Potential HIV-1 target cells in the human penis

Abstract

To study the distribution of HIV-1 receptors and degree of keratinization in the human penis.
Formalin-fixed penises were obtained from nine uncircumcised cadavers. Foreskins were obtained from 21 healthy adult men undergoing elective circumcision for social reasons. Uncircumcised penises were obtained within 24 h of death from eight men. All tissues were stained for keratin and HIV-1 receptors.
Penises from nine formalin fixed cadavers aged 64-80 years were obtained from the Department of Anatomy, University of Melbourne. Foreskins were obtained from 21 men aged 18-64 years following circumcision performed at either the Freemason's or Mercy Private Hospitals, Melbourne, Australia. Fresh penile necropsy specimens from eight uncircumcised men aged 23-63 years were obtained from the Victorian Institute of Forensic Medicine, Melbourne. The degree of keratinization was scored, and the distribution of HIV-1 susceptible cells was mapped in the glans penis, penile urethra, urethral meatus, frenulum and foreskin.
Cells with HIV-1 receptors were present in all penile epithelia, but Langerhans' cells were most superficial in the inner foreskin and frenulum. The inner foreskin had a significantly thinner keratin layer (1.8 +/- 0.1 units), than the outer foreskin (3.3 +/- 0.1), or glans penis (3.3 +/- 0.2), P < 0.05.
Superficial Langerhans' cells on the inner aspect of the foreskin and frenulum are poorly protected by keratin and thus could play an important role in primary male infection. These findings provide a possible anatomical explanation for the epidemiologically observed protective effect of male circumcision.

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Potential HIV-1 target cells in the human penis
Scott G. McCoombe
a
and Roger V. Short
b
Objectives: To study the distribution of HIV-1 receptors and degree of keratinization in
the human penis.
Design: Formalin-fixed penises were obtained from nine uncircumcised cadavers.
Foreskins were obtained from 21 healthy adult men undergoing elective circumcision
for social reasons. Uncircumcised penises were obtained within 24 h of death from
eight men. All tissues were stained for keratin and HIV-1 receptors.
Methods: Penises from nine formalin fixed cadavers aged 64– 80 years were obtained
from the Department of Anatomy, University of Melbourne. Foreskins were obtained
from 21 men aged 18–64 years following circumcision performed at either the Free-
mason’s or Mercy Private Hospitals, Melbourne, Australia. Fresh penile necropsy
specimens from eight uncircumcised men aged 23– 63 years were obtained from the
Victorian Institute of Forensic Medicine, Melbourne. The degree of keratinization was
scored, and the distribution of HIV-1 susceptible cells was mapped in the glans penis,
penile urethra, urethral meatus, frenulum and foreskin.
Results: Cells with HIV-1 receptors were present in all penile epithelia, but Langerhans’
cells were most superficial in the inner foreskin and frenulum. The inner foreskin had a
significantly thinner keratin layer (1.8 0.1 units), than the outer foreskin (3.3 0.1), or
glans penis (3.3 0.2), P<0.05.
Conclusions: Superficial Langerhans’ cells on the inner aspect of the foreskin and
frenulum are poorly protected by keratin and thus could play an important role in
primary male infection. These findings provide a possible anatomical explanation for
the epidemiologically observed protective effect of male circumcision.
ß2006 Lippincott Williams & Wilkins
AIDS 2006, 20:1491–1495
Keywords: circumcision, HIV, Langerhans’ cells, mucosa, penis
Introduction
The most recent World Health Organization estimates
show that there are currently 18.7 million men infected
with HIV-1 [1]. Approximately 80–90% of these men
were infected following heterosexual intercourse [1,2],
yet very little is known about the precise routes of HIV-1
entry into the male reproductive tract. Previous studies
[3–6] have observed HIV-1 susceptible cell populations
in the foreskin of adult men, but to date there have been
no studies of the other penile epithelia.
It is generally agreed that keratin provides an imperme-
able barrier to HIV-1 [7]. Two studies have shown that
the glans penis is heavily keratinized in both circumcised
and uncircumcised men [2,8]. Therefore, it is unlikely
to be involved in primary infection unless the keratin
layer is compromised by lesions, inflammation or micro-
trauma. The inner aspect of the foreskin is poorly
keratinized [4,5], but to date there have been no studies
on keratinization of the urethral meatus, penile urethra or
frenulum.
Recent epidemiological evidence collected from over
37 observational studies proposes that male circumcision
reduces the relative risk of acquiring HIV-1 by 1.8– 8.
2-fold [9– 12]. The protective effects of circumcision still
remain even when potentially confounding social practices
From the
a
Department of Zoology, The University of Melbourne, Victoria, Australia, and the
b
Department of Obstetrics and
Gynaecology, The University of Melbourne, Victoria, Australia.
Correspondence to S. McCoombe, Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern
University, Chicago, Illinois, 60611, USA.
Tel: +1 312 503 1142; fax: +1 312 503 0222; e-mail: s-mccoombe@northwestern.edu
Received: 3 November 2005; accepted: 1 January 2006.
ISSN 0269-9370 Q2006 Lippincott Williams & Wilkins 1491

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
such as religion, number of sexual partners and condom
usage are taken into consideration [13]. Perhaps the most
compelling evidence for the protective benefit of male
circumcision was obtained from a recently completed
prospective randomized control trial in South Africa.
Following over 3000 participants for 21 months, Auvert
and colleagues observed a 65% protective benefit amongst
the men they circumcised compared to the uncircumcised
control group [14]. Thus, it seems likely that the foreskin
plays a major role in HIV-1 transmission in uncircumcised
men.
Potential HIV-1 target cells in the mucosa include
Langerhans’ cells, subepithelial dendritic cells, macro-
phages and CD4 T cells [5,15]. The role of Langerhans’
cells in the mucosa is to sample foreign antigens and
migrate to regional lymph nodes where they present the
processed antigens to naive T cells [16,17]. Langerhans’
cells are the most superficial of all HIV-1 susceptible cells
in the absence of disease or trauma, and have also been
shown to express the c-type lectin langerin, which may
play a complimentary role in HIV-1 dissemination to
regional lymph nodes [18]. It is probable that Langerhans’
cells whose dendritic processes are closest to the epithelial
surface will be first to come in contact with HIV-1 in
mucosal secretions from the man’s infected partner. This
primary infection is most likely to occur when there is
little or no overlying protective layer of keratin.
Other cells with HIV-1 receptors including T cells,
dendritic cells and macrophages are all present in the inner
and outer foreskin [4,5], but are commonly found deeper
within the submucosa. Therefore, HIV-1 is less likely to
encounter these cell types in the healthy male genital
mucosa. If however there is a loss of epithelial integrity by
trauma during intercourse, ulcerative sexually transmitted
infection (STI) or inflammation then these cells are much
more likely to encounter infectious HIV-1 virions. It is
well recognized that some STI show an epidemiological
synergy with HIV-1 and may result in a significant
increase in susceptibility to HIV-1 infection [19]. This is
especially true of infections that cause epithelial lesions
such as syphilis, chancroid and genital herpes [19,20],
which expose the underlying target cells deeper within
the epidermis and dermis.
The purpose of this study was to locate HIV-1 susceptible
cells in all the epithelia of the human penis and to quantify
the thickness of the overlying layer of keratin in order to
evaluate potential sites of HIV-1 entry into the penis.
Methods
Tissue
The formalin fixed penises of nine uncircumcised cadavers
of mean age 77.4 years were obtained from the Depart-
ment of Anatomy, The University of Melbourne. Fresh
foreskins were obtained from 21 healthy, consenting men
of mean age 28.9 years following elective male circumci-
sions performed at either the Freemasons Hospital or the
Mercy Hospital in Melbourne, Australia. Penile necropsy
specimens were obtained within 18 h of death from eight
men of mean age 30.9 years from the Victorian Institute of
Forensic Medicine with next-of-kin consent. All samples
were obtained from HIV-seronegative individuals. The
study was approved by all relevant institutional ethics
committees.
Keratin staining
Cross-sections 1-cm thick were taken from the midpoint
of each glans penis from the nine cadavers, and embedded
in paraffin prior to sectioning and staining. These were
then sectioned at 8 mm and stained for keratin with both
the hematoxylin– eosin and the Ayoub–Shklar methods.
Sections were examined under light microscopy at 200 –
400 magnification.
Immunocompetent cell staining
Immediately following circumcision or autopsy, fresh
tissue was immersed in sterile saline and transported on ice
to the laboratory. Each foreskin was separated using blunt
dissection into inner and outer aspects. The frenulum,
urethra, urethral meatus and glans penis were dissected out
from the autopsy specimens. All tissues were then snap
frozen in Jung tissue freezing medium (Leica Microsys-
tems, Wetzlar, Germany). Frozen blocks were sectionedby
cryostat and stained for immunocompetent cells using
monoclonal antibodies targeting CD1a, CD4, HLA-DR,
DC-SIGN, CXCR4 and CCR5. Sections were stained
specifically for Langerhans’ cells using anti-CD1a hybri-
doma supernatant (OKT6, American Type Culture
Collection, Manassas, Virginia, USA). Other potential
HIV-1 target cells were specifically stained using anti-CD4
and anti HLA-DR hybridoma supernatants (American
Type Culture Collection), and anti-DC-SIGN, anti-
CCR5 and anti-CXCR4 (BD Bioscience, San Jose,
California, USA). Sheep anti-mouse immunoglobulin
conjugated with fluorescein isothiocyanate (Silenus Labs
Pty Ltd, Boronia, Australia) was diluted 1 : 100 and used to
label all positively stained cells. Nuclear counterstaining
with 0.25 mg/ml propridium iodide (Sigma Chemical
Co., St. Louis, Missouri, USA) enabled cell position within
the epithelium to be examined. Digital images were
obtained with a Zeiss Axioplan 2 confocal microscope
(Carl Zeiss Inc., Thor nwood, New York, USA) attachedto
am-Radiance confocal scanning system (Bio-Rad
Laboratories, Hercules, California, USA). Images were
collected at 100– 200 magnification for density analysis
and under oil immersion at 630–1000 for individual
morphological analysis. Lasersharp 2000 software (Bio-
Rad Laboratories) was used for all measurements.
Statistical analyses
Post-mortem autolysis prior to perfusion fixation of the
cadavers made it difficult to obtain objective quantitative
1492 AIDS 2006, Vol 20 No 11

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measurements of keratin thickness. Therefore, three
experienced microscopists subjectively estimated keratin
thickness in randomized sections. Three microscopic
fields of each glans penis, inner foreskin, and outer
foreskin were randomly selected from each of the nine
penis samples. Keratin thickness was subjectively assessed
on a scale of 0–5 arbitrary units, where 0 corresponded to
no keratin, and 5 to maximum keratinization (keratin
thickness 20 mm). Two-tailed, two-sample unequal
variance t tests were used to analyse the data.
Cellular densities were calculated for all samples by
averaging the number of positively stained cells within
three 500-mm
2
fields from the epithelial surface to the
dermis. Values were converted into cells per square
millimeter (cells/mm
2
). Individual cellular analysis was
performed on 20 randomly chosen cells from all fresh-
frozen sections. A cell was included only if the nucleus
was visible. Three-dimensional digital reconstructions of
individual Langerhans’ cells showed the number of
dendritic processes originating from each cell, and how
close to the epithelial surface these processes extended.
Two-tailed, paired t tests were used to determine
differences between Langerhans’ cell populations in the
various penile epithelia tested.
Results
Keratin measurements
In the cadaveric samples, the inner foreskin (mean
thickness, 1.8 units; SE, 0.1) was significantly less
keratinized than the outer foreskin (mean thickness,
3.3 units; SE, 0.1) or glanspenis (mean thickness, 3.3 units;
SE, 0.2; P<0.05). There was no difference in kera-
tinization between the outer aspect of the foreskin and the
glans penis. These results were confirmed by studying the
distribution of keratin in all penile epithelia collected at
autopsy. The frenulum and the urethral meatus were
poorly keratinized, and there was no keratin observed in
the penile urethra.
Immunocompetent cell localization
CD1a positive Langerhans’ cells were clearly visible in the
outer and inner foreskin, the glans penis, urethral meatus
and frenulum, although none were observed in the penile
urethra (Fig. 1). The majority were observed in the
superficial layers of the epidermis. Most Langerhans’ cells
had dendritic projections extending up between the
keratinocytes towards the epithelial surface (Fig. 2).
CD4 positive cells including T cells, macrophages and
dendritic cells were found in all epithelia but were
generally located within the dermis. CCR5 and CXCR4
were expressed in a minority of superficial Langerhans’
cells but were found on a greater proportion of cells
deeper in the dermis. DC-SIGN was expressed in low
levels in dermal dendritic cells and was localized
predominantly near the basal lamina.
Cell density and distribution
The highest density of Langerhans’ cells was in the
outer foreskin (85.5 cells/mm
2
; SE, 4.1) followed in
descending order by the inner foreskin (61.3 cells/mm
2
;
SE, 5.0), frenulum (56 cells/mm
2
;SE,8.3),glanspenis
Potential HIV target cells in the penis McCoombe and Short 1493
Fig. 1. Distribution of Langerhans’ cells (green) in the outer
foreskin, well beneath the keratinised epithelium (200 T
magnification).
Fig. 2. A single Langerhans’ cell (green) in the outer foreskin
with dendritic processes extending towards the epithelial
surface (630 Tmagnification).

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
(41 cells/mm
2
; SE, 10.0), and urethral meatus (14 cells/
mm
2
; SE 6.4). None were observed in the penile urethra.
Densities in each epithelium for CD4, CXCR4, CCR5
and DC-SIGN are shown in Table 1.
The depth of each HIV susceptible cell type from the
epithelium was measured (Table 2). Measurements were
taken from the cell surface or dendritic process nearest the
epithelial surface. Langerhans’ cells (61 mm) were
significantly more superficial across all epithelia than
CD4 (102 mm), CCR5 (94 mm), CXCR4 (92 mm),
DC-SIGN (77 mm) or HLA-DR (128 mm) expressing
cell types P<0.0001. Dendritic processes from Langer-
hans’ cells were particularly superficial on the inner aspect
of the foreskin (23 mm) and frenulum (34 mm). In the
inner foreskin, dendritic processes came within 4.8 mmof
the epithelial surface, whereas in the outer foreskin they
rarely came within 20 mm of the epithelial surface due to
the thicker layer of keratin.
Discussion
This study has shown that both the inner aspect of the
foreskin and the frenulum are poorly keratinized, and are
richly supplied with HIV-1 susceptible cells. Of the cell
types tested, Langerhans’ cells are the most likely to be
encounteredas theyare most superficial and have dendritic
processes sampling a large epithelial surface area. Langer-
hans’ cells of the inner foreskin and frenulum are protected
by a much thinner layer of keratin than those in the glans
penis or outer foreskin. The urethral meatus and penile
urethra, although poorly protected by keratin, contained
very few Langerhans’ cells. The highest density of Lang-
erhans’ cells was in the outer foreskin, but these were
covered by a thicker protective layer of keratin. The
dendritic processes of the Langerhans’ cells in the inner
foreskin were significantly more superficial due to decrea-
sed epithelial keratinization. The presence of c-type lectins
in the male genital mucosa was also observed and may play
an important role in the binding, internalization and
subsequent transport of HIV-1 to regional lymph nodes.
DC-SIGN was confined to dendritic cells near the basal
lamina. These findings provide a possible anatomical
explanation for the protective effect of male circumcision
against HIV-1 infection.
The position of T cells, macrophages and dendritic cells
other than Langerhans’ cells suggested that in healthy
individuals they are less likely to be involved in HIV-1
sexual transmission as they are predominantly dermal.
Many of these cells contain the specific receptors required
for HIV-1 infection, and are likely to become involved in
viral entry if the integrity of the overlying epithelium is
disrupted by STI or trauma. The presence of these cells in
the dermis may therefore help to explain the increased
susceptibility to HIV-1 infection in men with ulcerative
STI [19].
During penile erection, the turgid glans penis is well
protected by its thick overlying layer of keratin. However,
the delicate, vascular inner aspect of the foreskin is
stretched halfway down the penile shaft (Fig. 3), further
attenuating its thin protective layer of keratin that is
1494 AIDS 2006, Vol 20 No 11
Table 1. Mean density of HIV-1 susceptible cell types in penile
epithelia (cells/mm
2
).
CD1a CD4 CCR5 CXCR4 HLA-DR DC-SIGN
Outer foreskin 85 126 33 12 103 17
Inner foreskin 61 108 28 2 116 11
Glans 56 104 23 20 137 18
Frenulum 41 57 16 19 89 12
Urethra 0 22 0 1 21 0
Urethral meatus 14 47 7 11 43 2
Table 2. Mean depth of HIV-1 susceptible cell types in penile
epithelia (mm).
CD1a CD4 CCR5 CXCR4 HLA-DR DC-SIGN
Outer foreskin 43 121 103 179 176 106
Inner foreskin 19 78 71 67 94 48
Glans 67 144 89 122 147 136
Frenulum 31 108 82 100 102 108
Urethra – 77 – 89 73 –
Urethral meatus 40 49 102 91 129 80
Outer foreskin
Inner foreskin
Urethral meatus
Frenulum
Glans penis
HIV entry No HIV entry
Outer foreskin Inner foreskin Frenulum
(b)
(a)
Fig. 3. (a) Flaccid uncircumcised penis. (b) Erect uncircum-
cised penis with the foreskin retracted showing likely sites of
HIV-1 entry.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
directly exposed to vaginal secretions. The highly
keratinized outer foreskin is reflected down to the base
of the penis and much of it may not even come in contact
with the vaginal epithelium. In circumcised men where
the foreskin has been removed and the frenulum has
atrophied, the whole of the penile shaft is covered with a
thickly keratinized epithelium. Following intercourse,
the preputial cavity may also provide an environment
conducive to increased viral survival and thus increase
transmission in uncircumcised men. Male circumcision
has been shown to confer protection against genital
herpes, syphilis, candidiasis, gonorrhea and genital ulcer
disease [20,21]. Male circumcision also provides signifi-
cant protection to men, and their female partners against
human papilloma virus infection, and the resultant penile
and cervical carcinoma [22]. With the South African
randomized controlled trial showing a 61% protective
benefit of male circumcision against HIV infection [14],
the procedure must now be seriously considered as an
adjunct to existing prevention strategies. To be most
beneficial, the procedure must be conducted in a safe and
sterile manner and involve thorough education of the
participants to minimize the effects of disinhibition.
Acknowledgements
We thank Mr. David Webb MB, MS, DRCOG, FRACS,
Associate Professor Laurie Cleeve MBBS, FRACS and
The Victorian Institute of Forensic Medicine for providing
tissue, Dr. Paul Cameron for advice, Amanda Handley
for laboratory assistance, Bruce Abaloz for histological
processing, Professor Tom Hope for reviewing the
manuscript and David Paul for assistance with figures.
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