untreated coe, liacs (Stevens et al, 1975).

Auto-antibody formation increases with advancing age (Hooper et al, 1972) and the patients with auto-antibodies in this series were somewhat older than the remainder of their respective groups. Hyposplenism did not result in the de- velopment of auto-antibodies at a

354 O'Grady et al.

younger age. Females are repor ted to be espec ia l l y prone to develop auto- ant ibod ies (Hooper et at, 1972) but th is was not ev iden t in th is ser ies with the except ion of the coe l iac pat ients, where the f ind ing is par t ly exp la ined by the female excess in this populat ion. This study conf i rms the lack of ef fect of the HLA-B8 ant igen on anto-ant ibody forma- t ion prev ious ly repor ted in coe l iac pat- ients (Bul len et al, 1981; Kumar e t al, 1981) and ex tends th is observat ion to include, both normal and sp lenec tomised subjects.

We conc lude from this study that sp lenic funct ion does not influence, auto- ant ibody fo rmat ion in e i ther cce l i ac or non-coel iac populat ions, in keeping with the recent suggest ion that ce l l -media ted immuni ty is spared in hypcsple,nic state,s. The HLA-B8 ant igen also does not ef fect the product ion of auto-ant ibodies.

We thank the Medical Research Council of Ireland for generous support. We also thank Ms. M. Bourke, M. Brennan and M. Kearns for tech- nical assistance.

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