| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
¶
From the Departments of Pathology*
and
Medicine,
, Salt Lake Veteran’s
Administration, Salt Lake City, Utah; the Departments of
Pathology
and Medicine,¶
University of Utah School of Medicine, Salt Lake City, Utah; the
Division of Gastroenterology,
St.
Luke’s/Roosevelt Hospital and Columbia University, New York, New York;
and the Faculté des Sciences
St-Jérôme,||
Institut Mediterranéen de
Recherche en Nutrition, Marseilles, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The major HIV co-receptors CCR5 and CXCR4 are both present in enteric epithelium as well as HT-29 cells, but in vivo they are present mainly at and near the luminal surface.12 However, most productively HIV-infected cells in intestinal mucosa are superficial lamina propria macrophages.13 A co-receptor present on the basolateral surface of the enteric epithelium would be a more plausible mediator of this effect, but, to our knowledge, none have previously been found.
The orphan G-protein coupled receptor GPR15/Bob (hereafter termed
"Bob") is a previously little studied co-receptor for HIV and
simian immunodeficiency virus.14,15
Although Bob is a
frequently used simian immunodeficiency virus co-receptor (ie,
promoting viral fusion and infection), HIV-1 co-receptor study results
have varied with the sensitivity of the assay used, generally showing
either very rare or no Bob usage or common but extremely inefficient
Bob usage of uncertain clinical significance.16-19
Although gp120 is the HIV-1 envelope surface protein, most of it is
shed from infected cells rather than incorporated into
virions.20
It is thus a plausible mediator of toxic
effects in uninfected cells. The principal HIV co-receptors (CXCR4 and
CCR5) mediate gp120-induced calcium signaling, but previous studies
demonstrated calcium signaling using these receptors only at relatively
high gp120 concentrations (
200 pmol/L).21-23
Almost all
previous studies, however, focused on the main co-receptors; the
possibility that inefficient minor co-receptors could mediate
significant HIV-induced activation has, to our knowledge, not
previously been explored. Although the gp120 content probably varies
widely in different tissue compartments, blood gp120 content in
HIV-infected patients is very low (0.075 to 0.80 pmol/L), and is mostly
in immune complexes.24
The goal of this study was to find
a HIV co-receptor meeting the following criteria for a putative cause
of HIV enteropathy: 1) the receptor is expressed on the basal surface
of small intestinal epithelium, where the main exposure to HIV
proteins would likely be; 2) it mediates the gp120-induced effects seen
in HT-29 cells and either interacts with galactosyl ceramide and/or
cross-reacts with anti-galactosyl ceramide antibodies; and 3) it
induces calcium signaling at the low gp120 concentrations anticipated
in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following reagents were obtained through the AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, National Institutes
of Health: gp120IIIB and
gp120MN from DAIDS, NAIAD, produced by
ImmunoDiagnostics; gp120CM235 from Protein
Sciences Corporation; gp12093TH975 from Steve
Showalter, Maria Garcia-Moll, and the Division of AIDS (DAIDS),
National Institute of Allergy and Infectious Diseases (NIAAD);
gp120SF2 from Ms. Margarita Quiroga; anti-CXCR4
and anti-CCR5 antibodies from DAIDS, NIAID, produced by R&D Systems,
Minneapolis, MN; Ghost (3) cells and pBABE-Bob plasmid from Dr. Vineet
KewalRamani and Dr. Dan Littman; RANTES, and MIP-1ß from DAIDS,
NIAAD, produced by Piprotech Inc.; MIP-1
from R&D Systems;
anti-CXCR4 from Dr. James Hoxie; and anti-CCR5 from DAIDS, NIAID,
produced by PharMingen, La Jolla, CA.
Primary Antibodies
Two sets of three peptide antigens from the human GPR15/Bob sequence were synthesized, namely HAEDFARRRKRSVSL, DKEASLGLWRTGSFLCK, and MDPEETSVYLDYYYATS; and SGLRQEHYLPSAILQ, RELTLIDDKPYCAEKKAT, and KNYDFGSSTETSDSHLTK. Rabbits were injected with either the first three or second three peptides, and immune sera were affinity purified with the corresponding antigen peptides. These antibodies were named anti-Bob37 and anti-Bob39, respectively. This work was performed at Research Genetics, Huntsville, AL. Mouse monoclonal anti-acetylated tubulin (5 µg/ml; Sigma Chemical Co., St. Louis, MO), monoclonal anti-CXCR4 and anti-CCR5 (5 µg/ml; AIDS Research and Reference Reagent Program), polyclonal anti-galactosyl ceramide (Sigma), and monoclonal anti-galactosyl ceramide (Chemicon, Temecula, CA) were also used.
Western Blotting
Western blots were made from tissue or cultured cells; the soluble fractions of homogenates were prepared in Triton X-100-containing lysis buffer25 at 4°C. Electrophoresis in 10% polyacrylamide gels was done with 50 µg protein per lane, and the protein was electroblotted onto nitrocellulose. The blots were blocked with 20 mmol/L Tris (pH 7.6), 140 mmol/L NaCl, 0.1% Tween 20, and 5 g/100 ml nonfat dry milk for 1 hour, then treated with 0.5 µg/ml of primary antibody in the same buffer for 1 hour. Blots were visualized either by chemiluminescence using ECL reagents (Amersham/Pharmacia, Arlington Heights, IL) or by alkaline phosphatase-conjugated secondary antibodies with NBT/BCIP staining. Negative controls included omitting the primary antibody and overnight preabsorption of the primary antibody with 500-fold excess of the three appropriate peptides.
The HT-29 lysates were immunoprecipitated with 2 µg/ml of Bob37 or polyclonal anti-galactosyl ceramide (1:100 dilution), then incubated with protein A/G Sepharose (1.5% v/v) for 2 hours (both steps on a rocker at 4°C). The Sepharose beads were washed, then boiled and used in Western blotting. These Western blots were stained with anti-Bob37, anti-Bob39, or monoclonal anti-galactosyl ceramide antibodies at 0.5 µg/ml.
RNA in Situ Hybridization
RNA in situ hybridization was done with a digoxygenin-labeled cRNA antisense riboprobe prepared as follows: polymerase chain reaction amplification was made of the pBABE-Bob plasmid (courtesy of Dr. Dan Littman and the AIDS Research and Reference Reagent Program) using primers described by Deng and colleagues14 to which a T7 RNA polymerase site had been added at the 5' end of the downstream primer. This yielded a product of expected size (562 bp) and sequence. This was used with T7 polymerase in the presence of digoxygenin-labeled UTP (Dig-RNA labeling kit; Boehringer Mannheim/Roche, Indianapolis, IN) to make the antisense probe. Probe specificity was confirmed by Southern blot analysis of pBABE-BOB and comparing our Northern blot analysis of a human MTCII panel (Clontech, Palo Alto, CA), with results similar to those of Deng and colleagues.14
In situ hybridization was performed as previously described,26 except that formalin-fixed, paraffin-embedded colon tissue was used, and protease digestion was performed with pepsin (2 mg/ml in 0.1 N HCl for 1 hour at room temperature). After inhibition of endogenous alkaline phosphatase by 20% acetic acid for 15 seconds at 4°C, immunodetection was performed with alkaline phosphatase-conjugated anti-digoxygenin and overnight NBT/BCIP staining. Slides treated with RNase before in situ hybridization, but otherwise treated identically, were used as negative controls.
Immunostaining
Except for the microtubule staining mentioned below and the CXCR4 and CCR5 staining (for which air-dried, unfixed sections were used), all indirect immunofluorescent staining was done on acetone-fixed frozen tissue sections. Cultured cells for microtubule staining were fixed in paraformaldehyde phosphate-buffered saline (PBS) as previously described.7 The Bob37 and Bob39 antibodies were preincubated with 100-fold excess of the peptides DKEAS... or KNYDF... , respectively, which was found to increase staining specificity. These were incubated in 10% normal goat serum, then the primary antibody at 5 µg/ml for 1 hour, then washed and incubated with Cy-3-conjugated goat anti-rabbit or anti-mouse IgG (1:100 dilution; Jackson ImmunoResearch, West Grove, PA) as appropriate, all at room temperature. Similar preparations omitting the primary antibody were made as negative controls.
Cell Culture Studies—Calcium Measurements and Microtubules
HT-29 cells (from ATCC, Rockville, MD) were grown in Dulbecco’s modified Eagle’s medium (DMEM)-F12 with 10% bovine calf serum, and were used 24 to 48 hours after plating. The Ghost (3) cells were obtained from the AIDS Research and Reference Reagent Program, and were grown in DMEM with 10% bovine calf serum supplemented with 500 µg/ml G418, 100 µg/ml hygromycin, and 1 µg/ml puromycin.27 These cells were used 24 to 96 hours after plating.
Calcium studies were done on HT-29 cells and Ghost (3) cells loaded
with Fluo 4. The cells were placed in Locke’s buffer containing 5
µg/ml Fluo 4 a.m. ester (Molecular Probes) for 20 to 30 minutes,
then in Locke’s buffer for an additional 30 minutes before testing.
Various concentrations of gp120IIIB (X4 trophic),
gp120CM235 (R5 trophic),
gp120MN (X4 trophic),
gp12093TH975 (nonsyncytium-inducing primary
isolate from an asymptomatic subject, trophism undetermined but likely
R5), and gp120SF2 (X4R5 trophic), ranging from 10
nmol/L to less than 1 pmol/L, were added in Locke’s
buffer.6
The gp120 proteins were obtained from the AIDS
Research Reagent Referral Program. Some HT-29 cells were given one of
the following pretreatments: 50 µg/ml anti-Bob37 overnight, 1 µg/ml
pertussis toxin overnight, or 4.6 µg/ml U73122 for 1 hour. With the
HT-29 cells only, similar tests were also performed with SDF-1,
MIP-1, MIP-1ß, and RANTES rather than gp120. The cells were
visualized every 4 seconds in a confocal microscope, using 488-nm argon
laser excitation and detection filters appropriate for fluorescein-like
dyes.
To examine the microtubules, HT-29 cells were treated with 10 nmol/L of gp120CM235 or 50 pmol/L of gp120IIIB, then fixed 1 hour later in 4 g/100 ml paraformaldehyde PBS, then stained with mouse anti-acetylated tubulin (5 µg/ml, Sigma) by a method similar to that given above. Some cells were pretreated with 50 µg/ml of anti-Bob37 overnight, 1 µg/ml of pertussis toxin overnight, or 4.6 µg/ml of U73122 for 1 hour before the gp120 was added. Untreated cells were also stained.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In Western blots, homogenates of small bowel and colonic mucosa,
lymph node, prostate, testis, and liver, both antibodies stained bands
at 36 kd (Figure 1A)
, slightly less than
the 40.8-kd molecular weight of Bob estimated from the mRNA sequence.
No similar bands were observed in homogenates of brain, placenta, lung,
uterus, heart, pancreas, or skeletal muscle. The 36-kd band was absent
in Western blots omitting the anti-Bob primary antibody, and the bands
were greatly reduced if the anti-Bob was pretreated with excess of the
appropriate peptides (not shown).
|
. This suggests that both
Bob antibodies, formed to different epitopes, recognize the same
protein and that both polyclonal and monoclonal anti-galactosyl
ceramide antibodies also bind this protein. RNA in Situ Hybridization and Immunostaining
RNA in situ hybridization using a Bob antisense
riboprobe showed granular cytoplasmic staining of both surface and
crypt epithelial cells and lamina propria mononuclear cells in colonic
mucosa (Figure 2A)
. Indirect
immunofluorescent staining with anti-Bob antibodies showed extensive
staining of intestinal epithelium and lamina propria mononuclear cells.
In essentially all small intestinal epithelial cells and in a few
colonic crypt cells, there was granular membranous staining, mostly of
the basal membrane and just under the brush border of the luminal
surface (Figure 2B)
. The staining was similar throughout the villi and
crypts. In colonic epithelium, Bob staining was mostly granular and
cytoplasmic (Figure 2C)
, although a few crypt cells had membranous
staining resembling that of small bowel.
|
. Both the small bowel and the colonic epithelium
had weak staining for CXCR4 and CCR5, which was mostly at or near
the luminal surface. Small bowel epithelial cell staining was very weak
for both CXCR4 and CCR5. Calcium Flux Studies
Gp120IIIB and
gp120CM235 induced an identical, immediate
(within 4 seconds) increase in free calcium in HT-29 cells (Figure 3A)
. The signals lasted from 
12
seconds to several minutes, and varied from cell to cell. The signals
were generally longer when more gp120 was added. Serial dilution
studies showed that the minimal concentration causing calcium signaling
was 1 pmol/L for gp120IIIB and 2.5 nmol/L for
gp120CM235. Gp120MN,
gp120SF2, and gp12093TH975
induced no calcium signaling, even at 10 nmol/L concentrations.
|
, or MIP-1ß, and 10 ng/ml RANTES, induced similar
calcium signaling. Calcium signaling induced by either gp120 or SDF-1
was inhibited by the selective G protein inhibitor pertussis toxin and
by the phospholipase inhibitor U73122 (Figure 3, B and C)
. Pretreatment
with anti-Bob37 antibodies inhibited the calcium signaling by both
gp120CM235 and gp120IIIB
(Figure 3D)
. In cells pretreated with anti-Bob, calcium signaling could
then be induced by 100 ng/ml of SDF-1, implying that the CXCR4
receptors and the G protein-induced calcium-signaling pathway remained
functional (Figure 3E)
. Although HIVIIIB is an X4
or T lymphocyte-trophic virus, gp120IIIB-induced
calcium signaling was not inhibited by anti-CXCR4 neutralizing
antibodies. Comparable results, demonstrating that the gp120-induced
signaling by the gp120s of two different viral strains were inhibited
by anti-Bob37 but not by anti-CXCR4 antibodies, were found
independently in a second laboratory (that of JF), using confluent,
polarized monolayers of the D4 clone of HT-29 (results not shown).
Furthermore, the polarized cells could be stimulated by
gp120IIIB from either the basal or top surface,
and anti-Bob37 neutralized activation from either side (results not
shown).
To confirm that Bob is the receptor mediating gp120-induced calcium
signals, gp120-induced calcium signals were also examined in Ghost (3)
cells transfected to express Bob, CXCR4, or CCR5, as well as the
parental cell line.27
Calcium signaling was induced by as
little as 0.015 pmol/L of gp120IIIB (Figure 3F)
or 8 pmol/L of gp120CM235 in Bob-transfected
Ghost (3) cells, whereas Ghost (3) CXCR4-transfected cells required at
least 150 pmol/L gp120IIIB and did not respond to
gp120CM235 at any concentration tested. Both
Ghost (3) CCR5-transfected cells and the parental cell line
(which expresses CXCR4 at low levels) required 1 nmol/L of
gp120IIIB and did not respond to
gp120CM235 at any concentration tested. Gp120
from each of the three viral strains (MN, SF2, and 93TH975) that did
not cause calcium signals in HT-29 cells similarly did not cause
calcium signals in the Ghost (3) Bob-transfected cells at any
concentration tested.
The calcium signals in the Ghost (3) cells were usually very intense
and often lasted for several minutes. At the minimal concentrations,
only some of the cells exhibited calcium signals (Figure 3F)
, but at
higher concentrations most cells exhibited signaling. However, at very
high gp120 concentrations, calcium signaling became weak or negative.
Microtubule Staining
Corresponding experiments were done to demonstrate gp120-induced
microtubule alterations in HT-29 cells. Either
gp120IIIB or gp120CM235
caused a marked loss of acetylated tubulin-staining microtubules
(Figure 4, A and B)
. This effect was most
apparent in clusters of several cells, large cell clusters (>20 cells)
often retained stainable microtubules. The loss of microtubule staining
was inhibited by pretreatment with anti-Bob (Figure 4C)
, pertussis
toxin, or U73122 under the same conditions used for calcium signaling,
except that the cells were in DMEM-F12 rather than Locke’s buffer. The
loss of microtubules induced by gp120IIIB, and
that this effect could be inhibited by anti-Bob, was confirmed with
differentiated, polarized HT-29 cells in a second laboratory (that of
JF, results not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Both in situ hybridization and immunostaining with two different antibodies demonstrate that Bob is present in intestinal epithelium and in lymphocytes. Also, Bob is abundantly expressed at the basal and apical surfaces of small bowel epithelium. In agreement with previous results12 and in contrast to Bob, CCR5 and CXCR4, although present in intestinal epithelium, are mostly at the luminal surface. In chronic HIV infection, most productively HIV-infected cells in intestinal mucosa are superficial lamina propria macrophages;13 intestinal epithelium is probably exposed to gp120 on the basal surface. Thus Bob, unlike CCR5 or CXCR4, is present at the correct site to mediate gp120-induced signaling.
The inhibition of the gp120-induced calcium fluxes and microtubule loss in HT-29 cells by pertussis toxin and U73122, as well as induction of similar calcium signals by the natural ligands of CCR5 and CXCR4, strongly implicate a G protein-coupled receptor/pertussis toxin-sensitive G protein/phospholipase mechanism. We have shown, for both of our strains of gp120 that induce these changes, that they are inhibited by affinity-purified anti-Bob antibodies. Also, picomolar or lower concentrations of gp120 of either strain induced calcium fluxes in Bob-transfected Ghost (3) cells but not in the parental cell line or in CCR5 or CXCR4-transfected Ghost (3) cells. All three gp120s of strains not inducing signaling in HT-29 cells also do not induce calcium fluxes in Bob-transfected Ghost (3) cells. Thus we believe that Bob mediates these gp120-induced effects in HT-29 cells, which are very similar to those of HIV enteropathy.7
Together, these findings suggest that Bob is present at the right site, that it induces physiological changes in HT-29 cells resembling HIV enteropathy, it cross-reacts with anti-galactosyl ceramide antibodies, and that, unlike the principal co-receptors CCR5 and CXCR4, it induces calcium signaling at extremely low gp120 concentrations. Recently, we also showed a decrease in acetylated tubulin staining in the intestinal epithelium of HIV-infected patients, suggesting decreased microtubule stability in vivo.8 Together, these findings strongly implicate gp120-induced Bob activation as a likely cause of the calcium signaling and microtubule loss resulting in HIV-associated enteropathy. However, further work is needed, particularly to correlate the presence of a HIV strain inducing Bob activation with HIV enteropathy in vivo.
We suspect that gp120 causes HIV enteropathy by the following mechanism. Gp120 activates intestinal epithelial cell Bob, inducing G protein and phospholipase activation, and thus inositol triphosphate and calcium signaling. Increased cytosolic calcium depolymerizes microtubules.29 Microtubule loss alters RhoA and Rac1 activation, increasing actin-myosin contraction30 near the tight junctions, causing increased paracellular permeability and diarrhea.31 Microtubule loss also reduces enterocyte lipid transport,9 causing lipid malabsorption.
Previous studies of co-receptor activation by gp120 focused almost exclusively on the major co-receptors CCR5 and CXCR4, with the implicit assumption that the activation was an incidental phenomenon with receptors for which viral fusion was the main goal. Finding extremely sensitive gp120-induced activation of Bob, a very inefficient co-receptor, by both an X4 trophic strain (HIVIIIB) and an R5 trophic strain (HIVCM235) indicates that different receptors can be activated than those used as main co-receptors for viral fusion. However, like co-receptor activity, activation with calcium signaling seems to be a viral strain-specific phenomenon. Correspondingly, Baik and colleagues32 showed that efficient co-receptor activity/fusion can occur without avid gp120 to co-receptor binding. Activation and viral fusion are distinct phenomena; the search for receptors activated by HIV should not be limited to the major co-receptors and might even include those with no co-receptor activity at all.
The extraordinary potency of gp120IIIB for Bob activation seems unlikely to be random and presumably was selected for. We speculate that this activation could give an HIV strain some selective advantage, although it seems implausible that it is because of the enteropathy. Possibly, gp120-induced Bob activation induces similar calcium signaling in other Bob-expressing cells such as lymphocytes, explaining the long known gp120-induced alterations in lymphocyte inositol phosphate metabolism.33 Potentially, Bob activation could inhibit the anti-HIV immune response or induce the gp120IIIB-induced, actin-mediated co-localization of CD4 and the major co-receptors advantageous for HIV infection.34 Further work is needed to explore this and the significance of Bob expression in other cell types.
In summary, both the effects of co-receptor ligands and inhibition studies suggest that HIV co-receptor activation causes calcium signaling and microtubule loss in HT-29 cells. These calcium and microtubule changes were previously shown to induce HIV enteropathy-like malabsorption and increased paracellular permeability. Unlike the major HIV co-receptors, the inefficient, little studied co-receptor Bob is abundant at the basal surface of small intestinal epithelium. Both neutralizing antibody studies and co-receptor-transfected cell studies demonstrate that Bob mediates the gp120-induced calcium signaling and microtubule loss. HIV gp120-induced Bob activation is a very plausible cause of HIV enteropathy. Further study is needed to explore the possibility that gp120-induced activation of Bob or similar receptors could hasten immunodeficiency or induce other, as yet poorly understood side effects of HIV infection.
|
Footnotes |
|---|
Supported by the Veteran’s Administration Merit Review, Associated Regional and University Pathologists, and Western Institute for Biomedical Research (to F. C.), the National Institute of Diabetes and Digestive and Kidney Disease (grant K08-DK-02531 to S. K. K.), and the National Institutes of Health (grant AI211414 to D. P. K.).
Accepted for publication August 10, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  K. Balabanian, J. Harriague, C. Decrion, B. Lagane, S. Shorte, F. Baleux, J.-L. Virelizier, F. Arenzana-Seisdedos, and L. A. Chakrabarti CXCR4-Tropic HIV-1 Envelope Glycoprotein Functions as a Viral Chemokine in Unstimulated Primary CD4+ T Lymphocytes J. Immunol., December 15, 2004; 173(12): 7150 - 7160. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
