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Blood First Edition
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24,For
2002;
DOIuse
10.1182/blood-2002-07-2348
CCR4 Versus CCR10 in Human Cutaneous Th Lymphocyte
Trafficking.
Dulce Soler1@, Tricia L. Humphreys2, Stanley M. Spinola2.3
and James J. Campbell4,5#.
1. Millennium Pharmaceuticals, Inflammation Department, Cambridge, MA 02139.
2. Department of Medicine, Indiana University School of Medicine, Indianapolis, IN
46202.
3. Departments of Microbiology & Immunology, and Pathology & Laboratory Medicine,
Indiana University School of Medicine, Indianapolis, IN 46202.
4. Department of Pathology, Harvard Medical School, Boston, MA, 02115.
5. Joint Program in Transfusion Medicine, Children’s Hospital, Boston, MA, 02115.
@Address Reagent Requests to:
Dulce Soler
Inflammation Department
Millennium Pharmaceuticals
75 Sydney Street
Cambridge, MA 02139
Tel: (617) 551-7993
[email protected]
#Address Correspondence to:
James J. Campbell, PhD
Children’s Hospital
Bader 401
300 Longwood Avenue
Boston, MA 02115
Tel: (617) 355-8319
Fax: (617) 732-6142
[email protected]
1
Copyright (c) 2002 American Society of Hematology
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ABSTRACT
The chemokine receptors CCR4 and CCR10, and the cutaneous lymphocyte antigen
(CLA), have each been proposed as critical mediators of skin-specific Th lymphocyte
homing in mouse and man. CLA initiates skin-homing by mediating E-selectindependent tethering and rolling within cutaneous venules, but the specific roles of CCR4
and CCR10 are unclear. We have generated an anti-human CCR10 MAb (1B5) to
illuminate the individual contributions of these molecules. This MAb allows us to
compare CCR10, CCR4 and CLA expression within human Th populations. The MAb
1B5 recognizes functional CCR10 expression, as chemotactic responsiveness to
CTACK/CCL27 (a CCR10 ligand) parallels the staining of Th subsets. We find CCR10
expressed by only a minority (~30%) of blood-borne skin-homing (CLA+/CCR4+) Th
cells. However, essentially all members of the relatively small “effector”
(CLA+/CCR4+/CD27-/CCR7-) skin-homing Th population express CCR10. Most skininfiltrating lymphocytes in allergic DTH and bacterial Chancroid skin-lesions express
both CCR4 and CLA, but only ~10% express CCR10. This suggests (for the two models
of Th skin-homing studied here) that CCR10+ Th cells have no advantage over other
CLA+/CCR4+ Th cells in homing to cutaneous sites. We conclude that the skin-homing
Th compartment is itself divided into distinct subpopulations, the smaller of which
expresses both CCR4 and CCR10, and the larger expresses only CCR4. Thus, CCR10 is
unlikely to be necessary for cutaneous homing of Th cells in the models studied here.
CCR10 may instead play a role in the movement of specialized “effector” cutaneous Th
cells to and/or within epidermal microenvironments.
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INTRODUCTION
Adhesion Molecules and Tissue-Specific Homing. Classical studies of tissue-specific
homing within the human circulating Th (CD4+) compartment suggest that the memory
(CD45RA-) population can be divided into tissue-specific subsets. Each subset traffics
through and engages in immunosurveillance of distinct domains of non-lymphoid tissues.
For example, memory lymphocytes expressing the α4β7-integrin-heterodimer (an
adhesion molecule that binds the mucosal addressin MAdCAM-1) contain antigen
specificities associated with the gut1,2, and tend to traffic through intestinal sites3,4. Th
cells that express the CLA carbohydrate (an adhesion molecule that binds E-selectin)
contain specificities for cutaneous antigens5,6, and tend to traffic through cutaneous
sites7,8. CLA-expressing cells are identified by MAb HECA-452 in man7,8 or by flow
cytometry with an E-selectin-Ig chimaera in either mouse or man9.
Chemokine Receptors and Tissue-Specific Homing. Differential expression of chemokine
receptors also correlates with tissue-specific homing. For example, expression of
chemokine receptor 9 (CCR9) is associated with a small-intestine (but not colon) homing
subset of Th cells10,11. Moreover, CCR7 function is necessary for homing of T cells to
secondary lymphoid organs through high endothelial venules (HEV)12-14.
Another chemokine receptor, CCR4, is robustly expressed by most skin-homing
(CLA+) Th cells in the circulation, but rarely (and at lower levels on the few positives) by
intestinal-homing Th cells15. CCR4 is also expressed at high levels by skin-infiltrating
lymphocytes (isolated directly from skin), but not by gut-infiltrating lymphocytes10.
Additionally, the CCR4 ligand TARC/CCL17 is associated with cutaneous (but not
intestinal) endothelial cells15.
CCR4 expression can easily distinguish between cutaneous (CLA+) and intestinal
(α4β7+) Th memory cells, but is also expressed by some circulating memory Th cells that
lack both CLA and α4β715. Some of these CLA-/CCR4+ cells may (like CLA+ cells)
have cutaneous roles, as VCAM-1/integrin interactions appear to be more important than
CLA/E-selectin interactions for deep-dermal homing16. However, there also appears to
be an additional, less well-understood role for CCR4 in pulmonary leukocyte homing17-19.
The expression of CCR4 by apparently non-cutaneous lymphocytes has led to the
proposal that CLA and CCR4 must work together to convey skin-specific homing15.
Alternatively, it has been proposed that there may exist another chemokine receptor, with
better specificity for cutaneous Th cells than CCR4.
In fact, after the associations among CCR4, TARC and cutaneous Th homing
were discovered, another chemokine: CTACK/CCL27 (originally cloned as ALP in
mouse20) was found to be expressed by cutaneous keratinocytes21. CTACK is not a
ligand for CCR4, but like TARC, CTACK preferentially attracts CLA+ Th cells from
peripheral blood in vitro21. The receptor for CTACK was identified as CCR10, which
has another ligand (MEC, CCL28) expressed within the colon and within secretory
tissues including salivary and mammary glands22. CCR10 is expressed by a subset of
CLA+ Th cells in peripheral blood23.
Thus, CCR4 and CCR10 are both associated with conventionally-defined skin
homing Th cells from peripheral blood, but the association is imperfect in both cases:
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CCR4 is present on essentially all skin-homing cells, but also present on other systemic
cells. In contrast, CCR10 is absent from non-skin-homing Th cells23, but only present on
a subset of skin-homing Th cells.
Peripheral blood Th lymphocytes lacking CD27 and CCR7. In addition to the tissuespecific divisions among Th subsets, there exist other axes of subdivision. For example,
a relatively small subset of Th cells does not express the TNF-receptor family member
CD2724-26 and/or the chemokine receptor CCR724,27. Such cells are found within the
CLA+/α4β7-, CLA-/α4β7+, and CLA-/α4β7- memory compartments, but not within the
naïve (CD45RA+) compartment (virtually all naïve cells express both CD27 and
CCR724). The Th phenotypes that lack these markers have been called “effectormemory,” because they are enriched in recently-seen antigen specificities, and they
respond more quickly to antigen than most other memory Th cells25-27. Absence of
CCR7 would preclude entry of such cells into lymphoid organs via HEV through the
known mechanisms13,14,27, suggesting that such cells might home through only those nonlymphoid tissues to which they have become dedicated. Indeed, effector-memory cells
are found within uninflamed or inflamed non-lymphoid tissues, often side-by-side with
cells that continue to express both CD27 and CCR724. The relationship between effector
phenotype and trafficking phenotype has not yet been thoroughly assessed.
In order to understand the interesting differences between CCR10 and CCR4
expression among human Th subpopulations, we have generated a monoclonal antibody
that recognizes functional expression of CCR10 by human lymphocytes. We have
performed extensive analyses to compare and contrast CCR10 versus CCR4 expression
by Th subsets associated with cutaneous homing. We have examined expression (and
functionality) of these receptors by several distinct subsets of peripheral blood Th
memory cells, including those classified as “effector” Th cells. Furthermore, we have
compared expression of these receptors by Th lymphocytes directly isolated from
inflamed cutaneous sites.
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MATERIALS AND METHODS
Generation of mAbs against CCR10. The murine pre-B lymphoma cell line L1/2 was
transfected with an expression vector (pcDNA3.1) containing the human CCR10 DNA
sequence, as previously described28.
Transfected pools were chemotaxed to
CTACK/CCL27 and/or to MEC/CCL28, and clones with the best chemotactic potential
were selected (L1/2 cells expressing human CCRs 1 through 9 were created in the same
manner). CCR10-specific monoclonal antibody 1B5 was generated by immunizing
C57Bl/6 mice intraperitoneally with 107 CCR10 L1/2 cell transfectants every 2 weeks for
5 to 6 times. The final inmmunization was performed intravenously 4 days prior to
extraction of the spleen for fusion with the SP2/O cell line as described24. CCR10specific antibody secreting hybridoma cell clones were identified by performing
immunofluorescence staining of WT and CCR10 L1/2 cells with cell supernatants
followed by a fluorescently-labeled anti-mouse secondary antibody. Fluorescent cells
were detected by flow cytometry. Antibody secreting clones were isolated by multiple
rounds of subcloning. For screening the CCR10 Mab against L1/2 cells transfected with
other CCR’s (Fig. 1), each transfectant was stained with a specific MAb to its respective
receptor, to ensure high expression levels of the transfected gene (not shown).
Peripheral Blood: Buffy coats from anonymous healthy donors were obtained from the
Children's Hospital, Boston blood bank. Mononuclear layers were prepared as previously
described12,15,17,24,29,30.
Skin Inflammation. DTH responses to Candida extract, and subsequent isolation of
lymphocytes from vacuum-blister fluid were performed as described previously10,24.
Experimental human chanchroid was induced by inoculation of healthy, HIV
seronegative volunteers at 3 sites with Haemophilus ducreyi 35000HP (a human passaged
isolate of 35000). Sites that evolved into pustules were biopsied 7 to 14 days after
inoculation. In some cases, multiple sites from one subject were biopsied, and then
pooled. Isolation of lymphocytes from biopsied skin using EDTA was performed as
described previously10,24. Informed consent was obtained from the subjects in accordance
with the guidelines for human experimentation of the U.S. Department of Health and
Human Services and the Institutional Review Board of Indiana University-Purdue
University at Indianapolis under grants AI31494 and AI27863 to S.M.S. and
M01RR00750 to the GCRC at IU. Enrollment procedures, exclusion criteria, preparation
of the bacteria, inoculation procedures and clinical outcomes are described in detail
elsewhere31-33
Six-color flow-immunocytometry: Immunostaining was performed with a 5-step method.
Cells were stained first with unconjugated MAb (or its isotype-matched control);
followed by polyclonal second-stage Abs; followed by blocking with 3mg/ml mouse IgG
(Technical grade, Sigma) and 0.3mg/ml mouse IgM (Technical grade, Sigma); followed
by directly-conjugated MAbs, followed by streptavidin conjugate.
Color 1: CLA-FITC (HECA-452, BD-Pharmingen) or CD27-FITC (M-T271, BDPharmingen)
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Color 2: CD27-PE (M-T271, BD-Pharmingen) or β7-Integrin-PE (FIB-504, BDPharmingen)
Color 3: CD45RA-PE-TR (2H4, Beckman-Coulter)
Color 4: IgG2a isotype control (UPC-10, Sigma), CCR4 (2B1015, Millennium
Pharmaceuticals), or CCR10 (1B4, Millennium Pharmaceuticals); Followed by goat-antimouse-IgG2a(γa)-biotin (Southern Biotechnology); Followed by Streptavidin-PE-Cy7
(Cedarlane Labs)
Color 5: CCR7 (3D924, Millennium Pharmaceuticals)
Followed by goat-anti-mouse-IgM(µ)-Cy5 (Jackson Immunoresearch)
Color 6: CD4-APC-Cy7 (S3.5, Caltag)
Data on stained cells was acquired on dual-laser MoFlo cytometer (Cytomation, Inc.)
configured for 6 colors: 1) FITC, 2) PE, 3) PE-TR, 4) PE-Cy7, 5) Cy5, 6 ) APC-Cy7.
Raw data was compensated and analyzed using Summit 3.0 software (Cytomation, Inc.)
Chemotaxis: Chemotaxis was performed as described12,15. Two chemotaxis wells of the
following concentrations were set up for each chemokine for each donor: 1000nM,
300nM, 100nM, 30nM, and data is shown in Fig. 4 for the optimal concentrations.
Recombinant SDF-1α, (PeproTech), recombinant TARC (R&D Systems) and synthetic
CTACK (Gryphon) were used for all experiments shown. Input and migrated cells were
stained with a 5-color protocol: CLA-FITC, CD27-PE (M-T271, BD-Pharmingen),
CD45RA-PETR, CD4-biotin (13B8.2, Beckman-Coulter) + streptavidin-PE-Cy7, CCR7
(7H12, Millennium) + Goat-anti-mouse IgG(H&L)-Cy5 (Jackson).
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RESULTS
Generation of a MAb to Human CCR10. A monoclonal antibody against human CCR10
was developed by immunizing mice with L1/2 cells (a murine pre-B lymphoma)
transfected with human CCR10 mRNA in a mammalian expression vector24. The
resulting MAb, 1B5, recognized CCR10-transfected (but not wild-type) L1/2 cells (Fig.
1). 1B5 did not recognize L1/2 cells transfected with any of the other known CC
chemokine receptors (CCR1 through 9 plus CCR11/GusB) (Fig. 1).
Expression of CCR10 by Peripheral Blood Th Cells. We first examined the CCR10
expression of naïve Th and the three peripheral blood memory Th subsets classically
defined by the homing receptors CLA or α4β7-integrin34,35 (Fig. 2a). Among the memory
Th populations, CCR10 expression was restricted to the CLA+/α4β7- “skin homing”
subpopulation (Fig. 2b). Interestingly, however, CCR10 was not expressed by ~70% of
the CLA+ Th population. CCR10 was absent from naïve Th cells (Fig. 2b).
Two-color plots of CCR10 or CCR4 versus CLA are shown for the memory Thgated population in Fig. 2c. CCR4 stains the vast majority of CLA+ Th cells with equal
intensity, and also stains a subset of CLA- Th cells (Fig. 2c, right panel). In contrast,
CCR10 appears to preferentially stain only those Th cells in the brightest half-to-onethird of the CLA+ distribution (Fig. 2c, left panel).
Characterization of the CLAhi-Enriched Memory Th Subset That Expresses CCR10, and
Comparison to CCR4 Expression. Another previously identified axis of subdivision
within the peripheral blood CLA+ Th memory compartment (as well as the other Th
memory compartments) involves CD27 and CCR7 expression24-27. We therefore asked
whether the observed patterns of CCR10 expression correlate with CD27 and/or CCR7
expression by using 6-color flow cytometry.
The upper panel in Fig. 3a shows that peripheral blood CLA+ Th cells are spread
among all four quadrants of a CD27 versus CCR7 plot. However, plots of CLA versus
CD27 or CCR7 distinguish the individual subsets more clearly (Fig. 3a, lower plots).
The latter type of plot was used to enumerate CLA+ subsets displaying the 4 permutations
of CD27 and CCR7 expression for ten normal donors (Fig. 3b). Interestingly, the CD27and CCR7- subsets of the CLA+ Th populations were enriched for the brightest one-thirdto-one-half of the CLA expressers, with striking similarity to the CLA pattern observed
for CCR10 expressers in Fig. 2c.
For simplicity, we have chosen to focus on the two most extreme phenotypes
identified here for more detailed analysis: the CD27-/CCR7- “double negatives” (which
meet all immunophenotypic criteria for being “effector” Th cells), and CD27+/CCR7+
“double positives,” (which meet none of the criteria for being “effector” Th cells). Figs.
3c and 3d compare the CCR4 and CCR10 expression patterns of CD27/CCR7-double
positive and -double negative subsets of CLA+, CLA- and α4β7+ memory Th populations,
as well as the naïve populationA. Fig.3c shows CCR10 staining (black lines, left column)
or CCR4 staining (black lines, right column) overlaid on isotype-matched controls for the
Please note that the CLA- memory population also contains the α4β7+ population as a
subset.
A
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same gated populations from a representative donor (gray lines, both columns). Fig. 3d
shows the mean CCR4 and CCR10 expression (and SD) for the same populations by 10
normal blood donors. Although CCR4 is expressed by the same proportion of cells and
(at equal levels) by both double positive and double negative CLA+ populationsB, CCR10
is expressed by a much greater proportion (and at much greater levels)by double negative
cells. In fact, CCR10 is expressed by virtually all double-negative CLA+ Th cellsB. The
CD27+/CCR7- and CD27-/CCR7+ populations (not shown) expressed intermediate
CCR10 levels (DS and JJC, unpublished data).
Responsiveness of Peripheral Blood Th Subsets to the CCR10 Ligand CTACK. Whenever
possible, it is important to confirm chemokine receptor flow cytometry results through an
independent method. We therefore set out to determine whether CCR10 expression
patterns (determined by 1B5) correlate with functional responses to CTACK/CCL27 (a
CCR10 ligand) in standard chemotaxis assays. Each of the populations examined express
very similar amounts of the SDF-1 receptor CXCR4 (JJC, unpublished data). This fact
allows normalization of their CTACK-mediated migration to that of SDF-1, to correct for
potential intrinsic differences in migration rates among the various populations (Fig. 4,
upper scale). Migration is also plotted (on the same graphs) as per cent of input (Fig. 4,
lower scale) with comparable outcome. Figure 4 clearly demonstrates that the double
negative CLA+ Th population responds significantly better to CTACK than any of the
other populations tested (including the double positive CLA+ Th population, Fig. 4, left
panel). There was no significant difference in TARC responsiveness between double
positive and double negative populations (Fig. 4, right panel).
CCR10 Expression by Tissue-Infiltrating Lymphocytes from Inflammed Skin. The data
indicate that while CCR4 is expressed by most CLA+ Th cells in blood, CCR10 is only
expressed by a minority subset. We therefore asked whether the CCR10+ subset might
have an advantage over other CLA+/CCR4+ Th cells in their ability to home to inflamed
cutaneous sites.
We chose to analyze the homing phenotypes of Th cells directly isolated from two
different types of cutaneous lesions. The first was a widely studied DTH model, in which
cutaneous inflammation is induced by intradermal injection of a sterile extract of Candida
albicans10. The second was an experimental cutaneous bacterial infection of human
volunteers with Haemophilus ducreyi. H. ducreyi causes chancroid, a sexually
B
Please note that CCR10 and CCR4 expression is enumerated in Fig. 3d by counting
those cells brighter than the brightest 1% of the isotype control. This results in an
underestimate of CCR4 expression by the CLA+ populations (and CCR10 by the
“effector” cutaneous population), as it is quite clear that the entire stained population is
shifted with respect to the control-stained population. Also, this method does not
consider the mean expression of receptor by positive cells, as exemplified by the
apparently modest difference in CCR4 expression between the CLA- and α4β7+
populations in Fig. 3d, which in truth have a dramatically different mean expression as
seen in Fig. 3c.
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transmitted genital ulcer disease that facilitates efficiency of HIV transmission by
disruption of epithelial barriers and infiltration of Th (CD4+) cells into the lesions36.
Essentially all of the Th cells isolated from DTH skin were of the CD45RAlo/neg
memory phenotype (Fig. 5a, top panel). Th cells from donor-matched peripheral blood
contained 40-50% CD45RAhi naïve cells (not shown), suggesting that the skin-derived
samples were not significantly contaminated with peripheral blood lymphocytes. The
number of cells expressing CLA was greatly enriched with respect to peripheral blood
memory Th cells, but the ratio of CD27/CCR7-double positives to -double negatives did
not significantly differ from that of peripheral blood memory CLA+ Th cells (Fig. 5b&c).
CCR4 was expressed by skin-infiltrating Th cells at greatly enriched levels, but,
interestingly, CCR10 was not (Fig. 5d&e).
The identical flow cytometry was performed on a total of 3 DTH and 3 H. ducreyi
lesions, and the data presented as bar graphs (Fig. 6). For both types of lesion, the ratio
of memory to naïve cells was significantly different from that of the peripheral blood Th
population from matched donors (not shown). The proportion of CLA+ and CCR4+ Th
cells was significantly increased with respect to the peripheral blood memory population.
In contrast, the ratio of CD27/CCR7 double positives to double negatives was not
significantly different from that of peripheral blood memory Th cells. Most importantly
for the present study, the proportion of CCR10+ cells was not significantly enriched with
respect to peripheral blood CLA+ memory Th cells.
DISCUSSION
We have performed a detailed analysis of functional CCR4 and CCR10
expression by peripheral blood- and skin-derived human Th cells. These studies are part
of an ongoing effort to understand the role of chemokines and their receptors in
cutaneous (and other tissue-specific) lymphocyte homing.
Peripheral blood contains a heterogeneous mixture of Th (and other)
lymphocytes, many with distinct trafficking patterns34,35. Therefore, within a given tissue,
the specific enrichment of cells bearing a particular homing molecule suggests the
molecule’s potential importance in homing to that tissue.
We confirm that lymphocytes expressing CLA and CCR4 are greatly enriched
within the cutaneous lesions studied here, which is not the case for other tissues10,15.
CCR10 is slightly enriched in skin when compared the total circulating memory Th pool.
However, CCR10 is not enriched in cutaneous sites when compared to CLA+ memory T
cells from peripheral blood. Thus CLA+/CCR4+ Th cells that express CCR10 have no
apparent advantage over their CCR10- counterparts in homing to inflamed cutaneous
sites.
One should consider the caveat that cell-surface CCR10 might indeed be
necessary for skin-infiltration, but becomes down-regulated upon entry. This is clearly
not the case for CCR4, which is expressed at comparable levels by both CLA+ blood Th
cells and skin-infiltrating Th cells10. Further, we demonstrated that CCR10 expression is
greatly enriched on cells lacking CD27 and/or CCR7 in peripheral blood (figs 3c&d), and
that the CD27/CCR7 phenotype of cutaneous-infiltrating cells is not significantly
different from that of peripheral blood CLA+ memory Th cells. Therefore, as the
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CD27/CCR7-negatives are not enriched in cutaneous sites, there is no reason to propose
that CCR10 expression has been underrepresented by this assay.
Why Are There Two “Cutaneous” Chemokine Receptors? Both CCR10 and CCR4 have
qualities that suggest a role in Th homing to cutaneous sites. However, our data suggest
that, unlike CCR4, CCR10 is not a necessary component of cutaneous homing for most
Th cells. Within the skin-homing Th population, CCR10 is associated with the “effector”
phenotype. Virtually all those cells displaying the most extreme effector phenotype (i.e.
CD27/CCR7-double-negatives) express CCR10.
In vitro studies make it clear that effector Th cells are functionally distinct from
conventional memory Th cells25-27. However, it is not clear how such cells actually
contribute to in vivo immune responses. It is possible that such effector Th cells play a
regulatory role in coordinating immune responses for the tissues through which they
traffic. For example, CLA+ cutaneous “effector” Th cells may coordinate the responses
of conventional cutaneous CLA+ memory Th cells, either positively or negatively. Part
of this role may involve coordinating the traffic of conventional Th cells through the skin,
perhaps by regulation of TARC/CCR4 interactions. If this scenario proves true, effector
Th cells would require a TARC/CCR4 independent mechanism for skin entry.
CTACK/CCR10 interactions could provide such a mechanism.
This hypothesis would predict that cells entering the skin in the absence of
TARC/CCR4 interactions (i.e., in CCR4-deficient mice, as discussed below37) would be
greatly enriched in effector Th cells. The potential effect that blocking CTACK/CCR10
interactions would have on cutaneous inflammation is less straightforward to predict. If
effector cells tend to positively regulate the intensity of skin inflammation, such blocking
might be expected to dampen the response. If, in contrast, effector cells tend to
negatively regulate inflammation, blocking of CCR10 might actually exacerbate skin
inflammation.
CCR10 and Other Types of Skin Inflammation. Recent immunohistochemistry studies
suggest that CCR10 is expressed by many cells within allergic dermatitis and psoriatic
lesions23. However, it is not clear (from this type of data) what proportion of Th cells
express CCR10 within these lesions. Therefore, it is not clear that these findings differ
significantly from our own. However, if CCR10 is indeed expressed by many Th cells in
such lesions (as suggested by the authors23), then CCR10 may play a more pervasive role
in homing to allergic dermatitis and psoriatic sites than played in the DTH and bacterial
skin lesions studied here. In such a case, one would expect the allergic dermatitis and
psoriatic sites to have more CD27/CCR7-double-negative cells than DTH and bacterial
skin lesions.
Murine Models. Two recent murine studies of DNFB-induced DTH responses have
demonstrated an inhibitory effect of anti-CTACK polyclonal antibodies on cutaneous
lymphocyte homing. In the first model (on the C57Bl/6 background37), treatment with
anti-CTACK inhibited cutaneous lymphocyte homing in CCR4-deficient but not WT
mice. This implies that signaling through both CCR4 and CCR10 must be blocked to
influence cutaneous homing through the chemokine system. In the second model (on the
Balb/c background23), treatment with very large amounts of anti-CTACK polyclonal Ab
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alone influenced cutaneous Th homing in WT mice. Such classical murine DNFBinduced models of atopic dermatitis, however, raise concerns when used to study
cutaneous Th homing in this particular case: Although large numbers of Th cells do
indeed enter such lesions, skin swelling itself (as measured by changes in ear thickness
after application of DNFB) is not T cell dependentC. Thus, antibodies to CTACK must
have a not-yet-understood influence on some component of the intrinsic immune system
to explain their effects on T-independent swelling.
Therefore, the observed influence of anti-CTACK on cutaneous lymphocyte
homing should not necessarily be interpreted as a direct effect on T cell trafficking. It is
equally plausible that the observed reduction of T cell infiltrates within DNFB-treated
skin is secondary to anti-CTACK-mediated prevention of the inflammatory response
itself, through effects on the intrinsic immune system. Further studies will be required to
resolve this issue, such as reproducing the finding in strictly T cell-dependent models of
cutaneous inflammation.
Although this manuscript addresses only the putative skin-homing Th aspects of
CCR10/CTACK interactions, it should be noted that another CCR10 ligand
(MEC/CCL28) is expressed by exocrine tissues22. This suggests that CCR10 may have
other roles in leukocyte trafficking yet to be discovered.
Conclusions. Our data support the notion that CLA and CCR4 are intimately associated
with the process by which most memory Th cells specifically enter the skin. We suggest
that CCR10 may be part of an alternative cutaneous homing pathway utilized primarily
by the less numerous “effector” subset of the Th lymphocyte pool.
C
We have demonstrated that the classical DNFB-induced ear thickness procedure (used
in both of the murine studies under discussion) produces swelling in all T-cell-deficient
mouse strains tested, including homozygous nude, scid, and Rag-1 or Rag-2 deficient
strains). In such experiments, the increases in ear thickness were indistinguishable from
those induced in wild-type mice of the same genetic background (JJC, unpublished data).
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ACKNOWLEDGEMENTS
We thank Nasim Kassam and Meghan Wells from Millennium Pharmaceuticals for
invaluable technical assistance. We thank Drs. L. Silberstein, L. Jopling, M. Wurbel and
E. Baekkevold for critical reading of the manuscript and useful discussions of the data.
We also thank Rob Hromas for instigating the Spinola Lab/Campbell Lab collaboration.
This work was supported by NIAID grant AI46784 to JJC, and AI31494, AI27863 to
SMS and M01RR00750 to the GCRC at IU. T.L.H. was supported by T32AI07367 from
the NIAID.
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FIGURE LEGENDS
Figure 1. MAb 1B5 recognizes CCR10-expressing L1/2 cell transfectants but not other
CC chemokine receptor transfectants. Cytometry histograms showing the indicated L1/2
CC chemokine receptor transfectants (CCR1-CCR11/GusB) stained with anti-CCR10
MAb 1B5. Each cell line was stained with a MAb to its corresponding receptor to ensure
high expression (not shown). Except for CCR10 cells, 1B5 staining was indistinguishable
from the isotype-matched control (IgG2a) for all other transfectants (not shown). 1B5
also recognized CCR10 expressed by CHO cells (not shown).
Figure 2. CCR10 Expression by Peripheral Blood Memory Th Subsets Defined by
Memory Markers and Tissue-Specific Adhesion Molecules. a) Cytometry plots showing
gated populations from 5-color experiment analyzed in b. Left plot: CD45RA vs. CD4
(on lymphocyte scatter gate) shows gating of naïve Th (CD4+/CD45RA+) and memory
Th (CD4+/CD45RA-). Right plot: expression of the adhesion molecules CLA and β7integrin by memory Th cells, showing cutaneous (CLA+/β7-), intestinal (CLA-/β7+) and
undefined (CLA-/β7-). b) CCR10 expression by each of the populations defined in 2a on
a representative normal healthy blood donor. CCR10 staining (black line) overlaid on
isotype-matched control staining (gray line). c) Cytometry plots showing CLA vs.
CCR10 (left plot) or CLA vs. CCR4 (right plot) in the memory Th lymphocyte gate for a
representative normal healthy donor.
Figure 3. Subpopulations of Peripheral Blood CLA+ (and other) Memory Th Cells
Express Different Levels of CCR10. a) CLA+ “cutaneous” Th cells can be further
divided into distinct subpopulations based on CD27 and CCR7 expression. Top: CD27
versus CCR7 expression on CLA+ memory Th cells. Bottom: CLA vs. CD27 (left plot)
or CLA vs. CCR7 (right plot) for a representative normal healthy donor. b) bar graph
showing proportion of CLA+ memory Th cells expressing the 4 possible permutations of
CD27 and CCR7 expression. Mean and SD of 10 normal donors shown. c) cytometry
plots of CCR10 or CCR4 expression by adhesion-receptor-defined memory subsets
further divided by CD27 and CCR7 expression on representative donor blood. CCR10
staining (black lines) overlaid on isotype-matched control staining (gray lines). Naïve,
CLA+ and CLA- plots are all from the same 6-color stain. The β7+ and β7- populations
are from a separate 6-color stain of the same donor. d) Statistical analysis of CCR10 and
CCR4 expression on 10 normal donors by subsets gated as in 3b. The difference in
CCR10 expression between each of the two CLA+ populations and all other populations
is significant (p<0.05). There is no significant difference in CCR4 expression between
the two CLA+ populations.
Figure 4. Chemotaxis of Th Subsets to CCR10 and CCR4 Ligands. Peripheral blood
lymphocytes were attracted to optimal concentrations of CTACK or TARC through 5µm
pores, and 5-color immunostaining was performed. Migration is expressed as per cent of
input (lower scale) and per cent of response to optimal concentration of SDF-1α for each
subset (upper scale). Background migration for each subset (ranging from 0.5-3.0%) has
been subtracted. Mean and SEM is shown for CD4+ subsets from three normal, healthy
donors. For each donor, migration was performed in duplicate for medium alone, SDF-
13
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1α, CTACK and TARC.
The difference in CTACK responsiveness between
+
+
CD27 /CCR7 and CD27 /CCR7- cutaneous Th cells was s significant using the MannWhitney rank-sum test (p<0.05). The difference in TARC responsiveness between these
same two populations was not significant.
Figure 5. CCR10 Expression is Rare Among Skin-Infiltrating Lymphoctes Isolated from
Candida-Induced DTH Responses. a-c) Two-color cytometry plots of lymphocytes from
DTH skin. Panel a is gated only on the lymphocyte scatter gate. Panels b&c are gated
on CD4+ cells. d&e) Single color cytometry plots of CCR10 or CCR4 expression on the
same CD4+ cells. CCR10 or CCR4 staining (gray lines) overlaid on isotype-matched
control staining (gray lines). Results shown are from a single, typical DTH experiment.
Figure 6. Analysis of Th Phenotye from Two Different Types of Cutaneous Lesion. a)
Mean and SD from 3 separate DTH reactions as shown above. b) Similar analysis of
lymphocytes derived from biopsies of chancroid pustules from cutaneous Haemophilus
ducreyi infections of 3 individual patients. For each cutaneous lymphocyte donor,
peripheral blood was compared to the database of 10 normal donors presented in Figs
2&3. The composition of peripheral blood (for each subset shown in this figure) was not
significantly different from this database using the Mann-Whitney rank-sum test (not
shown). The difference between peripheral blood and skin-infiltrating cells was
significant for the CD45RA- (p<0.05), CLA+ (p<0.05) and CCR4+ (p<0.05) populations.
The difference between peripheral blood and skin-infiltrating cells was not significant for
the CD27+/CCR7+, CD27-/CCR7-, or CCR10+ populations.
14
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REFERENCES
1. Williams MB, Rose JR, Rott LS, Franco MA, Greenberg HB, Butcher EC. The
memory B cell subset responsible for the secretory IgA response and protective humoral
immunity to rotavirus expresses the intestinal homing receptor, alpha4beta7. J Immunol.
1998;161:4227-4235
2. Rott LS, Rose JR, Bass D, Williams MB, Greenberg HB, Butcher EC.
Expression of mucosal homing receptor alpha4beta7 by circulating CD4+ cells with
memory for intestinal rotavirus. J Clin Invest. 1997;100:1204-1208
3. Kuklin NA, Rott L, Darling J, Campbell JJ, Franco M, Feng N, Muller W,
Wagner N, Altman J, Butcher EC, Greenberg HB. alpha(4)beta(7) independent pathway
for CD8(+) T cell-mediated intestinal immunity to rotavirus. J Clin Invest.
2000;106:1541-1552
4. Mackay CR. Homing of naive, memory and effector lymphocytes. Curr Opin
Immunol. 1993;5:423-427
5. Santamaria Babi LF, Perez Soler MT, Hauser C, Blaser K. Skin-homing T
cells in human cutaneous allergic inflammation. Immunol Res. 1995;14:317-324
6. Santamaria Babi LF, Picker LJ, Perez Soler MT, Drzimalla K, Flohr P, Blaser
K, Hauser C. Circulating allergen-reactive T cells from patients with atopic dermatitis
and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous
lymphocyte-associated antigen. J Exp Med. 1995;181:1935-1940
7. Berg EL, Yoshino T, Rott LS, Robinson MK, Warnock RA, Kishimoto TK,
Picker LJ, Butcher EC. The cutaneous lymphocyte antigen is a skin lymphocyte homing
receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1. J Exp
Med. 1991;174:1461-1466
8. Picker LJ, Michie SA, Rott LS, Butcher EC. A unique phenotype of skinassociated lymphocytes in humans. Preferential expression of the HECA-452 epitope by
benign and malignant T cells at cutaneous sites. Am J Pathol. 1990;136:1053-1068
9. Maly P, Thall A, Petryniak B, Rogers CE, Smith PL, Marks RM, Kelly RJ,
Gersten KM, Cheng G, Saunders TL, Camper SA, Camphausen RT, Sullivan FX, Isogai
Y, Hindsgaul O, von Andrian UH, Lowe JB. The alpha(1,3)fucosyltransferase Fuc-TVII
controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand
biosynthesis. Cell. 1996;86:643-653
10. Kunkel EJ, Campbell JJ, Haraldsen G, Pan J, Boisvert J, Roberts AI, Ebert
EC, Vierra MA, Goodman SB, Genovese MC, Wardlaw AJ, Greenberg HB, Parker CM,
Butcher EC, Andrew DP, Agace WW. Lymphocyte CC chemokine receptor 9 and
epithelial thymus-expressed chemokine (TECK) expression distinguish the small
intestinal immune compartment: Epithelial expression of tissue-specific chemokines as
an organizing principle in regional immunity. J Exp Med. 2000;192:761-768
11. Zabel BA, Agace WW, Campbell JJ, Heath HM, Parent D, Roberts AI, Ebert
EC, Kassam N, Qin S, Zovko M, LaRosa GJ, Yang LL, Soler D, Butcher EC, Ponath PD,
Parker CM, Andrew DP. Human G protein-coupled receptor GPR-9-6/CC chemokine
receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal
lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated
chemotaxis. J Exp Med. 1999;190:1241-1256
15
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
12. Campbell JJ, Bowman EP, Murphy K, Youngman KR, Siani MA, Thompson
DA, Wu L, Zlotnik A, Butcher EC. 6-C-kine (SLC), a lymphocyte adhesion-triggering
chemokine expressed by high endothelium, is an agonist for the MIP-3beta receptor
CCR7. J Cell Biol. 1998;141:1053-1059
13. Warnock RA, Campbell JJ, Dorf ME, Matsuzawa A, McEvoy LM, Butcher
EC. The role of chemokines in the microenvironmental control of T versus B cell arrest
in Peyer’s patch high endothelial venules. J Exp Med. 2000;191:77-88
14. Stein JV, Rot A, Luo Y, Narasimhaswamy M, Nakano H, Gunn MD,
Matsuzawa A, Quackenbush EJ, Dorf ME, von Andrian UH. The CC chemokine thymusderived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine,
exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T
lymphocytes in peripheral lymph node high endothelial venules. J Exp Med.
2000;191:61-76
15. Campbell JJ, Haraldsen G, Pan J, Rottman J, Qin S, Ponath P, Andrew DP,
Warnke R, Ruffing N, Kassam N, Wu L, Butcher EC. The chemokine receptor CCR4 in
vascular recognition by cutaneous but not intestinal memory T cells. Nature.
1999;400:776-780
16. Petzelbauer P, Bender JR, Wilson J, Pober JS. Heterogeneity of dermal
microvascular endothelial cell antigen expression and cytokine responsiveness in situ and
in cell culture. J Immunol. 1993;151:5062-5072
17. Campbell JJ, Brightling CE, Symon FA, Qin S, Murphy KE, Hodge M,
Andrew DP, Wu L, Butcher EC, Wardlaw AJ. Expression of chemokine receptors by
lung T cells from normal and asthmatic subjects. J Immunol. 2001;166:2842-2848
18. Kawasaki S, Takizawa H, Yoneyama H, Nakayama T, Fujisawa R, Izumizaki
M, Imai T, Yoshie O, Homma I, Yamamoto K, Matsushima K. Intervention of thymus
and activation-regulated chemokine attenuates the development of allergic airway
inflammation and hyperresponsiveness in mice. J Immunol. 2001;166:2055-2062
19. Lloyd CM, Delaney T, Nguyen T, Tian J, Martinez AC, Coyle AJ, GutierrezRamos JC. CC chemokine receptor (CCR)3/eotaxin is followed by CCR4/monocytederived chemokine in mediating pulmonary T helper lymphocyte type 2 recruitment after
serial antigen challenge in vivo. J Exp Med. 2000;191:265-274
20. Hromas R, Broxmeyer HE, Kim C, Christopherson K, 2nd, Hou YH.
Isolation of ALP, a novel divergent murine CC chemokine with a unique carboxy
terminal extension. Biochem Biophys Res Commun. 1999;258:737-740
21. Homey B, Wang W, Soto H, Buchanan ME, Wiesenborn A, Catron D, Muller
A, McClanahan TK, Dieu-Nosjean MC, Orozco R, Ruzicka T, Lehmann P, Oldham E,
Zlotnik A. Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2
(GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ILC). J
Immunol. 2000;164:3465-3470
22. Pan J, Kunkel EJ, Gosslar U, Lazarus N, Langdon P, Broadwell K, Vierra
MA, Genovese MC, Butcher EC, Soler D. A novel chemokine ligand for CCR10 and
CCR3 expressed by epithelial cells in mucosal tissues. J Immunol. 2000;165:2943-2949
23. Homey B, Alenius H, Muller A, Soto H, Bowman EP, Yuan W, McEvoy L,
Lauerma AI, Assmann T, Bunemann E, Lehto M, Wolff H, Yen D, Marxhausen H, To
W, Sedgwick J, Ruzicka T, Lehmann P, Zlotnik A. CCL27-CCR10 interactions regulate
T cell-mediated skin inflammation. Nat Med. 2002;8:157-165
16
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
24. Campbell JJ, Murphy KE, Kunkel EJ, Brightling CE, Soler D, Shen Z,
Boisvert J, Greenberg HB, Vierra MA, Goodman SB, Genovese MC, Wardlaw AJ,
Butcher EC, Wu L. CCR7 expression and memory T cell diversity in humans. J
Immunol. 2001;166:877-884
25. De Jong R, Brouwer M, Hooibrink B, Van der Pouw-Kraan T, Miedema F,
Van Lier RA. The CD27- subset of peripheral blood memory CD4+ lymphocytes
contains functionally differentiated T lymphocytes that develop by persistent antigenic
stimulation in vivo. Eur J Immunol. 1992;22:993-999
26. Hintzen RQ, de Jong R, Lens SM, Brouwer M, Baars P, van Lier RA.
Regulation of CD27 expression on subsets of mature T-lymphocytes. J Immunol.
1993;151:2426-2435
27. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of
memory T lymphocytes with distinct homing potentials and effector functions. Nature.
1999;401:708-712
28. Campbell JJ, Qin S, Bacon KB, Mackay CR, Butcher EC. Biology of
chemokine and classical chemoattractant receptors: differential requirements for
adhesion-triggering versus chemotactic responses in lymphoid cells. J Cell Biol.
1996;134:255-266
29. Campbell JJ, Foxman EF, Butcher EC. Chemoattractant receptor cross talk as
a regulatory mechanism in leukocyte adhesion and migration. Eur J Immunol.
1997;27:2571-2578
30. Campbell JJ, Qin S, Unutmaz D, Soler D, Murphy KE, Hodge MR, Wu L,
Butcher EC. Unique subpopulations of CD56+ NK and NK-T peripheral blood
lymphocytes identified by chemokine receptor expression repertoire. J Immunol.
2001;166:6477-6482
31. Spinola SM, Wild LM, Apicella MA, Gaspari AA, Campagnari AA.
Experimental human infection with Haemophilus ducreyi. J Infect Dis. 1994;169:11461150
32. Al-Tawfiq JA, Thornton AC, Katz BP, Fortney KR, Todd KD, Hood AF,
Spinola SM. Standardization of the experimental model of Haemophilus ducreyi
infection in human subjects. J Infect Dis. 1998;178:1684-1687
33. Spinola SM, Orazi A, Arno JN, Fortney K, Kotylo P, Chen CY, Campagnari
AA, Hood AF. Haemophilus ducreyi elicits a cutaneous infiltrate of CD4 cells during
experimental human infection. J Infect Dis. 1996;173:394-402
34. Butcher EC, Williams M, Youngman K, Rott L, Briskin M. Lymphocyte
trafficking and regional immunity. Adv Immunol. 1999;72:209-253
35. Campbell JJ, Butcher EC. Chemokines in tissue-specific and
microenvironment-specific lymphocyte homing. Curr Opin Immunol. 2000;12:336-341
36. Spinola SM, Bauer ME, Munson RS, Jr. Immunopathogenesis of
Haemophilus ducreyi infection (chancroid). Infect Immun. 2002;70:1667-1676
37. Reiss Y, Proudfoot AE, Power CA, Campbell JJ, Butcher EC. CC chemokine
receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK)
in lymphocyte trafficking to inflamed skin. J Exp Med. 2001;194:1541-1547
17
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Prepublished online October 24, 2002;
doi:10.1182/blood-2002-07-2348
CCR4 versus CCR10 in human cutaneous Th lymphocyte trafficking
Dulce Soler, Tricia L Humphreys, Stanley M Spinola and James J Campbell
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