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Division of Immunology, Infection and Inflammation, Faculty of Medicine, University of Glasgow, Glasgow, United Kingdom
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression on Th1
cells however suggests preferential activation of the latter in chronic
lesions, e.g., in rheumatoid arthritis synovitis (5). A
role in innate responses is also proposed. IL-18 enhances NK cell
cytotoxicity and directly induces IFN-
production by NK cells
(6). Moreover, we recently demonstrated an important role
for IL-18 in activating neutrophils and in promoting their recruitment
to the peritoneal cavity in vivo (7). IL-18 can also
directly induce monokine production by macrophages that constitutively
express IL-18R (5).
IL-18 has been implicated as a critical factor in host responses in a
broad range of infectious and autoimmune diseases. Protective Th1
responses during various bacterial and viral infections may be
abrogated or enhanced by manipulation of IL-18 expression in
IL-18-intact mice (8, 9). IL-18-deficient mice exhibit
altered responsiveness to Mycobacterium bovis,
Propionibacterium acnes, and Leishmania major,
associated with modified T cell/NK cell function (6, 10).
IL-18 mRNA is up-regulated in nonobese diabetic mice and the murine
IL-18 gene maps to the idd2 susceptibility
locus (11). Similarly, IL-18-deficiency is associated with
altered myelin oligodendrocytic glycoprotein peptide-specific
autoreactive T cell responses and amelioration of autoimmune
encephalomyelitis (12). Several compelling data now
implicate IL-18 in the pathogenesis of human inflammatory disease
states including rheumatoid arthritis
(RA),3 psoriasis,
sarcoidosis, adult onset Still’s disease, and inflammatory bowel
disease (13, 14, 15, 16). In particular, we have recently proposed
that IL-18 exerts important proinflammatory activity in RA synovial
tissues (17). IL-18-deficient mice develop significantly
reduced incidence and severity of collagen-induced arthritis
compared with wild-type mice (18) and Ab-, or
IL-18-binding protein, mediated IL-18 neutralization suppresses
streptococcal cell wall-induced arthritis and collagen-induced
arthritis (19, 20). The mechanisms whereby IL-18 mediates
effects in synovium remain unclear, but likely include modulating
articular Th1 cell responses, directly promoting macrophage
TNF- production (5) and enhancing endothelial
activation and angiogenesis (21).
We have explored the hypothesis that IL-18 can contribute to acquired
immune response development and to the maintenance of chronic immune
stimulation in diseases such as RA by promoting chemotaxis of activated
T cells. The present report documents for the first time that IL-18
induces human CD4+ lymphocyte chemotaxis. IL-18
induced T cell polarization and migration into collagen gel matrices.
Moreover, we demonstrated that such activity resides primarily in human
Th1 cells that retain high levels of IL-18R expression. RA synovial
CD4+, but not CD8+ T cells
also migrate to IL-18, suggesting that our observations have
physiologic relevance. Finally, we have shown that IL-18 can induce
local mononuclear cell recruitment in vivo. Together these data show
that IL-18 is chemotactic for T lymphocytes and as such provide a novel
mechanism whereby IL-18 can promote and sustain inflammatory
responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PE-conjugated anti-human IL-18R Ab and anti-human IL-18 Ab were obtained from R & D Systems (Abingdon, U.K.). Recombinant human IL-18 was cloned, expressed, and purified as described previously (5). Murine recombinant IL-18 was obtained from PeproTech (London, U.K.). Cytokines used were free of LPS as assessed by the Limulus amebocyte assay (Sigma-Aldrich, Poole, U.K.). FITC-conjugated anti-human CD4, CD8, CD19, CD14, CD56, and PE- and FITC-conjugated isotype control mouse IgG1 were obtained from Sigma-Aldrich. The medium routinely used was RPMI 1640 supplemented with 25 µM 2-ME (Sigma-Aldrich), penicillin (100 IU/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), and FCS (10%) (all obtained from Life Technologies, Paisley, U.K.).
Cell preparation and culture.
Heparinized venous blood was withdrawn gently from the forearm of healthy donors. PBMCs were purified by standard procedures using density gradient separation medium (Lymphoprep; Nycom Pharma, Oslo, Norway). The cells were washed extensively and either resuspended in RPMI 1640–10% FCS and used immediately for polarization assay or cultured overnight at 37°C in 5% CO2 in 1 ml of medium (2 x 106 cells/ml) in 24-well dishes in the presence or absence of staphylococcal enterotoxin B (SEB, 1 µg/ml; Sigma-Aldrich) as described previously (22). After washing, most of the cells reverted from the locomotive to spherical morphology. Human umbilical cord blood was obtained during full-term cesarean deliveries under appropriate ethical consent (North Glasgow Trust Ethical Committee, Glasgow, U.K.). CD4+ T cell populations were negatively purified from human umbilical cord blood by high-gradient magnetic sorting using the Viro MACS magnetic columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s recommendations. The depletion mixture consisted of hapten-conjugated Abs (CD8, CD11b, CD16, CD19, CD36, and CD56). Purity of CD4+ T cells was >97% as judged by flow cytometry analysis using anti-CD14, CD3, CD19, CD4, CD56, and CD8 mAbs. To prepare Th1 cell populations, freshly purified CD4+ T cells were cultured in 25-ml tissue culture flasks (Nunc, Roskilde, Denmark) at 0.5 x 106 cells/ml in the presence of PHA (1 µg/ml; Sigma-Aldrich), rIL-2 (100 ng/ml; R & D Systems), rIL-12 (2 ng/ml; BD PharMingen, San Diego, CA), and neutralizing anti-IL-4 Ab (200 ng/ml; BD PharMingen). Three days later, IL-2 (10 ng/ml) was added and the cells were harvested on day 5 for analysis. Synovial fluid was obtained from six patients with RA satisfying the American College of Rheumatology criteria (23). Mononuclear cells were separated as described above. The cells were used immediately after separation.
Flow cytometry
Surface markers of the cells were analyzed by FACSCalibur flow cytometry using CellQuest software (BD Biosciences, Mountain View, CA). To prevent nonspecific binding, all samples were preincubated for 30 min with human IgG (Sigma-Aldrich) to block the FcR. Aliquots of fresh cells or cultured cells (3 x 105 cells/tube) were suspended in buffer containing 2% FCS and 0.02% sodium azide in PBS (staining buffer) and stained for 20–30 min with specific FITC- or PE-conjugated mAbs. Cells labeled with FITC- or PE-conjugated mouse isotype-matched Abs were used as controls.
Immunofluorescence microscopy
This allows a direct phenotype visualization of polarized cells. The cells were fixed with 1% paraformaldehyde (Sigma-Aldrich) for 10–15 min to retain locomotor morphology, then attached to poly-L-lysine-coated coverslips (2 mg/ml; Sigma-Aldrich), and stained with appropriate FITC- or PE-labeled markers. Marker-positive and -negative, polarized and spherical cells were counted using a x40 oil phase-contrast objective of a Zeiss Axioskop fluorescence microscope (Zeiss, Oberkochen, Germany).
Cytospin staining
Cell morphology using Giemsa-stained preparations is a useful preliminary to phenotype synovial cells (n = 6 patients). The morphology of these cells was observed under the optical microscope: neutrophils (77.50 ± 1.50%), lymphocytes (17.33 ± 1.23%), and monocytes (5.17 ± 0.6%).
Polarization assay
This assay measures the change from a spherical shape to the shape change characterized by head-tail polarity, typical of locomotive cells (24). Freshly isolated or overnight-cultured PBMCs were resuspended in RPMI 1640 medium supplemented with 10% FCS and mixed with human IL-18 or IL-15 at concentrations ranging from 0.1 to 1 µg/ml. Experiments were conducted using polystyrene round-bottom tubes (110 x 16 mm; Sterilin, Stone, U.K.). The tubes, containing 200 µl of cell suspension at a concentration of 2 x 106/ml, were incubated for 30 min at 37°C. A basal control value was established using RPMI 1640-FCS alone. The cells were fixed using 200 µl of 2.5% glutaraldehyde (Sigma-Aldrich) and the proportions of cells showing head-tail polarization typical of locomotive cells were determined. The proportion of cells scored as either spherical (nonmotile) or polarized (motile) was counted directly using a x40 phase-contrast objective. Cells 250–300(250–300) were counted blind, and polarized cells were expressed as a percentage of viable cells. Data presented are values after subtracting the background values (without cytokine), which ranged from 5 ± 0.8% for freshly isolated cells, 18 ± 1.7% for cells cultured with medium alone overnight, and 23 ± 2.3% for cell cultured with SEB.
Collagen gel invasion assay
Rat tail collagen (type I) was prepared in solution from freshly obtained rat tail tendons by established methods (25). Gels were formed by bringing soluble collagen (1.5–2 mg/ml) in dilute acetic acid solution back to physiologic pH and osmolarity. IL-18 or FCS in culture medium was added before gelatinization. The mixture was then transferred into 24-well dishes (Sterilin) and allowed to gel. Cells were prepared and activated with appropriate activators as described above. They were washed and overlaid on the gel in 24-well dishes and incubated at 37°C for 20 h (unless otherwise stated) to allow cells to invade the gel. The proportion of invading cells was determined with an inverted microscope by scoring the number of cells remaining on top of the gel and the number of cells that had penetrated the gel, counting a minimum of 200 cells. The proportion of locomotor cells was calculated from the ratio of the numbers of invasive and noninvasive cells. To distinguish between chemotaxis and chemokinesis, IL-18 was also added on top of the collagen gel and also in the gel and incubated for 20 h.
Measurement of IL-5, IFN-, and IL-18
IL-5 and IFN- concentrations in culture supernatants were
determined by ELISA using paired Abs (BD PharMingen) and developed with
tetramethylbenzidine peroxidase substrate (Insight
Biotechnology, Middlesex, U.K.) according to the manufacturer’s
recommendations. Sensitivity of the assay was 10 pg/ml. IL-18 in
synovial fluid was similarly determined using paired Abs (Diaclone
Research, Besancon, France). Sensitivity of the assay was 45 pg/ml.
IL-18 levels in RA synovial fluids were up to 155.4 ± 28.7 pg/ml
(mean ± SEM, n = 6).
IL-18-induced cellular migration in vivo
This was conducted as previously described (26, 27). Briefly, male DBA/1 mice (Harlan Olac, Oxon, U.K.) and IL-18-deficient mice of the DBA/1 background (10, 18), age 10 wk, were injected i.p. with 500 µg of P. acnes and 7 days later received 500 ng of murine rIL-18 or PBS in the hind footpad. Footpad swelling was measured at 0, 8, 15, and 24 h after injection using a constant pressure dial caliper (Kroeplin, Munich, Germany). Following the measurement, the mice were sacrificed and the footpads were removed, Formalin fixed, and analyzed histologically by H&E staining. All mice were kept in the Biological Service facilities at the University of Glasgow according to the U.K. Home Office guidelines.
Immunohistology of synovial membrane
Synovial membranes (arthoplasty specimens) were obtained from RA
patients satisfying the American College of Rheumatology diagnostic
criteria (n = 12). Formaldehyde-fixed paraffin sections
(3 µm) were stained by a standard streptavidin-HRP protocol using an
anti-IL-18 mAb (a kind gift from Dr. A. Jackson, Cancer Research
U.K., Leeds, U.K.) (5). Product was visualized with
3,3'-diaminobenzidine tetrachloride (Sigma-Aldrich) and counterstained
with hematoxylin (Sigma-Aldrich). IL-18 expression was quantified by
two independent observers using light microscopy. For verification of
specificity, the anti-IL-18 Ab was replaced by an isotype-matched
control (Sigma-Aldrich). Neutralization was performed by preincubation
of primary anti-IL-18 Ab with either 1 µg of rIL-18 or IL-1
(R
& D Systems) before tissue staining.
Statistical analysis
Values are expressed as mean ± SEM. Statistical analysis was performed using Student’s t test. A p < 0.05 was considered to be significant.
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Results |
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Lymphocyte shape change (polarization) and migration into collagen
gels represent useful indicators of chemokinetic activity. We therefore
determined the proportion of PBMCs exhibiting polarization in response
to increasing concentrations of IL-18. IL-18 induced no reproducible
effect on freshly isolated lymphocyte populations (Fig. 1a) reflecting low levels of
spontaneous IL-18R expression (data not shown). It has been shown
that SEB promoted cytokine-mediated lymphocyte polarization
(22). We therefore stimulated PBMCs with SEB to enhance
cell polarization. After overnight culture in SEB, PBMCs polarized to
IL-18 in a dose-dependent manner that was similar to the response
induced by IL-15, a recognized T cell chemoattractant
(23). Maximal polarization was seen with 100 ng/ml IL-18;
at this dose, 
20% of total PBMCs were polarized (Fig. 1a). Flow cytometric analysis of the PBMC fraction contained
65 ± 5.6% CD3+ cells (n =
5), 6.8 ± 0.7% CD19+ cells
(n = 5), 8.5 ± 0.5% CD14+
cells (n = 2), and 96% of the cells within the
lymphocyte gate expressed CD45 (data not shown). Immunofluorescence
microscopy analysis showed that the polarized cells were predominantly
CD3+CD4+ T cells, with few
CD8+ T cells (<5%) assuming polarized
morphology (data not shown). This is not
unexpected since CD4+ but not
CD8+ cells are responsive to SEB
(28) Commensurate with this, cells cultured for 16 h
in SEB, but not in medium alone, increased the expression of IL-18R
on CD4+ cells (up to 40% of total
CD4+ T cell population in PBMCs measured by flow
cytometry, data not shown). The polarized and nonpolarized cells are
shown in Fig. 1b. We also investigated the ability of the
mononuclear cells from human cord blood (which contained principally
naive lymphocytes) to polarize in response to IL-18. Cord blood cells
polarized in a similar manner as PBMCs in response to IL-18 following
activation by SEB, but did not respond to IL-18 when they were not
activated with SEB (Fig. 1c). A similar percentage of
polarized cells was obtained whether purified
CD4+ T cells or PBMCs were examined (data not
shown). As specificity control, we further showed that
neutralizing monoclonal anti-human IL-18 Ab abolished the
chemotactic effects of IL-18 (Fig. 1d). SEB-activated
lymphocytes polarized within 5 min of IL-18 stimulation and reached
maximal levels after 30 min (Fig. 1e). Although measurement
of shape change is a reliable correlate of chemoattraction/locomotion,
the assay does not directly measure locomotion. To formally demonstrate
this, we used the collagen gel invasion assay. PBMCs were cultured
overnight in SEB, washed to remove the supernatant, and overlaid on a
collagen gel previously impregnated with IL-18 (or diluent alone) in
medium/10% FCS. The percentage of cells migrating into collagen gels
in response to IL-18 after 20 h was significantly higher than that
in the control gels (Fig. 1f). Moreover, IL-12 (at a range
of concentrations) did not have any activity in both polarization and
gel invasion assays (data not shown), suggesting that this activity is
not a general property of innate response monokines. These data
therefore demonstrate clearly that IL-18 can rapidly induce not only
polarization but also invasive locomotion in PBMC subsets.
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Since we have previously shown that IL-18R is preferentially
expressed on committed Th1 cells (29), we next explored
the hypothesis that IL-18 induced locomotion in Th1 cells.
CD4+ cells purified from human cord blood were
cultured under Th1 differentiating conditions. Flow cytometry showed
that whereas naive cord blood CD4+ T cells
expressed a low level of IL-18R, 90% of committed Th1 cells
expressed the receptor (data not shown). Predominant IFN- (1.1
± 0.1 ng/ml), but not IL-5 (<30 pg/ml), production confirmed
functional Th1 differentiation (data not shown). IL-18
induced human Th1 cell migration into collagen gels in a dose-dependent
manner (Fig. 2a). However,
only 30% of the Th1 cells migrated into the gel in response to 200
ng/ml IL-18. This is consistent with earlier reports that lymphocytes
must enter G1 phase for full expression of a
locomotor phenotype (30). Cell cycle analysis by flow
cytometry showed that 60% of the committed Th1 cells were in the
G0-G1 phase at the time of
the migration assay (data not shown). Time course analysis comparing
the proportion and migration distance of cells into collagen gels
containing 200 ng/ml IL-18 or medium alone showed increasing responses
up to 18 h (Fig. 2, b and c). To confirm
that the migration was not due to random chemokinesis, the gel invasion
assay was performed with or without an IL-18 concentration gradient
(Fig. 3d). Maximal Th1 cell
migration occurred in a positive IL-18 concentration gradient between
the top of the gel and inside the gel (chemotactic condition). In
contrast, no significant migration was observed in the absence of an
IL-18 gradient (chemokinetic condition) or in the absence of IL-18
(spontaneous migration). These results therefore demonstrate that IL-18
induced primarily a chemotactic response. We also compared the relative
responsiveness of Th1 and Th2 cells to IL-18-induced polarization/cell
migration. CD4+ T cells from human cord blood
were driven to Th1 or Th2 lineages as described in Materials and
Methods. Although relatively pure Th1 lines were easily obtained,
a pure Th2 line proved difficult to establish. Viability was low after
two rounds of cultivation. The Th2 lines retained a substantial and
variable percentage of IL-18R+ IFN- producing
cells following a single round of driving under the Th2 condition.
These cells polarized and migrated in response to IL-18. However, there
was a marked decrease in the percentage of
IL-18R+ cells after the second round of driving
and this decrease was clearly reflected in the substantial decrease in
the percentage of cells migrating in response to IL-18 (data not
shown). It should be noted that although committed Th2 cells are
generally IL-18R-, Th0 cells and differentiating
Th2 cells express a low level of IL-18R (29). Therefore,
as much as IL-18R is preferentially expressed on committed Th1 but not
Th2 cells, IL-18 polarizes Th1 rather than Th2 cells. It should also be
noted that we have reported earlier that IL-18 attracts the migration
of neutrophils, which also express IL-18R (7).
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SEB represents only a surrogate for physiologic activation of
lymphocytes. We therefore performed locomotion assays on lymphocytes
that were activated in vivo. IL-18 expression is up-regulated in RA
synovial tissues (5), particularly in lymphocyte-rich
aggregates and adjacent to endothelia (Fig. 3). Mononuclear cells were
prepared from synovial fluids freshly aspirated from RA patients and
tested for their ability to polarize and migrate in the presence of
graded concentrations of IL-18. Lymphocytes polarized in response to
IL-18 in a dose-dependent manner (Fig. 4a). Similar results were
obtained when lymphocytes were tested for their ability to migrate
through collagen gels (Fig. 4b). Synovial lymphocyte
fractions differ functionally and with respect to IL-18R expression.
The expression of IL-18R on CD4+ and
CD8+ synovial T cells was measured by FACS and
revealed that whereas >50% of CD4+ synovial T
cells expressed IL-18R, <10% of CD8+ T cells
were IL-18R+ (Fig. 4, c and
d). To investigate which cell subset(s) was the targets of
the IL-18-mediated chemotaxis ex vivo, immunofluorescence microscopy
was used to identify the responder cells. CD4+,
but not CD8+, synovial T cells polarize
significantly to IL-18 compared with control medium (Fig. 4e). Moreover, CD19+ synovial B cells
did not express IL-18R and did not respond to IL-18 in a
polarization assay (data not shown).
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The above data suggested that IL-18 was a potent chemoattractant for CD4+ T cells in vitro, raising the possibility that IL-18 may recruit T cells into local tissues during inflammatory responses. To investigate this, female DBA/1 mice were primed with P. acnes and then injected s.c. into a hind footpad with 500 ng of recombinant murine IL-18 or PBS alone. Mice injected with IL-18 developed significant footpad swelling compared with PBS-injected controls (Fig. 5). Moreover, IL-18 injection into footpads of IL-18-deficient mice, which express higher levels of IL-18R a priori (6, 10), led to significantly greater footpad swelling than IL-18-injected wild-type mice. Subsequent histologic analysis showed a substantial mononuclear infiltrate at the site of injection, whereas PBS-injected footpads were similar to noninjected controls (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression
as a phenotypic feature, although we cannot rule out similar effects
manifest upon differentiating Th2 cells that transiently retain
IL-18R expression through earlier stages of maturation.
"Resting" IL-18 expression at the mRNA and protein (at least as
pro-IL-18) levels has been observed in several tissues and cell types.
By virtue of its early presence following tissue insult, the
chemotactic properties demonstrated herein support a dual role for
IL-18 in T cell activation. It may function as an important mediator in
determining the nature of memory T cells recruited (preferentially
IL-18R+CD4+ Th1 cells)
at the same time as influencing, in synergy with IL-12 or IL-4, the
functional destination of naive T cells present within a lesion.
Importantly, we found no evidence of IL-18R expression on naive
human T cells derived from cord blood nor any motile response by such
naive cells to IL-18, and it is therefore unlikely that IL-18 has any
significant role in recruitment of such T cells. It will be important to determine the relationship that IL-18-induced T cell recruitment has to that mediated by other established chemokine receptor-mediated pathways, in particular those operating via CCR5, a putative Th1 cell marker (31). That IL-18-deficient mice generate inflammatory responses has been clearly demonstrated in several models (6, 10), indicating that IL-18 expression is not obligatory for host defense. Nevertheless, several reports suggest that IL-18 deficiency does prejudice host responses to some microbial species. Moreover, excess expression of IL-18 is sufficient to initiate or considerably enhance autoimmune responses. Transgenic cutaneous overexpression of caspase 1 induces high levels of IL-18 expression in the skin and leads to development of a marked local inflammatory infiltrate that is dependent in large part upon the presence of IL-18 (32). Similarly, IL-18 injection into male MRL/lpr mice considerably accelerates the development of autoimmune glomerulonephritis and intriguingly induces a pronounced cutaneous inflammatory infiltrate similar to that observed in caspase 1-transgenic mice (33). IL-18 administration is sufficient to induce inflammatory arthritis in collagen/IFA-primed DBA/1 mice (34). We now show that local introduction of recombinant mature IL-18 in vivo into a footpad is sufficient to induce a marked mononuclear cell infiltrate. The precise mechanisms whereby IL-18 mediates such effects in vivo require further analysis. Although the in vitro time course results (IL-18-induced T cell polarization within 5 min) presented herein render it unlikely, our data do not exclude the possibility that IL-18 could operate through inducing release of intermediate factors, including chemokines. This possibility is currently being investigated.
IL-18 may exert chemotactic responses in other cell lineages. Chemokinetic responses to IL-18 for endothelial cells may in part underpin the proangiogenic effects of IL-18 in several neovascularization models (21). We have also shown that IL-18 may promote neutrophil expression of adhesion molecule expression and recruitment to the peritoneal cavity in vivo (7). IL-18 has also been shown to up-regulate ICAM-1 expression on monocytes and on T cells, providing further mechanisms whereby IL-18 can promote T cell recruitment (35, 36).
Our data are of particular significance in determining the potential
activities of IL-18 in chronic inflammatory diseases such as RA. IL-18
detection in inflammatory sites is characterized by high levels of
expression in mononuclear cells adjacent to endothelial cells,
providing circumstantial evidence that IL-18 is released in a
geographically cogent area of an inflammatory lesion to facilitate
further T cell recruitment (5). However, IL-18 could also
operate within an inflamed tissue irrespective of
trans-endothelial recruitment. We, and others, have recently
proposed that recruitment of memory T cells and their subsequent
cytokine-mediated activation within inflamed synovial membrane can
drive downstream TNF- release and consequent pathology (5, 37). Critical to this are cell contact-dependent interactions
between activated T cells and macrophages that in turn promote
proinflammatory cytokine release (38). In unpublished
studies we have shown that IL-18 can considerably enhance T
cell/macrophage cell contact-mediated cytokine release (J. A.
Gracie and I. B. McInnes, unpublished data). It is attractive to
propose that IL-18 released by macrophages generates microgradients
within synovial tissue that then optimizes further T cell recruitment
and activation adjacent to the macrophage, thereby perpetuating the
chronic inflammatory process.
In conclusion, we provide here evidence that IL-18 has chemotactic activities in vitro and in vivo operating in particular upon human Th1 cells. Thus, in addition to its activities in functional regulation of naive T cell differentiation, IL-18 may have an important role in promoting recruitment of memory Th1 cells to an inflammatory lesion. This adds further to the portfolio of activities that render IL-18 an attractive therapeutic target in a variety of important autoimmune inflammatory disease states.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Foo Y. Liew, Department of Immunology and Bacteriology, Western Infirmary, University of Glasgow, Glasgow G11 6NT, U.K. E-mail address: f.y.liew{at}clinmed.gla.ac.uk
3 Abbreviations used in this paper: RA, rheumatoid arthritis; SEB, staphylococcal enterotoxin B.
Received for publication July 12, 2002. Accepted for publication November 8, 2002.
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B. A. Zabel, A. M. Silverio, and E. C. Butcher Chemokine-Like Receptor 1 Expression and Chemerin-Directed Chemotaxis Distinguish Plasmacytoid from Myeloid Dendritic Cells in Human Blood J. Immunol., January 1, 2005; 174(1): 244 - 251. [Abstract] [Full Text] [PDF]
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 L. A.B. Joosten, R. L. Smeets, M. I. Koenders, L. A.M. van den Bersselaar, M. M.A. Helsen, B. Oppers-Walgreen, E. Lubberts, Y. Iwakura, F. A.J. van de Loo, and W. B. van den Berg Interleukin-18 Promotes Joint Inflammation and Induces Interleukin-1-Driven Cartilage Destruction Am. J. Pathol., September 1, 2004; 165(3): 959 - 967. [Abstract] [Full Text] [PDF]
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 A. Kaser, S. Kaser, N. C. Kaneider, B. Enrich, C. J. Wiedermann, and H. Tilg Interleukin-18 attracts plasmacytoid dendritic cells (DC2s) and promotes Th1 induction by DC2s through IL-18 receptor expression Blood, January 15, 2004; 103(2): 648 - 655. [Abstract] [Full Text] [PDF]
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R. Gutzmer, K. Langer, S. Mommert, M. Wittmann, A. Kapp, and T. Werfel Human Dendritic Cells Express the IL-18R and Are Chemoattracted to IL-18 J. Immunol., December 15, 2003; 171(12): 6363 - 6371. [Abstract] [Full Text] [PDF]
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 P. E. Szmitko, C.-H. Wang, R. D. Weisel, J. R. de Almeida, T. J. Anderson, and S. Verma New Markers of Inflammation and Endothelial Cell Activation: Part I Circulation, October 21, 2003; 108(16): 1917 - 1923. [Full Text] [PDF]
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) or PBMCs cultured
overnight with SEB or medium alone (no added SEB) were stimulated with
graded concentrations of IL-18 or IL-15 for 30 min and fixed, and the
cell shape change was examined under phase-contrast microscopy as
described in Materials and Methods. Data are mean
± SEM of percent polarized cells of four experiments.
b, PBMCs were cultured overnight with SEB and activated
with 200 ng/ml IL-18 for 30 min and fixed, and the polarized (filled
arrow) and nonpolarized (open arrow) cells were scored under
phase-contrast microscopy. c, Mononuclear cells from
human cord blood cultured overnight with SEB or freshly isolated were
also tested for polarization in response to IL-18. d,
IL-18-induced polarization of SEB-cultured PBMCs can be blocked by
anti-IL-18 Ab. e, Polarization of SEB-cultured PBMCs
was evident 5 min after IL-18 (500 ng/ml) stimulation and peaked at 30
min. d and e, The background value was
not subtracted. f, SEB-cultured PBMCs also migrated in
significantly higher numbers into collagen gel containing IL-18 (500
ng/ml) than into gel containing medium alone. Data are the mean ±
SEM of three experiments. *, p < 0.05; **,
p < 0.01. More than 200 cells were scored in each
experiment.
