| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Centre for Inflammatory Diseases, Department of Medicine Monash Medical Centre, Monash University, Clayton, Victoria, Australia; and
Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., Fujisaki, Okayama, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-inducing factor) enhances Th1
responses via effects that are thought to be dependent on and
synergistic with IL-12. The potential for IL-18 to exert
IL-12-independent effects in delayed-type hypersensitivity (DTH)
responses was studied in a model of Th1-directed, DTH-mediated
crescentic glomerulonephritis induced by planting an Ag in glomeruli of
sensitized mice as well as in cutaneous DTH. Sensitized genetically
normal (IL-12+/+) mice developed proteinuria and crescentic
glomerulonephritis with a glomerular influx of DTH effectors
(CD4+ T cells, macrophages, and fibrin deposition) in
response to the planted glomerular Ag. IL-12p40-deficient
(IL-12-/-) mice showed significant reductions in crescent
formation, proteinuria, and glomerular DTH effectors. Administration of
IL-18 to IL-12-/- mice restored the development of
histological (including effectors of DTH) and functional glomerular
injury in IL-12-/- mice to levels equivalent to those in
IL-12+/+ mice. IL-18 administration to
IL-12-/- mice increased glomerular ICAM-1 protein
expression, but did not restore Ag-stimulated splenocyte IFN-,
GM-CSF, IL-2, or TNF-
production. Sensitized IL-12+/+
mice also developed cutaneous DTH following intradermal challenge with
the nephritogenic Ag. Cutaneous DTH was inhibited in
IL-12-/- mice, but was restored by administration of
IL-18. IL-12+/+ mice given IL-18 developed augmented
injury, with enhanced glomerular and cutaneous DTH, demonstrating the
synergistic effects of IL-18 and IL-12 in DTH responses. These studies
demonstrate that even in the absence of IL-12, IL-18 can induce in vivo
DTH responses and up-regulate ICAM-1 without inducing IFN-, GM-CSF,
or TNF- production. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-inducing factor) was described and cloned in
1995 (1). Mononuclear cells and APC produce IL-18 during
interactions with cognate T cells (2). There is growing
evidence to support a role for IL-18 in the expression of Th1 responses
(1, 3, 4, 5, 6). IL-18 induces the production of IFN- and
other proinflammatory cytokines. In an established Th1 clone, the
levels of IFN- induced by IL-18 were, in fact, higher than those
induced by IL-12, suggesting a potential role for IL-18 in the
maintenance of Th1 responses (1). IL-18 acts in synergy
with IL-12 to induce IFN- production in CD4+
cells (3), and both IL-12 and IL-18 are necessary for the
full expression of Th1 responses. Further support for this synergistic
effect comes from the observation that IL-12 and IL-18 use different
signaling pathways in inducing IFN- production (7).
IL-18-/- mice are deficient in IFN-
production, examined by ex vivo IFN- production by
CD4+ cells in response to established
Th1-inducing stimuli (5). The extent of their deficiencies
is comparable to that exhibited by
IL-12-/- mice.
It is not clear whether IL-18 alone is capable of inducing Th1
responses (of which the delayed-type hypersensitivity
(DTH)3 response is the
classical example (8)). However, most work indicates that
IL-18 cannot induce a Th1 response in the absence of IL-12. Culturing
naive cells from IL-12-/- mice in the presence
of IL-18 does not result in the development of IFN--producing Th1
effector cells (5). This supports other in vitro data that
implicate IL-18 as a cofactor rather than an initiator of Th1
development (2, 9).
The recruitment of leukocytes in DTH responses involves tethering and
migration of cells into the target organ. Adhesion molecules and
chemokines are important in the effector phase of DTH responses
(10, 11, 12, 13). IL-18 has the potential to induce the expression
and production of adhesion molecules and chemokines, which could alter
the expression and magnitude of the DTH response (14, 15).
In particular, IL-18 has been shown to induce ICAM-1 expression on a
human myelomonocytic line, an effect that is IFN- independent
(14).
The potential for IL-12-independent actions of IL-18 in inducing and
maintaining Th1-driven DTH-like responses in vivo was studied in
experimental crescentic anti-glomerular basement membrane (GBM)
glomerulonephritis (GN), a model of Th1-directed DTH-mediated immune
injury. Crescentic GN is the most aggressive and damaging form of GN
(16). In humans, crescent formation has features of DTH,
with the consistent observation of T cells, macrophages, tissue factor,
and fibrin in the glomerular lesion (17, 18). In the
murine model used in the current studies, glomerular crescent formation
is effector CD4+ (but not
CD8+) dependent and is independent of the
presence of autologous Ab (19, 20, 21). Studies in murine
models have demonstrated that this DTH-like lesion in the glomerulus is
Th1 directed. Glomerular crescent formation and the influx of
CD4+ T cells, macrophages, and fibrin are
associated with a Th1-predominant response in C57BL/6 mice
(19). Glomerular crescent formation is inhibited by IL-12
or IFN- neutralization (19, 22) or by the
administration of the counter-regulatory cytokines IL-4 and IL-10
(23, 24). IL-12 administration, or the absence of
endogenous IL-4 or IL-10, enhances renal injury and crescentic GN
(22, 25, 26). The relevance of adhesion molecules and
chemokines in this model has been demonstrated by studies showing that
severe injury and crescent formation in this and similar models are
ICAM-1 and monocyte chemoattractant protein-1 dependent
(27, 28, 29). Therefore, this model of crescentic glomerular
injury provides a relevant and appropriate tool with which to study the
IL-12-independent role of IL-18 in vivo.
The hypothesis that IL-18 administration would induce DTH in IL-12 p40-/- mice was addressed by inducing GN by planting an Ag, sheep globulin, in glomeruli of sensitized mice and studying the resultant renal injury, dermal DTH to sheep globulin, and systemic immune response in three groups of mice. These parameters were studied in IL-12+/+ mice given vehicle alone, in vehicle-treated IL-12-/- mice, and in IL-12-/- mice given IL-18. Administering IL-18 to IL-12+/+ mice with GN tested a second hypothesis, that IL-18 would exacerbate glomerular injury in genetically normal mice.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Sheep anti-mouse GBM globulin was prepared as previously described (30). Eight- to 10-wk old male C57BL/6 mice (Monash University Animal Services, Melbourne, Australia) were assigned to groups that received IL-18 (IL-12+/+ + IL-18; n = 9) or vehicle alone (IL-12+/+ Ctrl; n = 9). Two mice that received IL-18 had to be killed on days 5 and 6 of GN because of poor condition with nephrotic syndrome: these mice had ascites and peripheral edema and are included in analyses of glomerular injury. IL-12 p40 subunit-deficient mice (31) (IL-12-/- mice; obtained from The Jackson Laboratory (Bar Harbor, ME) and bred at Monash University) received either IL-18 (IL-12-/- + IL-18; n = 6) or vehicle alone (IL-12-/- Ctrl; n = 6). Glomerulonephritis was induced by sensitizing mice by s.c. injection of a total of 100 µg of sheep globulin in 100 µl of CFA in divided doses in each flank (day -10). Ten days later (day 0), GN was initiated by i.v. challenge with 2.5 mg of sheep anti-mouse GBM. Glomerular injury and immune responses were assessed after a further 7 days (day 7). IL-18 was diluted in sterile, filtered 0.1% BSA in PBS and administered as follows; 1 µg in 200 µl of 0.1% BSA/PBS was injected i.p. on each of days -10, -9, and -8. A further 0.5 µg in 100 µl of vehicle was injected i.p. daily on days 1–5 of disease. Control mice received equivalent volumes of 0.1% BSA/PBS alone. An additional three groups of mice were studied to assess the effect of IL-18 on glomeruli of mice without GN: normal mice without GN (n = 6), mice sensitized with sheep globulin but not challenged that received IL-18 in the same protocol as mice with GN (n = 4), and mice sensitized with sheep globulin but not challenged that were injected with vehicle alone (n = 3). Results are expressed as the mean ± SEM. The statistical significance of differences between groups was determined by the Mann-Whitney U test.
Assessment of renal injury
Kidney tissue was fixed in Bouin’s fixative and embedded in paraffin, and 3-µm tissue sections were cut and stained with periodic acid-Schiff’s reagent (PAS). A glomerulus was considered to exhibit crescent formation when two or more layers of cells were observed in Bowman’s space. A minimum of 50 glomeruli were assessed to determine the crescent score for each animal. For deposition of PAS+ material in glomeruli, a 0–4+ scale was used as follows: 0, no accumulation of PAS+ material; 1, minor accumulation to cover up to 25% of the glomerular tuft; 2, PAS+ material in 25- 50% of the tuft; 3, 50–75% of the tuft involved; and 4 <75% involved, with relative glomerular hypocellularity. Assessments were performed on coded slides. Urinary protein concentrations were determined by the method of Bradford (32) on 24-h urine collections from each mouse from days 1 and 2 of disease.
Immunohistochemistry for T cells, macrophages, NK cells, and ICAM-1
Kidney or footpad tissue was fixed in periodate lysine paraformaldehyde (PLP) for 4 h, washed in 7% sucrose solution, then frozen in liquid nitrogen. Tissue sections (6 µm) were stained to demonstrate CD4+ cells, CD8+ cells, and macrophages using a three-layer immunoperoxidase technique, as previously described (33, 34, 35). The primary mAb were GK1.5 (anti-mouse CD4, American Type Culture Collection, Manassas, VA), 53-6.7 (anti-mouse CD8, American Type Culture Collection), and M1/70 (anti-mouse Mac-1, American Type Culture Collection). For the detection of NK cells, sections were first incubated with 10% swine serum in 5% BSA/PBS. A rabbit anti-mouse/rat asialo-GM1 Ab (Cedarlane, Hornby, Canada) (15) was applied for 1 h (dilution, 1/200), followed by a swine anti-rabbit HRP-conjugated Ab (Dako, Carpinteria, CA; 1/100). To quantify glomerular CD4+ T cells, macrophages, and NK cells, a minimum of 20 glomeruli was assessed per animal, and results were expressed as cells per glomerular cross-section (c/gcs), as previously reported (23). ICAM-1 was detected on cryostat-cut, 6-µm-thick, unfixed frozen sections using a hamster anti-mouse CD54 mAb (clone 3E2, 1/100 dilution; PharMingen, San Diego, CA). The secondary Ab was biotinylated mouse anti-hamster IgG (clones G70-204 and G94-56, 1/100 dilution; PharMingen). Endogenous peroxidase was blocked by incubating sections with 0.03% H2O2 in PBS, and endogenous biotin was blocked with a commercially available kit (Dako, Carpinteria, CA). To assess glomerular ICAM-1 staining, at least 20 glomeruli were scored in each mouse, using a scale of 0–4+ on the basis of the intensity of staining, with 0 being an intensity similar to that seen in normal mice and 4 being heavy staining over the entire glomerular tuft.
Glomerular deposition of fibrin, sheep globulin, and mouse Ig
Tissue was embedded in Optimal Cutting Temperature Compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at -70°C. Immunofluo-rescence was performed on 4-µm cryostat cut tissue. Glomerular fibrin deposition was detected on a minimum of 30 glomeruli/mouse using an FITC-anti-mouse fibrin/fibrinogen polyclonal Ab (Nordic Immunological Laboratories, Berks, U.K.) at a dilution of 1/50. Fibrin deposition was scored semiquantitatively (0–3+) as follows: 0, no fibrin deposition; 1, fibrin occupying up to one-third of the glomerular cross-sectional area; 2, fibrin occupying one-third to two-thirds of the glomerulus; and 3, greater than two-thirds of the glomerular cross-section covered by fibrin.
Deposition of sheep globulin was evaluated using FITC-rabbit anti-sheep Ig (Nordic Immunological Laboratories) at a dilution of 1/1000, and sections were scored 0–3+ on the basis of fluorescence intensity. There were no differences in the deposition of the nephritogenic Ag, sheep globulin, in the glomeruli of IL-12+/+ mice (IL-12+/+ Ctrl, 1.7 ± 0.2; IL-12+/+ + IL-18, 1.9 ± 0.3) or IL-12-/- mice (IL-12-/- Ctrl, 1.8 ± 0.2; IL-12-/- + IL-18, 2.2 ± 0.3). Two methods were used for assessment of mouse Ig (FITC-sheep anti-mouse Ig; Silenus, Hawthorn, Australia) in glomeruli. Firstly, fluorescence intensity was assessed semiquantitatively (0–3+) using a dilution of 1/150 for mouse Ig. Secondly, serial dilutions of these Abs were made to determine the end-point positive titer in each animal, and results were expressed as the log2 of this end-point titer.
Cutaneous DTH to sheep globulin
To assess the cutaneous DTH response to sheep globulin, mice were challenged 24 h before the end of the experiment by intradermal injection of sheep globulin (500 µg in 50 µl of PBS) into the plantar surface of a hindfoot. Four mice from each of the IL-12+/+ mouse groups, and all six mice from each of the IL-12-/- groups were challenged with sheep globulin. The same dose and volume of an irrelevant Ag (horse globulin) were injected into the opposite footpad as a control. DTH was assessed 24 h later in a blinded fashion by measuring the difference between sheep globulin- and horse globulin-injected foot pads in each mouse using a micrometer. Footpads were dissected and fixed in PLP as described above.
Measurement of cytokine production by Ag-stimulated splenocytes
Spleens from diseased mice (IL-12+/+ Ctrl,
n = 9; IL-12-/- Ctrl,
n = 6; IL-12-/- + IL-18,
n = 6; IL-12+/+ + IL-18,
n = 6) were removed aseptically on day 7 and placed in
DMEM/5% FCS medium. Single-cell suspensions were prepared by gently
teasing tissue apart. Erythrocytes were lysed by incubation in Boyle’s
solution (0.17 M Tris/0.16 M ammonium chloride) for 1 min at 37°C.
Cell suspensions were washed in DMEM/5% FCS. Splenocytes (4 x
106 cells/ml, in DMEM/10% FCS) were incubated
for 72 h at 37°C in 5% CO2 in 48-well
tissue culture plates (1 ml/well) with protein G-purified normal sheep
IgG at a concentration of 10 µg/ml. IFN- and IL-4 in culture
supernatants were measured by ELISA as previously described
(23). The mAbs used were rat anti-mouse IFN- (R46A2
(PharMingen, San Diego, CA) and biotinylated XMG1.2 (PharMingen) for
the IFN- ELISA) and rat anti-mouse IL-4 (11B11; American Type
Culture Collection) and biotinylated BVD6 (DNAX Research Institute,
Palo Alto, CA) for the IL-4 ELISA. IL-2 was measured using a similar
protocol, with the mAbs being rat anti-mouse IL-2 (JES6-1A12 (DNAX)
and biotinylated JES6-5H4 (DNAX)). TNF- and GM-CSF were measured
using commercially available immunoassays (for TNF-: DuoSet,
Genzyme, Cambridge, MA; for GM-CSF: Quantikine M, R&D Systems,
Minneapolis, MN). The sensitivities of the assays were as follows:
IFN-, 48 pg/ml; IL-4, 31 pg/ml; IL-2, 15 pg/ml; TNF-, 28 pg/ml;
and GM-CSF, 1 pg/ml.
Circulating mouse anti-sheep globulin Ig, IgG1, and IgG2a
Titers of total mouse anti-sheep globulin Ab and anti-sheep globulin IgG1 and IgG2a were measured by ELISA in serum collected from each mouse at the end of each experiment as previously described (30). Samples were incubated at dilutions of 1/500 for total Ig and IgG1 and 1/50 for IgG2a. Bound mouse Ig was detected with HRP-conjugated Abs: sheep anti-mouse Ig (Amersham, Little Chalfont, U.K.; 1/2000) for total Ig and HRP-conjugated goat anti-mouse IgG1 or IgG2a Ab (Southern Biotechnology Associates, Birmingham, AL; 1/4000). Sera from six nonimmunized mice were tested to provide normal controls.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vehicle-treated C57BL/6 IL-12+/+ mice with
GN showed an influx of the effectors of DTH (CD4+
T cells, macrophages, and fibrin deposition in glomeruli; Fig. 1
). Vehicle-treated
IL-12-/- mice with GN developed a lesser degree
of injury, with reduced glomerular CD4+ T cell
and macrophage accumulation and reduced fibrin deposition (Fig. 1;
CD4+ cells, p < 0.05;
macrophages and fibrin, p < 0.01). The administration
of IL-18 to IL-12-/- mice restored the numbers
of infiltrating CD4+ T cells and macrophages and
the extent of glomerular fibrin deposition to values comparable with
those of IL-12-intact animals. IL-12+/+ mice with
GN given IL-18 developed increased accumulation of the effectors of DTH
(CD4+ T cells, macrophages, and fibrin) in
glomeruli compared with vehicle-treated IL-12+/+
mice (Fig. 1; p < 0.01), suggesting that IL-18 is
synergistic with IL-12 in enhancing glomerular injury.
CD8+ cells were present in glomeruli of
IL-12+/+ mice with GN (0.31 ± 0.05 c/gcs)
and were reduced in the absence of IL-12
(IL-12-/- Ctrl, 0.16 ± 0.06 c/gcs;
p = 0.04). However, IL-18 administration did not
increase glomerular CD8+ cell number in either
IL-12-intact or IL-12-deficient mice (IL-12-/-
+ IL-18, 0.18 ± 0.04 c/gcs; IL-12+/+ +
IL-18, 0.31 ± 0.06 c/gcs). Small numbers of NK cells were present
in glomeruli of IL-12+/+ mice with GN (0.11
± 0.03 c/gcs). Genetic deficiency of IL-12 or the administration of
IL-18 to either IL-12+/+ or
IL-12-/- mice did not significantly alter the
degree of NK cell infiltrate (IL-12-/- Ctrl,
0.12 ± 0.02 c/gcs; IL-12-/- + IL-18,
0.17 ± 0.05 c/gcs; IL-12+/+ + IL-18,
0.14 ± 0.04 c/gcs).
|
and 3, A and B),
control treated IL-12+/+ mice had developed
proliferative GN with accumulation of PAS+
material and crescent formation on day 7 of GN and significant
proteinuria (Figs. 2 and 3, D and E). Deficiency
of IL-12 (in vehicle-treated IL-12-/- mice)
significantly attenuated glomerular injury and proteinuria (Figs. 2 and 3, J and K). However, administration of IL-18 to
IL-12-/- animals resulted in an increase in
glomerular injury such that glomerular crescent formation, the
deposition of PAS+ material in glomeruli, and
proteinuria were similar to those observed in vehicle-treated
IL-12+/+ mice (Figs. 2 and 3, G and
H). Consistent with these findings of increased cellular
effectors in glomeruli of genetically normal mice given IL-18, renal
injury was increased in IL-12+/+ mice given IL-18
(Fig. 2). Glomerular crescent formation and deposition of
PAS+ material in glomeruli were significantly
increased, and there was a trend toward increased proteinuria
(p = 0.08). In the absence of GN, IL-18
administration or the injection of vehicle alone did not alter
glomerular histology, total glomerular cell number, or the number of
CD4+ cells or macrophages in glomeruli. NK cells
were not detected in glomeruli of mice without GN (Table I).
|
|
|
Immunostaining for ICAM-1 in IL-12+/+ mice
with GN revealed moderate staining within glomeruli, in some proximal
tubules and periglomerular regions (Fig. 3
F), and in the endothelium of
interstitial blood vessels compared with light staining in some
glomeruli and tubules in IL-12+/+ mice without GN
(Fig. 3C). ICAM-1 expression in vehicle-treated
IL-12-/- mice was not statistically
significantly diminished on semiquantitative assessment using a score
of 0–4+ for at least 20 glomeruli/mouse
(IL-12+/+ Ctrl, 1.1 ± 0.1;
IL-12-/- Ctrl, 0.8 ± 0.1; Fig. 3I). Administration of rmIL-18 to mice deficient in IL-12
enhanced the expression of ICAM-1 protein in glomeruli of
IL-12-/- mice with GN
(IL-12-/- Ctrl, 0.8 ± 0.1;
IL-12-/- + IL-18, 1.4 ± 0.1;
p < 0.005; Fig. 3L), demonstrating that
IL-18 can increase ICAM-1 in the absence of IL-12. Administering IL-18
to IL-12+/+ mice with GN further increased
glomerular ICAM-1 staining (IL-12+/+ Ctrl,
1.1 ± 0.1; IL-12+/+ + IL-18, 1.6 ±
0.1; p < 0.005; Fig. 3O). Administering
IL-18 or vehicle alone to mice without GN did not increase glomerular
ICAM-1 expression above that seen in normal, untreated mice without GN
(normal mice, 0.12 ± 0.04; no GN + vehicle, 0.10 ± 0.05; no
GN + IL-18, 0.14 ± 0.05).
IL-18 restores dermal DTH responses in IL-12-/- mice and enhances responses in IL-12+/+ mice
IL-12+/+ mice treated with vehicle alone
showed Ag-specific dermal DTH (Fig. 4),
with a prominent influx of CD4+ T cells and
macrophages (Fig. 5, A and
B). In vehicle-treated IL-12-/-
mice, DTH was significantly reduced, and a lesser influx of T cells and
macrophages was observed (Figs. 4 and 5, D and
E). In IL-12-/- mice given IL-18,
Ag-specific skin swelling was present to the same extent as in
vehicle-treated IL-12+/+ animals, with a similar
leukocytic cell influx as that in genetically normal mice (Figs. 4 and 5, G and H). Dermal DTH responses in
IL-12+/+ mice were increased by administration of
rmIL-18 (Fig. 4), with increases in CD4+ T cells
and macrophages (Fig. 5, J and K). There were no
differences between any groups in the relatively minor NK cell
infiltrates observed in the footpads of mice (Fig. 5, C,
F, I, and L).
|
|
All mice with GN developed Ag-specific Ab responses that were
measured in serum drawn at the end of experiments (Table II). Total serum Ag-specific Ig and IgG2a
titers were unchanged in IL-12-/- mice,
although IgG1 titers were significantly increased.
IL-12-/- mice given IL-18 had higher titers of
total Ig and both IgG subclasses than vehicle-treated
IL-12+/+ mice. IgG2a was increased in
IL-12-/- mice by the administration of IL-18
(p = 0.04). There were no significant changes
in serum Ag-specific Ig, IgG1, or IgG2a titers in
IL-12+/+ mice given IL-18. Glomerular deposition
of autologous Ab (measured either by end-point positive titer
measurements or semiquantitative scoring of fluorescence intensity) was
not affected by either IL-12p40 gene deletion or IL-18 administration,
suggesting that the changes in renal injury observed were not due to
alterations in Ab deposition (Table III).
|
|
IFN-, GM-CSF, TNF-, and IL-2 were detected in the
supernatants of splenocytes isolated from
IL-12+/+ mice with GN (Fig. 6). The production of these
Th1/proinflammatory cytokines was reduced in
IL-12-/- mice. Treatment with IL-18 did not
restore Ag-stimulated splenocyte production of IFN-, GM-CSF,
TNF-, or IL-2 in IL-12-/- mice. IL-4 was
detected in all groups of mice studied. There were no significant
alterations in IL-4 production in either vehicle- or IL-18-treated
IL-12-/- mice. IL-4 was not measurable (<31
pg/ml) in three of nine vehicle-treated IL-12+/+
mice, three of six vehicle-treated IL-12-/-
mice, four of six IL-12-/- IL-18-treated mice,
and two of six IL-12+/+ mice given IL-18.
However, Ag-stimulated splenocyte cytokine production was modified by
IL-18 administration. As expected, the production of GM-CSF was
increased, and there was a trend toward increased IFN-
production (p = 0.11) after 72 h of
culture (Fig. 6). TNF- production was unchanged, and IL-2 levels
fell. It is possible that the IL-2 results may relate to increased
expression of the IL-2R, as is the case with mice injected with IL-12
(22, 31). IL-4 levels were not affected, consistent with
previous data on the actions of IL-18 (2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production by
Th1 CD4+ T cells. The effects of IL-18 are
synergistic with those of IL-12. IL-12 has been considered to be
pivotal to the initiation of Th1 responses, and most in vitro work
suggests that IL-12 is required to establish a Th1-predominant
response. Moreover, IL-12 does induce Th1 cells in the absence of IL-18
(5). The ability of IL-18 to facilitate Th1 responses in
vivo in the absence of IL-12 is less well defined and has not been
studied at the tissue level in models of Th1-dependent injury. While
some conflicting data exist, overall consideration of in vitro data
suggests that IL-18 by itself is insufficient to induce effector Th1
responses when assessed by cytokine production by
CD4+ cells (2, 5, 9)
Although the actions of IL-18 have been extensively studied in
vitro and to some degree in immune responses where noncognate
mechanisms play a major role, there have been few studies in
Th1-dependent cognate immune responses. In particular, it is not known
whether IL-18 can increase DTH or Th1 responses in vivo independent of
IL-12. To address this question, IL-18 was administered to
IL-12-/- C57BL/6 mice in two models of DTH:
Ag-specific dermal DTH in sensitized mice and experimental
proliferative and crescentic GN (accelerated anti-GBM GN).
Dermal DTH is considered to be a classical Th1 response. Recent studies
have characterized experimental crescentic anti-GBM GN as akin to
DTH in the glomerulus and directed by the Th1 subset. Crescent
formation and the influx of CD4+ cells and
macrophages are effector CD4+ and IL-12- and
IFN--dependent (19, 22, 30), while crescentic GN can be
attenuated by IL-4 and/or IL-10 (23, 24).
Consistent with earlier published work on the role of endogenous IL-12
in this model (22), mice genetically deficient in IL-12
developed a lesser degree of renal injury and DTH effectors in
glomeruli compared with genetically normal mice. While clearly
diminished in the absence of IL-12, renal injury was still present, as
evidenced by glomerular cellular proliferation, abnormal numbers of T
cells and macrophages, autologous Ab deposition, and abnormal
proteinuria. Furthermore, skin DTH was reduced in
IL-12-/- mice, with reduced footpad swelling
and a diminution in the degree of CD4+ and
macrophage infiltration. In this system the absence of IL-12 did not
result in a default Th2 response, at least as measured by Ag-stimulated
IL-4 production, although IgG1 levels were increased in
IL-12-/- mice. Administering IL-18 to
IL-12-/- mice restored renal injury and
glomerular and skin DTH to levels seen in
IL-12+/+ mice injected with vehicle alone. There
were increases in effectors of DTH: CD4+ cells,
macrophages, and (in the glomerulus) fibrin. Other potential mechanisms
of glomerular injury (CD8+ cells, NK cells, and
Ab) were assessed. CD8+ cells were present in
glomeruli and were decreased in the absence of IL-12, but were not
affected by IL-18 administration. These findings together with the
observation that injury in this model is not CD8 dependent
(21) do not support a role for CD8+
cells in this study. NK cells were not present in greater numbers in
either the footpad or glomeruli of IL-18-treated mice. However, as
IL-18 stimulates NK cell IFN- production (that would not necessarily
have been measurable in Ag-stimulated splenocyte cultures), it is
possible that increased IFN- production by NK cells present in the
lesion may have contributed to the increased injury observed. There was
no increase in the deposition of autologous Ab in glomeruli, confirming
that humoral mediators were not responsible for the increased injury
observed with IL-18 administration.
These results demonstrate that IL-18 can induce DTH responses in vivo
in the absence of IL-12, and that the effectors present in the lesions
are those classically associated with the DTH response. IL-18 is
capable of inducing a number of proinflammatory cytokines that are
relevant to Th1 responses, including IFN-, IL-2 (2),
GM-CSF (36), and TNF- (37). In this study,
although there were increases in DTH effectors in both the glomerulus
and the skin, splenocyte production of any of these proinflammatory
cytokines was not induced by IL-18 in the absence of IL-12. It remains
possible that other proinflammatory cytokines, such as IL-1 or
lymphotoxin-, may have been induced by IL-18.
To effect a cell-mediated immune response cells are required to
localize within the target tissue, a process requiring both adhesion
molecules and chemokines. The relevance of a number of adhesion
molecules and chemokines has been demonstrated in crescentic
glomerulonephritis (27, 28, 29) and dermal DTH
(11, 12, 13). In the absence of any demonstrable effect by
IL-18 in IL-12-/- on proinflammatory cytokines
such as IFN-, GM-CSF, and TNF-, the expression of the adhesion
molecule ICAM-1 was studied within glomeruli. IL-18 has been shown to
up-regulate ICAM-1 expression in an IFN--independent manner
(14), suggesting the potential for alterations in ICAM-1
(and indeed other adhesion molecules, such as VCAM-1, that has been
shown to act in synergy with ICAM-1 in contact hypersensitivity
(10)) to be relevant to the current studies. ICAM-1 is
functionally important in the development of proliferative and
crescentic glomerulonephritis (27, 28). Administering
IL-18 to IL-12-/- mice resulted in increased
glomerular ICAM-1 expression compared with that in
IL-12-/- control mice with GN. Mice without GN
that received IL-18 did not exhibit increased glomerular ICAM-1
expression, implying that in the absence of a local stimulus IL-18 does
not up-regulate ICAM-1 expression. These findings show that despite
reductions in Th1/proinflammatory cytokines found in control
IL-12-/- mice and the failure of IL-18 to
restore these deficits, at a tissue level Th1 responses (in this case
the DTH response) can be restored by IL-18 via the up-regulation of
chemoattractants or adhesion molecules such as ICAM-1. Furthermore,
they imply that the increase in ICAM-1 expression observed in
IL-12-/- mice given IL-18 is a direct effect of
IL-18, although the mechanism by which ICAM-1 expression is increased
by IL-18 could be indirect, involving other inflammatory mediators
themselves affected by IL-18. Increased glomerular ICAM-1 expression in
IL-12-/- mice given IL-18 demonstrates the
principle that adhesion molecules can be up-regulated by IL-18 in an
IL-12-independent manner in DTH responses. These studies do not address
whether IL-18 induces T cell or macrophage integrin expression, or
chemokine and chemokine receptor expression, both of which have the
potential to contribute to injury in DTH. While these studies have
demonstrated IL-12-independent effects of IL-18 in target tissues and
have not shown effects on the systemic immune response (via cytokines
and Ab production), IL-18 may play an important IL-12-independent role
in the initiation and generation of the immune response (for instance,
at Ag presentation and during T cell proliferation and
differentiation).
Genetically normal mice that were given IL-18 showed enhanced DTH
responses, renal injury, and skin swelling compared with those in
vehicle-treated IL-12+/+ mice. These results
demonstrate that although IL-18 can facilitate DTH responses in the
absence of IL-12, when IL-18 is administered to IL-12-intact mice, a
synergistic enhancement of DTH responses and injury occurs, with
increased DTH effectors within the lesion. This increased injury
observed in normal C57BL/6 mice given IL-18 was associated with
increased splenocyte production of GM-CSF together with a trend toward
increased IFN-. Glomerular ICAM-1 expression was also increased.
Therefore, the enhancement of Th1 response by IL-18 in normal animals
is likely to be mediated both by increases in the generation of Th1
effector cytokines and by induction of adhesion molecules.
The effects of IL-18 on Ag-specific Ab responses and on IgG subclasses
in vivo have not, to our knowledge, previously been studied. In vitro
studies have demonstrated that the combination of IL-12 and IL-18
suppress IgG1 and enhance IgG2a levels (38). However,
studies of murine graft-vs-host disease suggest that IL-18 lacks this
discriminatory effect and, in fact, suppresses total Ig, IgG1, and
IgG2a levels in vitro (39). While IL-18 would be expected
to induce IgG2a production in vivo (given its role in inducing
IFN-), there were no significant changes in Ag-specific total Ig,
IgG1, or IgG2a titers when IL-18 was administered to
IL-12+/+ mice.
In summary, these studies demonstrate that IL-18 is capable of substituting for IL-12 in the generation of in vivo cell-mediated DTH-like responses, in this case experimental crescentic glomerulonephritis and dermal DTH. These effects were not demonstrated to be due to alterations in patterns of cytokine synthesis and secretion. However, IL-12-independent effects of IL-18 on adhesion molecules were demonstrated, suggesting direct effects of IL-18 in enhancing cellular recruitment. In animals with an intact IL-12/IL-12R system, IL-18 enhances DTH-like responses via CD4+ and macrophage-mediated cellular mechanisms. In the presence of IL-12, the effects of IL-18 are likely to be multifactorial and include effects on proinflammatory cytokines and adhesion molecules. The current studies show that IL-18 is capable of inducing effector Th1 responses in the absence of IL-12 and point toward an important role for IL-18 in DTH and DTH-like inflammatory responses.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Richard Kitching, Centre for Inflammatory Diseases, Department of Medicine, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia.
3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; c/gcs, cells per glomerular cross-section; Ctrl, control group; GN, glomerulonephritis; GBM, glomerular basement membrane; PAS, periodic acid-Schiff’s reagent; PLP, periodate lysine paraformaldehyde.
Received for publication November 24, 1999. Accepted for publication July 26, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production by T cells. Nature 378:88.[Medline]
-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J. Immunol. 158:1541.[Abstract]
-inducing factor in enhanced production of IFN-. J. Immunol. 159:2125. production. J. Immunol. 161:3400. promoter in primary CD4+ T lymphocytes. J. Immunol. 160:3642. production and activates IRAK and NF
B. Immunity 7:571.[Medline]
. J. Immunol. 162:1648.-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells. J. Leukocyte Biol. 64:519.[Abstract]
(MIP-1)-dependent pathways. J. Clin. Invest. 187:1.
mediates crescent formation and cell-mediated immune injury in murine glomerulonephritis. J. Am. Soc. Nephrol. 10:752. production and type 1 cytokine responses. Immunity 4:471.[Medline]
-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J. Immunol. 156:4274.[Abstract]
-inducing factor) induces IL-8 and IL-1ß via TNF production from non-CD14+ human blood mononuclear cells. J. Clin. Invest. 101:711.[Medline]
production from activated B cells. Proc. Natl. Acad. Sci. USA 94:3948.This article has been cited by other articles:
|
|
 |
|
  H.-T. LEE, W.-S. CHEN, K.-H. SUN, C.-T. CHOU, and C.-Y. TSAI Increased Spontaneous But Decreased Mitogen-stimulated Expression and Excretion of Interleukin 18 by Mononuclear Cells in Patients with Active Systemic Lupus Erythematosus J Rheumatol, September 1, 2009; 36(9): 1910 - 1916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Miyashita, Y. Obase, K. Ouchi, K. Kawasaki, Y. Kawai, Y. Kobashi, and M. Oka Clinical features of severe Mycoplasma pneumoniae pneumonia in adults admitted to an intensive care unit J. Med. Microbiol., December 1, 2007; 56(12): 1625 - 1629. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-W. Chen, H.-T. Kuo, C.-Y. Chai, J.-L. Ou, and R.-C. Yang Pretreatment of curcumin attenuates coagulopathy and renal injury in LPS-induced endotoxemia Innate Immunity, February 1, 2007; 13(1): 15 - 23. [Abstract] [PDF]
|
|
|
|
 |
|
 P. G. Tipping and S. R. Holdsworth T Cells in Crescentic Glomerulonephritis J. Am. Soc. Nephrol., May 1, 2006; 17(5): 1253 - 1263. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. R. Young, M. T. Borchers, H. L. Allen, R. S. Gibbons, and F. X. McCormack Lung-Restricted Macrophage Activation in the Pearl Mouse Model of Hermansky-Pudlak Syndrome J. Immunol., April 1, 2006; 176(7): 4361 - 4368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Kitching, A. L. Turner, G. R.A. Wilson, T. Semple, D. Odobasic, J. R. Timoshanko, K. M. O'Sullivan, P. G. Tipping, K. Takeda, S. Akira, et al. IL-12p40 and IL-18 in Crescentic Glomerulonephritis: IL-12p40 is the Key Th1-Defining Cytokine Chain, Whereas IL-18 Promotes Local Inflammation and Leukocyte Recruitment J. Am. Soc. Nephrol., July 1, 2005; 16(7): 2023 - 2033. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Q. Gao, Y. Tsuda, K. Katayama, T. Nakayama, Y. Hatanaka, Y. Tani, H. Mizuguchi, T. Hayakawa, O. Yoshie, Y. Tsutsumi, et al. Antitumor Effect by Interleukin-11 Receptor {alpha}-Locus Chemokine/CCL27, Introduced into Tumor Cells through a Recombinant Adenovirus Vector Cancer Res., August 1, 2003; 63(15): 4420 - 4425. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tanaka, M. Narita, S. Teramoto, T. Saikai, K. Oashi, T. Igarashi, and S. Abe Role of Interleukin-18 and T-helper Type 1 Cytokines in the Development of Mycoplasma pneumoniae Pneumonia in Adults* Chest, May 1, 2002; 121(5): 1493 - 1497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Wang, C. Feliciani, B. G. Howell, I. Freed, Q. Cai, H. Watanabe, and D. N. Sauder Contribution of Langerhans Cell-Derived IL-18 to Contact Hypersensitivity J. Immunol., April 1, 2002; 168(7): 3303 - 3308. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
