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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 91, Num. 3, 1996, pp. 367-369
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 91(1), May/June
1996
Mucosal Immunology and Models of Mucosal HIV Infection
GE Griffin*, LRR Castello-Branco/^+, MB Ortig o-de-Sampaio, R
Shattock*
Departamento de Imunologia, Instituto Oswaldo Cruz, Av. Brasil
4365, 21045-900 Rio de Janeiro, RJ, Brasil *Division of
Infectious Diseases, St. George's Hospital Medical School,
London, UK
Code Number: OC96073
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The mucosa associated lymphoid tissue regulates and
coordinates immune responses against mucosal pathogens. Mucosal
tissues are the major targets exposed to HIV during transmission.
In this paper we describe in vitro models of HIV mucosal
infection using human explants to investigate target cells within
this tissue.
Key words: mucosal immunology - HIV - immunity to HIV - mucosa
associated lymphoid tissue
COMMON MUCOSAL-ASSOCIATED LYMPHOID TISSUE
The concept of a common mucosal-associated system regulating
and coordinating immune responses at mucosal surfaces has been
an important advance in our understanding of protection against
mucosal pathogens. This system, called the mucosa associated
lymphoid tissue (MALT), is based on primed T and B lymphocytes
that migrate from the site of antigen presentation via the
lymphatics and blood to selectively 'home' to lymphoid tissue at
distant sites in gastrointestinal, respiratory, genitourinary and
other mucosa-associated regions.
The majority of human pathogens are encountered at a mucosal
surface, and therefore, in addition to providing protection
against naturally acquired pathogens, MALT has great attraction
for the development of mucosally protective vaccines. The gut in
particular, with its great surface area and huge population of
immune cells (gut-associated lymphoid tissue) and specific
antigen sampling M cells overlying Peyer s patches, provides an
attractive target for immunization. Novel vaccines to protect
inaccessible human mucosal surfaces and secretions (such as the
genital tract or breast milk) might therefore be delivered to the
gut or nasal tract and protection disseminated throughout MALT.
Although evidence exists, however, for the trafficking of primed
B (Lewis 1991) and T (Castello-Branco 1994) cells after oral
antigen delivery, the characterization of a common mucosal immune
system in humans is incomplete.
When compared with trafficking B cells induced by systemic
immunization, mucosally derived B cells expressed more advanced
markers of B cell maturation, were phenotypically more
homogeneous, and lacked the adhesion molecule L-selectin which
is associated with homing to peripheral lymph nodes (Quiding
1995b). This study demonstrated for the first time in humans
clear phenotypic differences between mucosal and systemic B-cell
responses, and further studies, particularly with reagents to
detect mucosa-specific adhesion molecules, may begin to unravel
the complex mechanisms regulating the differential homing seen
after immunization of various mucosal sites.
A key addressin appears to be the integrin alpha4beta7, which
is expressed on mucosal T and B cells and is the dominant
receptor for the glycoprotein mucosal vascular addressin (MAdCAM-
1). Pals et al. (1994) found that malignant lymphomatous
polyposis tumors (a gastrointestinal variant of mantle cell
lymphoma characterized by multiple lymphomatous polyps in the
gastrointestinal tract) express alpha 4 beta 7 integrin, in
contrast with control cases of lymph node mantle cell lymphoma.
This suggests that alpha4beta7 may act as a mucosal addressin for
these human gastrointestinal tumours. Boll et al. (1995) studied
the phenotypic nature of gastrointestinal T cells in the rodent
model, and found differential expression of alpha4beta7-related
integrins by large and small intestinal lymphocytes, suggesting
that either the specific homing of T cell subsets to different
parts of the gut, or the modulation of phenotypic differential
characteristics by the regional microenvironment of the gut may
be regulated by expression of adhesion molecules. Quiding et al.
(1995a) also found sub-compartmentalization within MALT in a
study of human volunteers receiving nasal or intratonsillar
immunization. Although nasal immunization resulted in a more
disseminated response, response to tonsillar immunization was
localized to the upper aerodigestive tract. The fine detail of
MALT compartmentalization in humans will need to be explored if
these novel routes of immunization are to be exploited
effectively.
HIV AND THE MUCOSA
Mucosal tissues are the major targets exposed to the HIV during
transmission. In the majority of subjects the initial acquisition
of HIV involves passage of virus across a mucosal surface. The
sexual route is the most important route of transmission in: (1)
homosexuals where lymphoid cells are likely to be the prime
target and (2) heterosexuals where the genital tract provides the
virus access to lymphoid cells. In children, the upper
gastrointestinal tract mucosa seems to participate in acquisition
of infection in vertical transmission through swallowing of (1)
HIV-infected amniotic fluid in utero, (2) HIV-infected
blood and cervical secretions intrapartum, and (3) HIV-infected
breast milk post-partum.
In order to define more clearly the possible effect of HIV on
intestinal mucosa, we developed in vitro models of HIV
infection using human foetal explants. In addition we developed
a model of cervical explant culture to investigate target cells
within this tissue.
IN VITRO STUDIES OF INFECTION OF HUMAN MUCOSAL TISSUE
BY HIV
Intestine - The large intestine and rectum in male
homosexuals, and the upper intestine in children being breastfed
by HIV-infected mothers, are potential mucosal surfaces for HIV
infection. However, debate exists as to the intestinal mucosa
cells that are targets for infection with HIV. HIV-infected cells
detected within intestinal mucosa in late stages of disease may
represent trafficking of lymphocytes or macrophages from distant
sites of the reticuloendothelial system. Results of an initial
study of intestinal mucosal biopsy samples (Nelson et al. 1988)
from patients with AIDS suggested that HIV infection of
epithelial argentacromaffin was etiologic in HIV enteropathy.
However, other studies have failed to detect HIV genome or
proteins within epithelial cells but within small numbers of
cells resembling macrophages in the lamina propria, and using
immunocytochemistry we have detected HIV p24 antigen only within
reticuloendothelial cells of European and African small
intestinal biposy samples (unpublished observations).
In order to shed light on possible target cells for HIV in the
intestine, we have developed in vitro mucosal explant
culture systems using human foetal intestine. Adult intestinal
biopsy samples disintegrate within 24 hr of in vitro
culture and are therefore not suitable for detailed investigation
over several days. In contrast, human foetal mucosal explants of
small and large intestine survive in in vitro culture for
up to two weeks. Using this system, we found that epithelial
cells are not targets for HIV infection per se, but that
CD4+ cells of both macrophage and lymphocyte phenotype were
productively infected with virus (Fleming et al. 1992). Although
atypical of the in vivo situation, this simple clearly
excludes productive infection of enterocytes within the limits
of detection used. We have analyzed HIV-infected mucosal explants
using an antibody to a nuclear protein (PCNA) present in the
mitotic spindle of actively replicating epithelial cells and have
shown that there is an increase in epithelial cells expressing
this protein, indicating that HIV infection induces epithelial
cell proliferation (Batman et al. 1995). Such effects are clearly
indirect since HIV cannot be detected within epithelial cells in
this system, and we are investigating the possibility of cytokine
production by HIV-infected immune cells.
Cervix - The mucosal route of infection in Africa is
undoubtedly via heterosexual intercourse, and the paradox of the
apparently relative infrequency of this route of transmission in
the developed world remains a fundamental enigma. Seminal and
vaginal secretions contain HIV, and it is therefore not
unreasonable to assume that cells within the reproductive tract
of men and women are targets for infection. Cervical biopsy
samples from HIV-infected women have been shown to contain HIV-
infected macrophages, but again this may represent trafficking
from distant sites. We have recently investigated this using a
simple in vitro culture system of normal ectocervix
dissected from hysterectomy specimens. Such explants can be
maintained in culture for up to 12 days. We find that cervical
macrophages are the primary targets of HIV when macrophage-tropic
strains of the virus are used. Surprisingly, using this explant
system which contains both Langerhans cells (CD1a) and
lymphocytes (CD3) neither of these cell types became infected
with HIV. We are now using the system to investigate factors
regulating HIV gene transcription in cervical cells, as well as
agents-such as cytokines-that may be locally active in women with
vaginal or cervical infection. Such sexual transmitted infections
of the female genital tract are likely to influence
susceptibility to HIV infection by attracting a population of CD4
bearing cells as part of the immune response and the production
of ulcers in the epithelium exposing such cells to HIV.
CONCLUSION
We have shown that gut and cervical immune cells are potential
targets for direct HIV infection and have begun to study control
mechanisms involved in subsequent mucosal immunopathological
events with in vitro systems. We have demonstrated that
humans respond to oral immunogens until late HIV disease with
antibody and circulating B-cell responses. These findings raise
the possibility of contriving immunity against HIV-related
pathogens with novel mucosal vaccines. A better understanding of
in vitro and in vivo interactions between HIV and
the mucosal immune system will also be fundamental in vaccines
against mucosal transmission of HIV. Ideally, the integration of
cellular and molecular studies with an in vivo patient-
centered approach will lead to the final understanding and
control of these interactions, and it is this fundamental
principle that directs our current and future studies.
REFERENCES
Batman PA, Fleming SC, Sedgwick PM, MacDonald TT, Griffin GE
1995. HIV infection of human fetal intestinal explant cultures
induces epithelial cell proliferation. AIDS 8: 161-167.
Boll G, Rudolphi A, Spiess S, Reinmann J 1995. Regional
specialization of intraepithelial T cells in the murine small and
large intestine. Scand J Immunol 41: 103-113.
Castello-Branco LRR, Griffin GE, Poulton TA, Dougan G, Lewis DJM
1994. Characterization of the circulating T-cell response after
oral immunization of human volunteers with cholera toxin B
subunit. Vaccine 12: 65-72.
Fleming SC, Kapembwa MS, MacDonald TT, Griffin GE 1992. Direct
in vitro infection of human intestine with HIV-1. AIDS
6: 1099-1104.
Lewis DJM, Novotny P, Dougan G, Griffin GE 1991. The early
cellular and humoral response to primary and booster oral
immunization with cholera toxin B subunit. Eur J Immunol
21: 2087-2094.
Nelson JA, Wiley CA, Reynolds-Kohler C 1988. HIV detected in
bowel epithelium from AIDS patients with gastrointestinal
symptoms. Lancet 1: 259-262.
Pals ST, Drillenburg P, Dragosics B, Lazarovits AI, Radaszkiewicz
T 1994. Expression of the mucosal homing receptor alpha4 beta7
in malignant lymphomatous polyposis of the intestine.
Gastroenterol 107: 1519-1523.
Quiding JM, Granstrom G, Nordstrom I, Czerkinsky C 1995a.
Induction of compartmentalized B-cell responses in human tonsils.
Infect Immun 63: 853-857.
Quiding JM, Lakew M, Nordstrom I, Banchereau J, Butcher E,
Holmgren J, Czerkinsky C 1995b. Human circulating specific
antibody-forming cells after systemic and mucosal immunizations:
differential homing commitments and cell surface differentiation
markers. Eur J Immunol 25: 322-327.
^+Corresponding author. Fax: 55-21-280.1589
Received 7 December 1995
Accepted 10 January 1996
Copyright 1996 Fundacao Oswaldo Cruz
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