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*
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, and
Howard Hughes Medical Institute, New York, NY 10016; and
Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Members of the chemokine receptor family also serve as coreceptors, in conjunction with the CD4 molecule, for entry of HIV and SIV into target cells (11, 12, 13, 14, 15, 16). CCR5 is the major coreceptor for R5 strains (previously referred as M-tropic) of HIV-1 and most SIV strains, while CXCR4 allows entry of X4 strains (previously referred as T-tropic) of HIV-1 (11, 12, 13, 14, 15, 16). Additionally, CCR2, CCR3, CCR8 and CX3CR1 have been reported to be used by some of the HIV/SIV isolates, albeit at lower efficiencies (17, 18, 19). Several chemokine-receptor-like orphan receptors have also been shown to function as coreceptors for HIV and SIV strains (20, 21, 22, 23). One of these seven-transmembrane domain orphan receptors, Bonzo (also named STRL33 or TYMSTR), was identified as a principal coreceptor for several strains of SIV (including SIVagm and SIVsm family viruses) as well as some HIV-2 and HIV-1 strains (20, 21, 22, 23). Bonzo is a putative chemokine receptor based on sequence homology with other chemokine receptor family members. However, its biologic function is not understood, and no natural ligand has yet been identified among the known human chemokines (21) (T. Schall, unpublished observation). Expression of Bonzo mRNA is restricted to lymphoid tissues, PBMC, and placenta (20, 21, 22, 23). Interestingly, the Bonzo gene maps to human chromosome 3 close to the region where genes encoding CC family chemokine receptors are clustered (21). Little is known about the cell subset-specific expression and regulation of Bonzo. The in vivo distribution of Bonzo-expressing cells and the modulation of this expression may be important in determining the role of Bonzo in SIV and HIV pathogenesis. To address this issue we have used a mAb specific for Bonzo to demonstrate that expression of this receptor is highly restricted to the memory subset of resting human T cells. We further show that, similar to CCR5, Bonzo expression is up-regulated by the cytokines IL-2 and IL-15. However, in contrast to CCR5, upon PMA treatment or TCR stimulation Bonzo was not down-modulated. A similar expression pattern was observed on mouse CD4+ T cells of mice in which a green fluorescent protein (GFP)4 gene was knocked into the endogenous Bonzo locus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An mAb against Bonzo/STRL33 was raised at R&D
Systems (Minneapolis, MN) by immunizing BALB/c mice with a syngeneic
mouse myeloma (NSO) transfected with full-length human Bonzo/STRL-33
with a polyhistidine fused to the C-terminal end of the receptor
sequence. The polyhis serves as an epitope tag that can be
used to identify (via intracellular staining in FACS or via Western
blot) transfected clones that appear to be expressing high levels of
the gene of interest. An immunization protocol (24) for
soluble protein was adapted for use with whole cells as the immunogen.
The priming immunization was performed by mixing the cell suspension in
PBS with an equal volume of emulsified MPL/TDM adjuvant (Ribi);
subsequent boosts used cells in PBS alone. Following immunization,
lymph node cells were used for polyethylene glycol-mediated fusion
following conventional protocols. After 7 days of culture, supernatants
were screened for Abs that could bind to paraformaldehyde-fixed
NSO/STRL-33/polyHis cells used for immunization. Cultures that were
positive in this primary screen were then tested for binding to NSO
cells that had been transfected with an irrelevant gene (GDF-9) also
expressed as a polyhis construct. One clone was chosen and subcloned
based on strong binding of its supernatant to the unfixed relevant
transfectants. This hybridoma secretes an IgG2b 
mAb that was
purified and used in subsequent experiments. This Ab is designated
MAB699.
Preparation of human PBMC and resting T cells
PBMC were separated from buffy coats of healthy donors (New York Blood Bank, New York, NY) through Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Purification of T cells was performed as previously described (25). Briefly, monocytes were first removed from PBMC by plastic adherence for 2 h at 37°C. Nonadherent cells were incubated with anti-CD4 or anti-CD8 conjugated with Dynabeads (Dynal, Oslo, Norway) at a 1:4 target/bead ratio. The bead-bound cells were recovered using a magnet (Dynal) washed at least four times to remove unbound cells. The CD4+ or CD8+ cells were detached from the beads using Detachabead according to the manufacturer’s instructions (Dynal). These cells were then incubated with anti-HLA-DR Ab followed by Dynabeads conjugated with goat anti-mouse IgG for magnetic removal of preactivated T cells and contaminating dendritic cells or macrophages.
Media, reagents, and T cell cultures
The culture medium used in all experiments was RPMI (Life
Technologies, Grand Island, NY) supplemented with 10% FCS (HyClone,
Logan, UT), penicillin (50 U/ml; Life Technologies), streptomycin (50
µg/ml), sodium pyruvate (1 mM; Life Technologies), and glutamine (2
mM; Life Technologies). T cell lines were prepared by activation of
purified resting T cells with allogeneic PBMC and were treated with 50
µg/ml mitomycin C (Sigma) for 30 min at 37°C and 5 µg/ml PHA
(Sigma). Cells were split 3 days postactivation, expanded, and
maintained in culture medium supplemented with 200 U/ml recombinant
IL-2 (Chiron). T cell lines were maintained by restimulating the cells
every 2 wk with PHA and mitomycin C-treated allogeneic PBMCs. Culture
of T cells with cytokines has been previously described (26, 27). Cytokines IFN-
, IL-4, IL-7, IL-12, and IL-15 and
chemokines RANTES, macrophage inflammatory protein-1
(MIP-1),
MIP-1ß, and stromal cell-derived factor 1 (SDF-1) were all
obtained from R&D Systems.
Ab staining and FACS analyses
Cells were incubated with the relevant Ab on ice for 30 min in
PBS buffer with 2% FCS and 0.1% sodium azide. For staining of Bonzo,
cells were incubated with anti-Bonzo mAb at 3 µg/ml; after two
washes, cells were incubated with goat anti-mouse IgG conjugated to
PE or tricolor (TC) (Caltag, South San Francisco, CA). The cells were
washed twice again and blocked with excess mouse IgG (100 µg/ml)
followed by staining with directly conjugated Abs against the relevant
cell surface molecules. The Abs used for staining were PE, FITC, TC, or
peridin chlorophyll protein conjugates of anti-human CCR5 and
CXCR4; anti-mouse CD3, CD4, CD8, CD44, CD45RB, and 
TCR
(PharMingen, San Diego, CA); anti-human CD4 and CD45RO, and
secondary Abs goat anti-mouse PE or FITC (Caltag); anti-human
CD3, CD8, CD14, CD16, CD19, CD45RA, HLA-DR, and TCR (all from
Becton Dickinson, Palo Alto, CA); or anti-human CCR6 (R&D Systems).
Staining was analyzed on a FACScan using CellQuest software (Becton
Dickinson). Live cells were gated based on forward and side scatter.
Intracellular staining was performed using Cytofic/Cytoperm solution
according to the manufacturer’s protocol (PharMingen). To perform
FACS analyses on fresh human thymocytes, thymi were obtained from 7- to
9-mo-old pediatric heart surgery cases. Thymic tissue was disrupted by
mincing and forcing through stainless steel mesh. Thymocytes were
incubated twice at 37°C for 30 min in complete medium to remove
adherent cells. Single-cell suspensions were then placed on ice and
stained for FACS analysis as described above.
Gene targeting in embryonic stem cells and generation of mice
A 129/Sv mouse genomic DNA library was screened with human Bonzo cDNA and a 16-kb DNA fragment was isolated. Sequence analysis identified a 1.2-kb intronless open reading frame (ORF) homologous to that of the human Bonzo gene. To generate an EGFP (Clontech) knockin targeting vector, three DNA fragments were sequentially inserted into a pBS-KS+ plasmid: 1) a GFP expressing cassette followed by SV40 poly(A)n sequence, 2) a neomycin resistance cassette (neoR) flanked with loxP sites (28), and 3) a HSV thymidine kinase cassette (HSV-TK). Subsequently, an 8-kb ApaI-NotI genomic fragment downstream of the Bonzo ORF was inserted between the neoR and HSV-TK cassettes, whereas a 1.6-kb genomic fragment upstream of the Bonzo ORF and containing the 5'-untranslated region was inserted 5' of the GFP ORF. The resulting targeting vector was linearized with ClaI and electroporated into 129/Sv-derived E14 embryonic stem (ES) cells. G418-resistant ES cell clones were then screened for homologous recombination. To eliminate possible interference from the neo gene, correctly targeted clones were electroporated with a Cre recombinase-expressing vector (pCMV-Cre) to delete the neoR cassette, which was confirmed by sensitivity to G418 and by PCR or Southern blotting for the absence of the neoR-coding region. The resulting clones were microinjected into C57BL/6 blastocysts. Chimeric mice were mated with wild-type C57BL/6 mice to produce heterozygous progeny. Six- to 8-wk-old littermates from the mating of heterozygous mice were then analyzed.
RT-PCR analysis
Total RNA was extracted from mouse lymph nodes and spleen using Trizol reagent (Life Technologies). The RNA was further treated with RNase-free DNase I (Roche, Indianapolis, IN). RNA (1 µg) was used in an Access RT-PCR system (Promega, Madison, WI) with 50 pmol of sense (GCT TGC TCA TTT GGG TG) and antisense (CGC CGC GTC GAC CTT CTC TAA GTG TGG CAA GGC) Bonzo primers. RT was performed for 45 min at 48°C, and cDNA amplification was conducted for 40 cycles at 94°C for 30 s, 58°C for 1 min, and 68°C for 2 min. To exclude possible contamination with genomic DNA, control reactions in which reverse transcriptase was omitted were performed in parallel.
Fluorescence microscopy
Mouse organs were fixed in 4% formaldehyde for at least 12 h and incubated in 15% sucrose in PBS for 1 h. The samples were embedded in HistoPrep (Fisher, Fairlawn, NJ) and later frozen with dry ice. Samples were cut into 15-µm sections with a cryostat. Fluorescence microscopy was used to visualize GFP expression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An mAb reacting against human Bonzo was obtained as described in
Materials and Methods. This Ab binds specifically to HOS
cells that stably express the Bonzo cDNA (Fig. 1
a) (20). Similar
results were obtained using Bonzo-transduced NIH-3T3 cells or a mouse
thymoma line (data not shown). In addition, 293T cells were transfected
with a plasmid encoding Bonzo-GFP fusion and stained with
anti-Bonzo Ab. Only Bonzo-GFP-expressing cells were positively
stained with anti-Bonzo mAb, and this correlated with GFP
expression. To exclude the cross-reactivity of the Ab with other
chemokine receptors, we also stained HOS cell lines stably expressing
CCR1, CCR3, CCR4, CCR7, CCR8, APJ, and V28. None of these cell lines
was stained with anti-Bonzo mAb (data not shown). We then
determined the expression pattern of Bonzo on PBMC of normal donors
using multicolor FACS analysis. Expression of Bonzo was found to
segregate primarily to CD3+ T cells (Fig. 2a). Both
CD4+ and CD8+ T cell
subsets expressed Bonzo (Fig. 2, b and c),
although the percentage of Bonzo+ cells was
always higher within the CD8+ subset in samples
obtained from different donors (data not shown). T cells can also be
subdivided into memory and naive subsets based on expression of
CD45 isoforms, CD45RO+RA-
and CD45RO-RA+,
respectively (29). Bonzo was exclusively expressed on
CD45RO+ T cells (Fig. 2d). About
30–40% of T cells also expressed Bonzo (Fig. 2f).
The few CD3-negative cells that expressed Bonzo were
CD16+ NK cells (Fig. 2e), and no
expression was detected on B cells, monocytes, or dendritic cells (Fig. 2, g, h, and i, respectively).
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, the expression profile of CCR5 on
resting T cells very closely parallels that of Bonzo. Indeed,
costaining of PBMC with Abs specific for Bonzo and CCR5 showed that
most Bonzo-positive cells were also CCR5+ (Fig. 4). PBMC from a healthy donor with a
mutation in the CCR5 gene that prevents its cell surface
expression (31) exhibited normal expression of Bonzo (Fig. 3b), demonstrating that absence of CCR5 from the cell
surface does not negatively influence Bonzo expression. Most
Bonzo-positive resting T cells also expressed CCR6 (Fig. 4), whereas
expression of Bonzo and CXCR4 was inversely correlated (Fig. 4). This
inverse expression pattern reflects lower levels of CXCR4 on memory T
cells (30) (data not shown), which preferentially express
Bonzo (Fig. 2
d).
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Regulation of Bonzo expression by cytokine stimulation of resting T cells
The concordant subset distribution of Bonzo and CCR5 suggested
that the surface expression of these molecules may be regulated by
similar mechanisms. It has been noted that IL-2 or IL-15 stimulation of
human T cells can up-regulate some of the CC chemokine receptors
(27, 32, 33, 34). We asked whether Bonzo is similarly
regulated by cytokines on resting human T cells.
CD4+ and CD8+ resting T
cells were purified from PBMC and cultured for 8 days in the presence
of various cytokines. Culture of CD4+ or
CD8+ T cells with IL-2 or IL-15 resulted in
up-regulated expression of Bonzo exclusively on
CD45RO+ memory T cells (Fig. 5), with expression on
CD8+ T cells usually greater than that on
CD4+ T cells. This result is consistent with the
staining pattern observed on freshly isolated PBMC. Little expression
was observed on CD4+ T cells cultured with IL-4
or IL-7 (Fig. 5), and none was found with IL-6, IL-12, or IFN- (data
not shown). However, significant up-regulation of Bonzo was observed on
CD8+ T cells cultured with IL-7 (Fig. 5, lower panel). IL-2 and IL-15 also up-regulated CCR5 and CCR6
expression on CD4+ T cells, and most
Bonzo-expressing cells also coexpressed these CC chemokine receptors
(Fig. 6). Similar results were obtained
with CD8+ T cells (data not shown). This result
recapitulates the coordinate expression profile of Bonzo with CCR5 or
CCR6 in freshly isolated PBMC (Fig. 4). Conversely, Bonzo-positive
cells had slightly lower CXCR4 expression than Bonzo-negative cells
after stimulation with IL-2 or IL-15 (Fig. 6). CXCR4 expression was
significantly up-regulated in the presence of IL-4, and there was also
a moderate increase in response to IL-2 or IL-15, in accordance with
previous reports (35, 36, 37).
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Most chemokine receptors contain a DRY sequence motif that appears
to be required for coupling to G proteins (38). In
contrast to other CC and CXC-family chemokine receptors, Bonzo
possesses a noncanonic DRY box sequence at the second intracellular
loop. This raised the possibility that in response to its physiologic
ligand Bonzo may not signal in a similar fashion to the other chemokine
receptors. Recently, it has been reported that chemokine receptors are
down-regulated through ligand-mediated endocytosis
(39, 40, 41, 42). Therefore, we reasoned that if any of the CCR5
ligands also bound to Bonzo, we may be able to detect this through
down-regulation of Bonzo in the absence of detectable signaling. Fig. 7a shows that none of the
known CCR5 ligands (MIP-1, MIP-1ß, and RANTES) had any effect on
Bonzo expression, whereas they completely down-modulated CCR5 at
the same concentrations. As expected, CXCR4 was down-modulated by its
ligand SDF-1, but not by CCR5 ligands (Fig. 7a).
Interestingly, stimulation of cells with phorbol ester (PMA) also did
not have any effect on Bonzo expression, although CCR5 and CXCR4
expression was completely down-modulated (Fig. 7b).
Similarly, upon restimulation of T cell lines with mitogen, CCR5
expression was down-modulated, but that of Bonzo was unaffected (Fig. 7c). To determine whether Bonzo is premade, stored in
intracellular compartments, and released to cell surface upon
signaling, we performed intracellular staining on resting or
cytokine-stimulated primary T cells with anti-Bonzo Ab. No
difference was observed between cell surface staining alone or in
conjunction with intracellular staining (data not shown).
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To gain insight into the expression pattern and function of Bonzo
in mice, we used a gene-targeting strategy that replaced the
Bonzo-coding sequence with that of EGFP (Fig. 8A) Germline transmission was
obtained with two independent ES cell clones. Deletion of the
Bonzo gene was verified by Southern blot analysis (Fig. 8B). In an RT-PCR assay, a 3' portion of Bonzo mRNA was
amplified from total RNA derived from lymph node and spleen of both
Bonzo+/-
(Bz+/-) and
Bonzo-/-
(Bz-/-) mice. No amplification was
observed from Bz-/- mice (Fig. 8C). Mating of heterozygous mice gave rise to
Bz-/- mice in Mendelian proportions.
Bz-/- mice were phenotypically
indistinguishable from Bz+/+ and
Bz+/- littermates in a specific
pathogen-free environment. Histologic analysis of organs displayed no
morphologic difference among the three genotypes. Flow cytometric
analysis of cells from lymphoid organs showed no alteration in various
cell populations (data not shown). Proliferation of T cells from
Bz+/- or
Bz-/- mice was assessed in response
to different concentrations of anti-CD3 Ab in the presence or the
absence of anti-CD28 Ab. No difference was observed between
Bz-/+ and
Bz-/- cells (data not shown).
Nucleoprotein-keyhole limpet hemocyanin-immunized
Bz+/- and
Bz-/- mice also displayed similar
levels of nucleoprotein-specific IgM and IgG1 production (data not
shown), suggesting that T cell help and B cell function were normal in
Bonzo-deficient mice.
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The in-frame substitution of EGFP for Bonzo
yields a sensitive tool to trace Bonzo expression and regulation in
vivo. Histologic analysis did not show any GFP expression in
nonlymphoid tissues. Strong green fluorescence was detected in spleen
and lymphoid nodes. In spleen, GFP-positive cells were clustered in the
periarteriolar lymphoid sheath area in the white pulp, where T cells
reside (Fig. 9). Notably, there was no
difference in expression pattern between
Bz+/- and
Bz-/- mice.
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Within the lymph nodes and spleen almost all GFP+
cells were CD3+ T cells (Fig. 10a). The mean fluorescence
of GFP-expressing Bz-/- cells was
slightly higher than that of Bz+/-
cells, most likely due to GFP expression from both targeted alleles
(data not shown). Few CD4+ T cells expressed GFP,
whereas the majority of CD8+ T cells were
positive, with a broad distribution of intensity of GFP expression. We
next analyzed the distribution of GFP expression on naive vs memory T
cells in Bonzo knockin mice. CD8+
cells were all CD44+, and it was difficult to
discriminate naive vs memory cells (Fig. 10b). T
cells from lymphoid organs or resident within the skin, reproductive
tract, or gut also expressed GFP (Fig. 10c). Interestingly,
GFP expression on CD4+ murine T cells was
restricted to the CD44+ and
CD45RBlow subset (Fig. 10d),
suggesting that, as in human CD4+ T cells, Bonzo
is primarily expressed in the memory subset of mouse
CD4+ T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been shown that CC family chemokine receptors CCR2, CCR5, and
CCR6 are expressed primarily on the CD45RO+ or
CD26+ memory subset of T cells (30, 44, 45, 46). The expression pattern of Bonzo was similarly
restricted to memory T cells. In this regard we found that most
Bonzo-positive cells also coexpressed CCR5 or CCR6 on peripheral blood
T cells. In contrast, Bonzo was not coexpressed with high levels of
CXCR4, reflecting lower CXCR4 expression on memory vs naive T cells
(30). Although Bonzo was expressed on
CD4+, CD8+ and
subsets of T cells, expression levels were usually higher on
CD8+ and TCR+ T
cells. Few NK cells were found to express Bonzo, and those that did had
relatively low levels of CD16 expression (Fig. 2e), possibly
representing recently activated NK cells.
The coexpression of Bonzo and CCR5 on memory T cells is notable from
the perspective of HIV coreceptor usage, because CCR5 is the major
receptor used by most strains of HIV and almost all SIVs. Expression of
CCR5 on the cell surface has been shown to be critical in transmission
of HIV-1 infection, because a homozygous 32-bp deletion in the
CCR5 gene prevents CCR5 expression and confers resistance
against HIV-1 infection (31, 47, 48). We showed that T
cells isolated from individuals who are homozygous for the
CCR5
32 mutation expressed normal levels of
Bonzo. Although Bonzo is a minor HIV-1 coreceptor, its role during
transmission of infection and pathogenesis is unclear. However, Bonzo
is a major SIV receptor and is also used by many HIV-2 strains in vitro
(18, 20). It is interesting to note that different species
of nonhuman primates vary widely in their responses to SIV infection,
and people infected with HIV-2 usually have a more delayed progression
to AIDS. It is possible that Bonzo may substitute for CCR5 usage during
infection with SIV and HIV-2 isolates and perhaps influences the course
of infection and pathogenesis. In contrast to CCR5, Bonzo was not
expressed by macrophages or dendritic cells. This expression pattern
may preclude its involvement during the initial phase of infection,
where macrophage or dendritic cell infection is thought to be necessary
for the virus to gain a foothold in the body (49).
Cytokine stimulation of T cells differentially regulates the surface expression of chemokine receptors. IL-2 has been described to potently up-regulate CCR1, CCR2, CCR5, CCR6, and CXCR3 (27, 30, 32, 50, 51). More recently, IL-15 has been shown to have similar effects on some of the CC chemokine receptors (27, 33). In contrast, CXCR4 expression is dramatically up-regulated in the presence of IL-4 (35, 36, 37). We have shown that Bonzo, similar to CC chemokine receptors, is induced through stimulation of highly purified resting T cells with IL-2 or IL-15 and is coexpressed with CCR5 or CCR6. The coordinated regulation of Bonzo and CCR5 on resting T cells by IL-2 or IL-15 recapitulates the ex vivo expression pattern.
Notably, the gene for Bonzo maps to chromosome 3, where most of the
chemokine receptors, especially those of the CC family, are localized
(21). Additionally, the amino acid sequence of Bonzo is
similar to that of other members of the chemokine receptor family.
Taken together with our results on its expression pattern and
cytokine-mediated regulation, these findings suggest that Bonzo is also
a chemokine receptor. However, Bonzo stands apart from other chemokine
receptors, because its expression is resistant to PMA-induced
down-modulation. This result contrasts with PMA-induced down-modulation
of CXCR4 (52, 53), CCR5 (Fig. 7b), or CCR6
(data not shown). However, Bonzo may still be down-modulated by its
ligand(s), because the mechanisms of ligand- and PMA-induced
down-modulation appear to be different (42, 52, 53). We
did not detect any ligand-induced down-modulation of Bonzo in the
presence of CCR5 ligands, but we cannot rule out that Bonzo is
resistant to ligand-induced endocytosis and thus may still be able to
bind to these chemokines.
Because there is no Ab yet available against mouse Bonzo, we used the targeted knockin of GFP in place of the Bonzo gene as an indicator of Bonzo expression. We found that murine Bonzo is expressed in cell subsets similar to human Bonzo, namely in T cells, particularly those displaying markers of effector/memory cells and those stimulated by IL-2 (data not shown). However, in both mouse and human only a subset of memory cells expressed Bonzo, similar to CCR5 expression. Chemokines regulate both the inter- and intraorgan migration patterns of human T cell subsets. Recently, human memory T cells were subdivided into two functionally distinct subsets based on CCR7 expression (54). CCR7- memory cells express receptors for migration to inflamed tissues and display immediate effector function. In contrast, CCR7+ memory cells express lymph node-homing receptors and lack immediate effector function. Interestingly, the CCR7- cells were enriched for expression of CCR5, CCR6, and CCR1 (54). Based on the similar expression patterns, we would predict that the majority of Bonzo+ T cells will be compartmentalized to the CCR7- memory subset, suggesting a role for Bonzo in recruiting memory T cells to sites of inflammation.
In homozygous Bonzo knockout mice we have not yet detected any functional defect. This may be due to redundancy in the chemokine receptor system. However, it will be important to perform more detailed analyses of migration patterns of T cells in response to a wide variety of pathogens and in particular to study T cell memory responses to the pathogens. Mutations in other chemokine receptor family members, such as CCR5 and CCR2, resulted in phenotypes revealed only after studies in responses to pathogen or in disease models (55, 56, 57, 58). It will also be interesting to establish multiple knockouts between the Bonzo mutant mice and mutants for other chemokine receptors expressed in memory T cells to identify potentially overlapping functions of these molecules.
Bonzo is unique in that its sequence bears a noncanonic DRY box motif that is thought to couple chemokine receptors to G proteins (18, 20). It is currently not known whether Bonzo elicits signals that are similar to those of other chemokine receptors. It will be critical to identify the natural ligand(s) of Bonzo to understand the receptor’s signaling function. In addition, mice in which the Bonzo gene has been replaced with EGFP may be of considerable utility for tracking the migration patterns of memory T cell in responses to inflammatory stimuli. New insight into the expression pattern and function of Bonzo may also help elucidate its role during SIV/HIV infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Derya Unutmaz, Department of Microbiology and Immunology, Vanderbilt University Medical School, AA-5216 Medical Center North, Nashville, TN 37215.
3 D.R.L. is an Investigator with the Howard Hughes Medical Institute.
4 Abbreviations used in this paper: GFP, green fluorescent protein; MIP, macrophage inflammatory protein; SDF-1, stromal cell-derived factor; TC, tricolor; ORF, open reading frame; ES, embryonic stem.
Received for publication June 16, 2000. Accepted for publication June 28, 2000.
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H. Stenstad, A. Ericsson, B. Johansson-Lindbom, M. Svensson, J. Marsal, M. Mack, D. Picarella, D. Soler, G. Marquez, M. Briskin, et al. Gut-associated lymphoid tissue-primed CD4+ T cells display CCR9-dependent and -independent homing to the small intestine Blood, May 1, 2006; 107(9): 3447 - 3454. [Abstract] [Full Text] [PDF]
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 Y. Huang, X.-Y. Zhu, M.-R. Du, X. Wu, M.-Y. Wang, and D.-J. Li Chemokine CXCL16, a scavenger receptor, induces proliferation and invasion of first-trimester human trophoblast cells in an autocrine manner Hum. Reprod., April 1, 2006; 21(4): 1083 - 1091. [Abstract] [Full Text] [PDF]
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J. L. Matsuda, Q. Zhang, R. Ndonye, S. K. Richardson, A. R. Howell, and L. Gapin T-bet concomitantly controls migration, survival, and effector functions during the development of V{alpha}14i NKT cells Blood, April 1, 2006; 107(7): 2797 - 2805. [Abstract] [Full Text] [PDF]
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X. Jiang, T. Shimaoka, S. Kojo, M. Harada, H. Watarai, H. Wakao, N. Ohkohchi, S. Yonehara, M. Taniguchi, and K.-i. Seino Cutting Edge: Critical Role of CXCL16/CXCR6 in NKT Cell Trafficking in Allograft Tolerance J. Immunol., August 15, 2005; 175(4): 2051 - 2055. [Abstract] [Full Text] [PDF]
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 B. Chandrasekar, S. Mummidi, A. J. Valente, D. N. Patel, S. R. Bailey, G. L. Freeman, M. Hatano, T. Tokuhisa, and L. E. Jensen The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling J. Biol. Chem., July 15, 2005; 280(28): 26263 - 26277. [Abstract] [Full Text] [PDF]
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V. Sordi, M. L. Malosio, F. Marchesi, A. Mercalli, R. Melzi, T. Giordano, N. Belmonte, G. Ferrari, B. E. Leone, F. Bertuzzi, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets Blood, July 15, 2005; 106(2): 419 - 427. [Abstract] [Full Text] [PDF]
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V. Monceaux, L. Viollet, F. Petit, R. H. T. Fang, M.-C. Cumont, J. Zaunders, B. Hurtrel, and J. Estaquier CD8+ T Cell Dynamics during Primary Simian Immunodeficiency Virus Infection in Macaques: Relationship of Effector Cell Differentiation with the Extent of Viral Replication J. Immunol., June 1, 2005; 174(11): 6898 - 6908. [Abstract] [Full Text] [PDF]
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 S. Tabata, N. Kadowaki, T. Kitawaki, T. Shimaoka, S. Yonehara, O. Yoshie, and T. Uchiyama Distribution and kinetics of SR-PSOX/CXCL16 and CXCR6 expression on human dendritic cell subsets and CD4+ T cells J. Leukoc. Biol., May 1, 2005; 77(5): 777 - 786. [Abstract] [Full Text] [PDF]
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 G Aust, M Kamprad, P Lamesch, and E Schmucking CXCR6 within T-helper (Th) and T-cytotoxic (Tc) type 1 lymphocytes in Graves' disease (GD) Eur. J. Endocrinol., April 1, 2005; 152(4): 635 - 643. [Abstract] [Full Text] [PDF]
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 H. Blaak, P. H. M. Boers, R. A. Gruters, H. Schuitemaker, M. E. van der Ende, and A. D. M. E. Osterhaus CCR5, GPR15, and CXCR6 Are Major Coreceptors of Human Immunodeficiency Virus Type 2 Variants Isolated from Individuals with and without Plasma Viremia J. Virol., February 1, 2005; 79(3): 1686 - 1700. [Abstract] [Full Text] [PDF]
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M. Heydtmann, P. F. Lalor, J. A. Eksteen, S. G. Hubscher, M. Briskin, and D. H. Adams CXC Chemokine Ligand 16 Promotes Integrin-Mediated Adhesion of Liver-Infiltrating Lymphocytes to Cholangiocytes and Hepatocytes within the Inflamed Human Liver J. Immunol., January 15, 2005; 174(2): 1055 - 1062. [Abstract] [Full Text] [PDF]
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T. Sato, H. Thorlacius, B. Johnston, T. L. Staton, W. Xiang, D. R. Littman, and E. C. Butcher Role for CXCR6 in Recruitment of Activated CD8+ Lymphocytes to Inflamed Liver J. Immunol., January 1, 2005; 174(1): 277 - 283. [Abstract] [Full Text] [PDF]
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 D. M. Wuttge, X. Zhou, Y. Sheikine, D. Wagsater, V. Stemme, U. Hedin, S. Stemme, G. K. Hansson, and A. Sirsjo CXCL16/SR-PSOX Is an Interferon-{gamma}-Regulated Chemokine and Scavenger Receptor Expressed in Atherosclerotic Lesions Arterioscler. Thromb. Vasc. Biol., April 1, 2004; 24(4): 750 - 755. [Abstract] [Full Text] [PDF]
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B. Chandrasekar, S. Bysani, and S. Mummidi CXCL16 Signals via Gi, Phosphatidylinositol 3-Kinase, Akt, I{kappa}B Kinase, and Nuclear Factor-{kappa}B and Induces Cell-Cell Adhesion and Aortic Smooth Muscle Cell Proliferation J. Biol. Chem., January 30, 2004; 279(5): 3188 - 3196. [Abstract] [Full Text] [PDF]
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B. Johnston, C. H. Kim, D. Soler, M. Emoto, and E. C. Butcher Differential Chemokine Responses and Homing Patterns of Murine TCR{alpha}{beta} NKT Cell Subsets J. Immunol., September 15, 2003; 171(6): 2960 - 2969. [Abstract] [Full Text] [PDF]
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T. Cilliers, J. Nhlapo, M. Coetzer, D. Orlovic, T. Ketas, W. C. Olson, J. P. Moore, A. Trkola, and L. Morris The CCR5 and CXCR4 Coreceptors Are Both Used by Human Immunodeficiency Virus Type 1 Primary Isolates from Subtype C J. Virol., April 1, 2003; 77(7): 4449 - 4456. [Abstract] [Full Text] [PDF]
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R. Mo, J. Chen, Y. Han, C. Bueno-Cannizares, D. E. Misek, P. A. Lescure, S. Hanash, and R. L. Yung T Cell Chemokine Receptor Expression in Aging J. Immunol., January 15, 2003; 170(2): 895 - 904. [Abstract] [Full Text] [PDF]
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P. Shashkin, D. Simpson, V. Mishin, B. Chesnutt, and K. Ley Expression of CXCL16 in Human T Cells Arterioscler. Thromb. Vasc. Biol., January 1, 2003; 23(1): 148 - 149. [Full Text] [PDF]
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M. Nishimura, H. Umehara, T. Nakayama, O. Yoneda, K. Hieshima, M. Kakizaki, N. Dohmae, O. Yoshie, and T. Imai Dual Functions of Fractalkine/CX3C Ligand 1 in Trafficking of Perforin+/Granzyme B+ Cytotoxic Effector Lymphocytes That Are Defined by CX3CR1 Expression J. Immunol., June 15, 2002; 168(12): 6173 - 6180. [Abstract] [Full Text] [PDF]
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 M Heydtmann and D H Adams Understanding selective trafficking of lymphocyte subsets Gut, February 1, 2002; 50(2): 150 - 152. [Full Text] [PDF]
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L. M. Ebert and S. R. McColl Up-Regulation of CCR5 and CCR6 on Distinct Subpopulations of Antigen-Activated CD4+ T Lymphocytes J. Immunol., January 1, 2002; 168(1): 65 - 72. [Abstract] [Full Text] [PDF]
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B. Lee, G. Leslie, E. Soilleux, U. O'Doherty, S. Baik, E. Levroney, K. Flummerfelt, W. Swiggard, N. Coleman, M. Malim, et al. cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor J. Virol., December 15, 2001; 75(24): 12028 - 12038. [Abstract] [Full Text] [PDF]
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A. Wilbanks, S. C. Zondlo, K. Murphy, S. Mak, D. Soler, P. Langdon, D. P. Andrew, L. Wu, and M. Briskin Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C Chemokines J. Immunol., April 15, 2001; 166(8): 5145 - 5154. [Abstract] [Full Text] [PDF]
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 A. Motsinger, D. W. Haas, A. K. Stanic, L. Van Kaer, S. Joyce, and D. Unutmaz CD1d-restricted Human Natural Killer T Cells Are Highly Susceptible to Human Immunodeficiency Virus 1 Infection J. Exp. Med., April 1, 2002; 195(7): 869 - 879. [Abstract] [Full Text] [PDF]
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, loxP
sites. H, HindIII; A, ApaI; RV,
EcoRI; C, ClaI. B,
Southern blot analysis of progeny of heterozygous intercrossing. The
probe used is depicted by the solid bar beneath the targeted structure
in A. The wild-type allele yields a 7-kb
EcoRV genomic fragment, whereas the targeted allele
yields a 6.6-kb fragment. C, RT-PCR analysis of
expression of Bonzo mRNA in mutant mice. The primers used are described
in Materials and Methods. In a TAE gel, a 590-bp DNA
fragment is amplified from wide-type (top panel,
lane 2) and heterozygous (lane 3) mice,
but is absent in Bz-/- mice (lane
4). A similar RT-PCR assay was conducted for GADPH mRNA, as a
loading control (lower panel).
