Immunological study of the rectal mucosa of men with and without human immunodeficiency virus infection - Gut
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Gut: first published as 10.1136/gut.28.12.1619 on 1 December 1987. Downloaded from http://gut.bmj.com/ on November 13, 2021 by guest. Protected by copyright.
Gut, 1987, 28, 1619-1624
Immunological study of the rectal mucosa of men with
and without human immunodeficiency virus infection
P E BISHOP, A McMILLAN, AND H M GILMOUR
From the Department of Genito-Urinary Medicine and Department of Pathology, University of Edinburgh,
Edinburgh
SUMMARY Biopsies of rectal mucosa were taken from 81 men and stained using cytochemical
methods for B and T lymphocytes, T cell subsets, immunoglobulin containing plasma cells and
mucosal mast cells. The patients studied included human immunodeficiency virus (HIV) infected
and non-infected heterosexual and homosexual men, and homosexual men with rectal gonorrhoea.
There were increased numbers of T lymphocytes in the lamina propria of the rectum in HIV
infected individuals regardless of whether the infection had been acquired through anal intercourse
or intravenous drug use. This increase resulted from a marked increase in the numbers of CD8+
suppressor T cells, there also being a reduction in the numbers of CD4+ helper T cells. In non-HIV
infected men with rectal gonorrhoea there were increased numbers of CD8+ T cells but no
significant difference in numbers of CD4+ cells. No difference was seen in numbers of
immunoglobulin containing plasma cells or mucosal mast cells between HIV infected and non-
infected men.
In Western Europe and the United States of America infected intravenous drug users (all with persistent
sexually active homosexual men constitute the pop- generalised lymphadenopathy, without history of
ulation group at most risk for acquiring the human homosexual contact) and 67 consecutive homosexual
immunodeficiency virus (HIV).'12 Most studies have male patients attending the department (Table 1).
shown a clear association between anal intercourse, All but three of the healthy homosexual men had
particularly penoreceptive, and acquisition of the been the recipient partners during anal intercourse.
virus.34 As the rectum seems to be the portal of entry After taking a careful history and completing a
of HIV it is surprising that there have been few general physical examination, material for micro-
reports of the immunohistology of the rectum in biological examination was obtained.' A sigmoid-
homosexual men. The present study was undertaken oscope, lightly lubricated with K-Y jelly (Johnson
to discover if the cellular content of the lamina and Johnson, Slough, UK) was passed and using
propria of the rectums of sexually active homosexual Patterson's forceps at least two biopsies were taken.
men was different from that of heterosexual men,
and to assess the effect of gonococcal infection on the LABORATORY METHODS
lymphocyte composition of the rectal mucosa. One biopsy was carefully orientated on a glass slide,
fixed in buffered formol sublimate and processed for
Methods paraffin sections for histological evaluation. A
second similarly orientated biopsy was snap frozen in
PATIENTS AND CLINICAL METHODS liquid nitrogen and subsequently embedded in OCT
The study was approved by the Ethics Committee of compound (Miles Scientific, Naperville, USA) for
the Lothian Health Board. Eighty one men were the preparation of cryostat sections. The block was
studied: nine healthy heterosexual men, five HIV sectioned immediately or stored at -70°C for up to
one week. A third biopsy, obtained from 24 con-
Address for correspondence: Dr A McMillan, Department of Genito-Urinary secutive patients, was fixed in Carnoy's fluid and
Medicine, The Royal Infirmary, Edinburgh EH3 9YW. processed for paraffin sections for staining for mast
Received for publication 8 May 1987. cells.
1619Gut: first published as 10.1136/gut.28.12.1619 on 1 December 1987. Downloaded from http://gut.bmj.com/ on November 13, 2021 by guest. Protected by copyright.
1620 Bishop, McMillan, and Gilmouir
Table 1 Diagnoses made in the 67 hom0osexual men ELISA or indirect immunofluorescence methods
studied with confirmation of positive results by western
blotting.9
lIiagniosis Metz affected (it)
STATISTICAL ANALYSIS
Syphilis (early) 2 The data obtained on the lymphocyte subset compo-
Rectal gonorrhoca 9 sition of the lamina propria were normally distri-
Non-gonococcal urethritis I
Chiamydial infection of rectum 1 buted and Student's t test was used in their analysis.
Anorectal herpes simplex infection 2 As the numbers of mucosal mast cells and immuno-
Perianal condylomaita acuminata 17 globulin containing plasma cells were not normally
Amochiasis
Giardiasis distributed, Wilcoxon's rank-sum test was used. The
Enterobiasis x2 test was used to compare the histological grading of
HIV infection 9t the rectal mucosa.
No infection identified 36
Results
* Some men had concurrent infections; t Eight men h.ad persistent
generalised lymphadenopathy. the other had no clinical evidence of
HIV infection. HISTOL OGICAL GRADING (Table 2)
The histology of the rectum was normal (grade A) in
IMMUNOCYTOCHEMICAL METHODS the 14 heterosexual men studied, including the five
Cryostat sections at 4 [tm were cut at two levels and intravenous drug users seropositive for HIV. A mild,
stained for lymphocyte subsets using an indirect non-specific proctitis was found in five of 14 HIV
immunoperoxidase method6 with the following infected and six of 36 non-infected homosexual men.
monoclonal antisera: Pan-B/M708 (CD22, mature B There was no significant difference in the prevalence
cells, Dako Ltd., High Wycombe, UK), anti-Leu 4 of inflammatory changes between HIV infected and
(CD3a, mature T cells), anti-Leu 2a (CD8, sup- non-infected men (X'= 1 79; p>0. 10).
pressor/cytotoxic T cells) and anti-Leu 3a (CD4,
helper/inducer T cells) (all from Becton Dickinson, I YMPHOCYTE SUBPOPUI ATIONS WITHIN THE
Laboratory Impex, Twickenham, UK). L AMINA PROPRIA (Figure)
Paraffin sections at 3-4 sim taken at two levels
from 15 consecutive blocks were stained for plasma CD22' B cells
cells containing IgA, IgG and IgM using a peroxidase- Using the antiserum M708, which is broadly reactive
antiperoxidase (PAP) method.7 with mature B cells, no staining was seen in the
lamina propria. All B cells reactive with this anti-
STAINING FOR MAST CELLIS serum were confined to organised lymphoid aggre-
Mucosal mast cells were stained with Astra blue gates.
(BDH Ltd., Poole, UK) and safranin as described by
Strobel et al.' Table 2 Histological grading of rectal biopsies in relation to
HIV infection and gonorrhoea
HISTOMETRY
Cells were counted using an eyepiece graticule Histological grade
marked with a lOx 10 grid. The sections were
examined under high power magnification (objective Group A B Total
x40, eyepiece x 10) and one edge of the grid aligned
with the basement membrane of the surface epi- Non-infected. non-intravcnous
drug-using heterosexual men 9 t) 9
thelium. At this magnification the grid edge covered
IIIV-infected heterosexual
a length of 0-283 mm, area 0 08 mm2. Where intravenous drug uscrs 5 ( 5
orientation of the sections allowed, five high power Non-infceted homoscxual men 311 5 36
fields were counted (minimum three) by two IiIV infected homoscxual men 9' 5 14
observers independently. Areas including, or im- hiomosexual men with rectal
mediately adjacent to lymphoid aggregates were gonorrhoea but no scrological
avoided. Interobserver variation wasGut: first published as 10.1136/gut.28.12.1619 on 1 December 1987. Downloaded from http://gut.bmj.com/ on November 13, 2021 by guest. Protected by copyright.
Rectal lymphocytes and HIV infection 1621
CD3a+ cel Is CD4+ cel Is
80-
60-
4
40-
I0
20- 4
1-
0 A B C D
80- 8-
CD8+ cells CD4+: CD8 cell ratio
60- 6-
20-
0 WZZ
A B
Iz
rz
C
fz wZgL-
D
Al
4-
2-
0 A
I
B
4;
rl~Z v le
C
-sz A
D
o o
Figure Numbers of T cells per unit area (0-08 mm2 ) and ratio of CD4 :CD8+ cells in the lamina propria of the rectum.
A=non-infected heterosexual men (n=5); B=non-infected homosexuial men (n=12); C=HIV infected homosexual men
(n =9); D=HIV infected heterosexual men (n=5); *errorbar
CD3a T cells HIV infected homosexual men (16-1 ± 6-3) was
Within the unit area of 0-08 mm) there was no lower than that of the non-infected homosexual men
significant difference in the number of T cells be- (30.3 ± 9-4): this difference was highly significant
tween non-infected heterosexual (44-0 ± 11.8) and (t=3-91; p0- 1). Similarly, the mean numbers of T cells in the non-infected (47.6 ± 28-1) heterosexual men
mucosa of HIV infected homosexual men (67.3 ± (t=2.75; p0-5). The mean CD8' cells
numbers of CD3a' cells in the lamina propria of HIV The mean number of CD8+ cells in the lamina
infected homosexual men and intravenous drug users propria was significantly greater in the HIV infected
were significantly higher than those of the non- (50-4 ± 26-1) than in the non-infected (8-2 ± 2-8)
infected men (t=5.15; pGut: first published as 10.1136/gut.28.12.1619 on 1 December 1987. Downloaded from http://gut.bmj.com/ on November 13, 2021 by guest. Protected by copyright. 1622 Bishop, McMillan, and Gilmtnoiur CD4': CD8 cell ratio Discussion As can be seen from the Figure, the CD4+: CD8' cell ratio was significantly lower in the two HIV This study has shown that in HIV infected indi- infected groups (0.4 ± 0(3) compared with the non- viduals, regardless of how the infection had been infected groups (3-9 ± 1-1) (t=11 3; p). I p>0. I
Gut: first published as 10.1136/gut.28.12.1619 on 1 December 1987. Downloaded from http://gut.bmj.com/ on November 13, 2021 by guest. Protected by copyright.
Rectal lymphocytes and HlV infection 1623
cells between Rodgers and colleagues"' and our study potentiation of eosinophil and macrophage cyto-
is not immediately apparent. Among sexually active toxicity." Although increased numbers of mucosal
homosexual men, infection with Epstein-Barr virus mast cells were noted in men with AIDS associated
(EBV) and cytomegalovirus (CMV) is universal,'2 diarrhoea," we did not find a difference in the median
and there are data to suggest that HIV infection may numbers of cells per unit area between HIV and non-
reactivate EBV.'" Reactivation of these viruses is HIV infected men.
sometimes associated with increased numbers of Secretory IgA is important in the host defence
CD8-bearing lymphocytes in the peripheral blood.' against infection at mucosal surfaces."1 As CD4+ cells
Unfortunately facilities for viral culture from our play a part in the regulation of IgA responses,'7 HIV
patients were not available. Although the number of infection might result in impaired mucosal immunity.
CD8' cells in the peripheral blood of homosexual Indeed, intestinal infection of such individuals
men infected with HIV is often greater than normal,' with organisms such as Crypto-sporidium spp that
it is difficult to know whether this represents a direct generally produce a transient diarrhoeal illness is well
effect of that infection or the result of reactivation of recognised.2" Although we did not find a significant
latent viruses such as CMV or EBV. After acute HIV difference in the median numbers of IgA containing
infection, there is an increase in the number of CD8 plasma cells within the lamina propria of HIV
cells in the peripheral blood.4 The only patient in our infected and non-infected men, a functional impair-
series who had a mononucleosis like illness followed ment of IgA secretion is not precluded.
by seroconversion had the most marked infiltration In conclusion, our findings confirm that abnor-
of the lamina propria with CD8 cells of all the malities of lymphocyte subset distribution within the
patients studied. The mean number of CD4' cells (33 lamina propria of the rectums of HIV infected men
per unit area) was the highest of the HIV infected are common, but their significance with respect to
homosexual men but within one standard deviation mucosal immunity is still uncertain.
of the mean value for non-infected men. As a result
of the large increase in CD8X cells the CD4':CD8, We would like to thank Dr A E Dewar, Department
ratio was low (0-4). None of the other patients, of Pathology, University of Edinburgh, for her
however, were studed at this stage of infection helpful advice. This work was supported by Scottish
(median duration of lymphadenopathy 12 months, Home and Health Department, Biomedical Re-
range five to 24 months). search Committee grant K/MRS/50/C685.
Although in most studies receptive anal inter-
course has been shown to be a risk factor for the References
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