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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
A. Richard Kitching,* Amanda L. Turner,* Gabrielle R.A. Wilson,* Timothy Semple,*
Dragana Odobasic,* Jennifer R. Timoshanko,* Kim M. O’Sullivan,* Peter G. Tipping,*
Kiyoshi Takeda,
†
Shizuo Akira,
†
and Stephen R Holdsworth*
*Centre for Inflammatory Diseases, Monash University Department of Medicine, Monash Medical Centre, Clayton,
Victoria, Australia; and
†
Department of Host Defense Research, Institute for Microbial Diseases, Osaka University,
Suita, Osaka, Japan
Experimental crescentic glomerulonephritis (GN) is characterized by T helper 1 (Th1) directed nephritogenic immune
responses and cell-mediated glomerular injury. IL-12p40, the common cytokine chain for both IL-12 and IL-23, is important in
the generation and potentially the maintenance of Th1 responses, whereas IL-18 is a co-factor for Th1 responses that may have
systemic and local proinflammatory effects. For testing the hypothesis that both endogenous IL-12p40 and endogenous IL-18
play pathogenetic roles in crescentic GN, accelerated anti– glomerular basement membrane GN was induced in mice
genetically deficient in IL-12p40 (IL-12p40
ⴚ/ⴚ
), IL-18 (IL-18
ⴚ/ⴚ
), or both IL-12p40 and IL-18 (IL-12p40
ⴚ/ⴚ
IL-18
ⴚ/ⴚ
). Compared
with wild-type C57BL/6 mice, IL-12p40
ⴚ/ⴚ
mice failed to make a nephritogenic Th1 response and developed markedly reduced
crescent formation and renal leukocytic infiltration, despite renal production of chemoattractants and adhesion molecules.
IL-18
ⴚ/ⴚ
mice developed an intact antigen-specific systemic Th1 response, a similar degree of crescent formation, but fewer
glomeruli affected by other severe histologic changes and fewer leukocytes in glomeruli and interstitium. IL-18 was expressed
within diseased kidneys. Local production of TNF, IL-1

, IFN-
␥
, CCL3 (MIP-1
␣
), and CCL4 (MIP-1

) was reduced in IL-18
ⴚ/ⴚ
mice, demonstrating a local proinflammatory role for IL-18. Combined deletion of IL-12p40 and IL-18 did not result in
synergistic effects. Consistent with the hypothesis that inflammation leads to fibrosis, all three groups of deficient mice
expressed lower levels of intrarenal TGF-

1 and/or
␣
1(I) procollagen mRNA. These studies demonstrate that in severe
experimental crescentic GN, IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 has local proinflammatory roles.
J Am Soc Nephrol 16: 2023–2033, 2005. doi: 10.1681/ASN.2004121075
C
rescentic glomerulonephritis (GN) is the most severe
and rapidly progressive subset of GN. Kidneys from
patients with human crescentic GN demonstrate the
presence of effectors of delayed type hypersensitivity (DTH),
particularly T cells and macrophages (1–3). These data, with
other findings regarding the nature of the immune response in
these patients (4), suggest a role for T helper cell 1 (Th1)-
directed nephritogenic immune responses in the pathogenesis
of crescentic GN. The relevance of these observations has been
confirmed in experimental murine crescentic GN (5,6). Severe
crescentic glomerular injury is Th1 directed, effector CD4⫹ cell
mediated (6,7), and regulated by endogenous and exogenous
Th2 cytokines IL-4 and IL-10 (8 –10). Although it is clear that
humoral immune mediators are capable of inducing injury
(11,12), crescent formation can occur in the absence of autolo-
gous antibody (13–15).
Previous work, using anti–IL-12p40 antibodies and IL-
12p40
⫺/⫺
mice, has demonstrated that in experimental murine
models, glomerular crescent formation is directed by IL-12p40
(7,16–18). IL-12 is a heterodimeric cytokine, consisting of a p40
subunit that binds to the IL-12R

1 and a p35 subunit that binds to
the IL-12R

2. Recently, a related cytokine was cloned, IL-23 (19),
also a heterodimer, that shares the IL-12p40 subunit and its bind-
ing to IL-12R

1 but has a different subunit, the IL-23p19 subunit,
that binds to the IL-23R. Although there is some overlap in their
biologic effects, studies in experimental autoimmune encephalo-
myelitis (EAE) have shown that whereas IL-12 primes cells for
IFN-
␥
production, IL-23 has significant proinflammatory effects
on both memory T cells and macrophages (20).
IL-18, originally described as IFN-
␥
–inducing factor (21),
both is a co-factor for IFN-
␥
and other Th1 cytokine production
and induces expression of leukocyte chemoattractants and ad-
hesion molecules (16,22). Exogenous IL-18 can enhance im-
mune responses and increase the severity of experimental cres-
centic GN (16), independent of IL-12p40. Intrarenal IL-18
expression has been documented in murine lupus nephritis
Received December 12, 2004. Accepted April 5, 2005.
Published online ahead of print. Publication date available at www.jasn.org.
Address correspondence to: Dr. A. Richard Kitching, Centre for Inflammatory
Diseases, Monash University Department of Medicine, Monash Medical Centre,
246 Clayton Road, Clayton, Victoria 3168, Australia. Phone: 61-3-9594-5520; Fax:
61-3-9594-6495; E-mail: richard.kitching@med.monash.edu.au
Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1607-2023
(23), rat experimental crescentic GN (24), and experimental
ischemia reperfusion injury (25), playing a functional role in the
last disease (26). However, the role of endogenous IL-18 in
experimental crescentic GN, as well as any synergism or dif-
ferential effects with IL-12p40, is unknown. Using mice that are
genetically deficient in IL-12p40, IL-18, or both IL-12p40 and
IL-18, we sought to define the relative roles and nature of the
contributions of endogenous IL-12p40 and IL-18 in experimen-
tal crescentic GN.
Materials and Methods
Experimental Design
IL-12p40
⫺/⫺
mice (27) (C57BL/6 background) were obtained from
Jackson Laboratories (Bar Harbor, MA). IL-18
⫺/⫺
mice (C57BL/6 back
-
ground) were created as described previously (28). Mice were bred at
Monash University (Clayton, Victoria, Australia). Combined IL-12p40–
and IL-18–deficient (IL-12p40
⫺/⫺
IL-18
⫺/⫺
) mice were created by crossing
IL-12p40
⫺/⫺
mice with IL-18
⫺/⫺
mice, then interbreeding IL-12p40
⫹/⫺
and IL-18
⫹/⫺
offspring. Progeny were selected as founders for IL-
12p40
⫺/⫺
IL-18
⫺/⫺
mice. Colony founders, breeders, and experimental
animals in the colonies were genotyped by a PCR-based protocol.
Figure 1 shows a representative experiment confirming the disruption
of the IL-18 gene and/or the absence of the wild-type (WT) IL-12p40
gene in appropriate mice. Anti-mouse glomerular basement membrane
(GBM) globulin was prepared as described previously (29).
For inducing crescentic GN, 8- to 10-wk-old male WT C57BL/6 (n ⫽
20), IL-12p40
⫺/⫺
(n ⫽ 5), IL-18
⫺/⫺
(n ⫽ 16), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 13) mice were sensitized by subcutaneous injection of 500
gof
sheep globulin (SG) in 100
l of Freund’s complete adjuvant (FCA).
After 10 d, GN was initiated by intravenous injection of 12 mg of sheep
anti-mouse GBM globulin. Renal injury was studied after an additional
10 d. For assessing intrarenal leukocytes, cytokines, and chemokine
production, another experiment was performed in WT C57BL/6 (n ⫽
7), IL-12p40
⫺/⫺
(n ⫽ 6), IL-18
⫺/⫺
(n ⫽ 5), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 7) mice using a similar experimental protocol but antisera from a
different sheep. Studies adhered to the National Health and Medical
Research Council of Australia guidelines for animal experimentation.
Histologic examinations were performed on coded slides. Results are
expressed as the mean ⫾ SEM. The significance of differences between
groups was determined by ANOVA.
Assessment of Systemic Immune Responses
For experiments in dermal DTH, WT C57BL/6 (n ⫽ 4), IL-12p40
⫺/⫺
(n ⫽ 5), IL-18
⫺/⫺
(n ⫽ 4), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice (n ⫽ 7) and
were sensitized with 2 mg of SG in 100
l of FCA, boosted with 1 mg
of SG in 100
l of FCA after 7 d and after an additional 7 d challenged
by intradermal injection of 300
gofSGin30
l of PBS into the ear (30).
Horse globulin was injected in the opposite ear as a control. DTH was
assessed after 24 h by measuring the difference between the SG and
horse globulin injected ear thicknesses using a micrometer. For cyto-
kine production, splenocytes from mice with GN were cultured for 72 h
with 10
g/ml normal sheep IgG. IFN-
␥
, IL-4, IL-1

, TNF, and GM-CSF
in supernatants were measured by ELISA as described previously (16).
IL-5 was measured by ELISA using the rat anti-mouse IL-5 mAb TRFK5
(0.5
g/ml; R&D Systems, Minneapolis, MN) for capture and biotinyl-
ated TRFK4 (100 ng/ml; R&D Systems) for detection, with rmIL-5 as a
standard (R&D Systems) and streptavidin– horseradish peroxidase
(HRP; 1:1000; Chemicon, Boronia, Victoria, Australia). Circulating lev-
els of mouse anti-SG immunoglobulins were measured by ELISA as
described previously (31) on serum collected at the end of experiments.
For IgG1 assessment (serum dilution 1:100), HRP-conjugated goat anti-
mouse IgG1 antibodies (Southern Biotechnology Assoc., Birmingham,
AL; 1:4000) were used. For IgG3 (serum dilution 1:50), biotinylated rat
anti-mouse IgG3 (R40 – 82; Pharmingen, San Diego, CA; 2
g/ml) then
streptavidin-HRP complex (Silenus, Victoria, Australia) were the de-
tecting antibodies.
Assessment of Glomerular Injury, Leukocyte Accumulation,
and Adhesion Molecules
Glomerular crescent formation (two or more layers of cells observed
in Bowman’s space) was assessed on periodic acid-Schiff–stained par-
affin sections. The proportion of glomeruli that were severely affected
(crescent formation, evidence of accumulation of cells in Bowman’s
space that did not satisfy the criteria for crescent formation, ⬎50% of
the glomerular tuft affected by necrosis, or severe proliferative changes)
was assessed, according to a modification of a previously published
method (32). A minimum of 50 glomeruli were assessed in each animal.
Tissue sections of periodate lysine paraformaldehyde–fixed kidneys
were stained to demonstrate CD4
⫹
cells, CD8
⫹
cells, and Mac-
1(CD11b)
⫹
macrophages/neutrophils using a three-layer immunoper
-
oxidase technique. The primary antibodies were GK1.5 (anti-mouse
CD4; American Type Culture Collection [ATCC], Manassas, VA),
53-6䡠7 (anti-mouse CD8; ATCC), and M1/70 (anti-mouse CD11b;
ATCC). A minimum of 20 consecutively viewed glomeruli and a min-
imum of 10 high-power cortical interstitial fields (excluding perivascu-
lar regions) were assessed per mouse, and results were expressed as
cells per glomerular cross-section (c/gcs) or cells per high-power field
(c/hpf). Renal deposition of P-selectin and intercellular adhesion mol-
ecule-1 (ICAM-1) was assessed as described previously (33) and scored
semiquantitatively—0, background staining; 1, lowest clearly positive
staining; 2, moderate staining; and 3, intense deposition—in 20 ran-
domly selected glomeruli and 20 randomly selected tubulointerstitial
areas at medium power.
Assessment of IL-18 in Nephritic Kidneys
Tissue sections of periodate lysine paraformaldehyde–fixed kidneys
were stained to demonstrate the presence of IL-18, using a polyclonal
rabbit anti-mouse IL-18 antibody (a gift of Dr. M. Kurimoto, Fujisaki
Institute, Okayama, Japan) at a concentration of 11
g/ml, followed by
HRP-conjugated swine anti-rabbit antibodies (Dako, Glostrup, Denmark;
1:100), then HRP-conjugated rabbit anti-peroxidase globulin (Dako; 1:100),
then diaminobenzidine. Sections of spleen were positive controls. Nega-
tive controls included substituting normal rabbit immunoglobulins for the
primary antibody, preincubating the primary antibody with excess
rmIL-18 (16) (final concentration 2
g/ml; Dr. M. Kurimoto), and immu-
nostaining sections of an IL-18
⫺/⫺
mouse with GN.
Figure 1. Confirmation of IL-12p40 and IL-18 gene disruption.
Lanes 1 to 4, IL-12p40
⫺/⫺
mice; lanes 5 to 8, IL-18
⫺/⫺
mice;
lanes 9 to 12, combined IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice; and lanes
13 to 16, wild-type (WT) C57BL/6 mice. (A) Presence of the WT
IL-18 gene product (291 bp) in IL-12p40
⫺/⫺
and WT mice and
the disrupted 320-bp allele in IL-18
⫺/⫺
and IL-12p40
⫺/⫺
IL-
18
⫺/⫺
mice. (B) Absence of an intact IL-12p40 gene (465 bp) is
demonstrated in IL-12p40
⫺/⫺
and IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice.
2024 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2023–2033, 2005
Assessment of Intrarenal Chemokine, Cytokine, and Collagen
mRNA Expression
Total kidney RNA was extracted with TRIzol reagent (Invitrogen,
San Diego, CA) according to the manufacturer’s protocol from ran-
domly selected mice from a single experiment (n ⫽ 4 to 5 for each group
with GN). Multiprobes incorporating [
␣
-
32
P]UTP were transcribed
from the template set mCK-5c (RiboQuant System, Pharmingen), a
modification of mCK-2 (IL-12p40, IL-4, IL-13, IL-18, IL-1

, IL-10, IFN-
␥
,
and MIF probes; Pharmingen), and a custom template (lymphotoxin-
␣
[LT-
␣
], lymphotoxin-

, TNF, IL-13, IFN-
␥
,
␣
1(I) procollagen, TGF-

1,
and TGF-

3 probes; Pharmingen) using T7 RNA polymerase in vitro
transcription. After DNase I treatment, riboprobes were isolated by
phenol/chloroform extraction and precipitation with 4 M ammonium
acetate and ethanol. Incorporation of [
␣
-
32
P]UTP was determined by
Cherenkov activity, probes were diluted to 3.5 ⫻ 10
5
cp/m
l, then
added to 20
g of kidney RNA. Hybridization and isolation were
conducted according to the RiboQuant manual. RNA hybrids were
separated by electrophoresis (5% polyacrylamide/8 M urea). The gel
was dried, then exposed to the phosphoimager imaging plate (FLA-
2000; Fuji Photo Film Co., Tokyo, Japan). Image Gauge software (ver-
sion 3.46; Fuji Photo Film Co.) was used to evaluate the gel image. Gene
expression was measured and normalized to the housekeeping gene
L32.
For measurement of IFN-
␥
mRNA by real-time PCR, 1
g of RNA
(n ⫽ 6 to 7 for each group with GN) was treated with 1 unit of
amplification-grade DNase I (Invitrogen) then primed with 500 ng of
Oligo(dT)
12-18
(Roche, Mannheim, Germany) and reverse transcribed
(Super Script II; Invitrogen). Gene-specific primers for murine IFN-
␥
and murine

-actin were designed (Vector NTI software; Invitrogen).
IFN-
␥
cDNA was amplified using the PCR primers (F 5-GAAAGA-
CAATCAGGCCATCA-3⬘ andR5⬘-TTGCTGTTGCTGAAGAAGGT-3⬘)
to produce a product of 78 bp, whereas the

-actin cDNA was ampli-
fied with primers (F 5⬘-AGGCTGTGCTGTCCCTGTAT-3⬘ andR5⬘-
AAGGAAGGCTGGAAAAGAGC-3⬘) to produce a 388-bp product. Re-
al-time PCR was performed on a Rotor Gene RG-3000 (Corbett
Research, Mortlake, New South Wales, Australia) using Faststart DNA
master, Sybr Green I (Roche). IFN-
␥
and

-actin mRNA expression was
quantified using serial dilutions of an exogenous standard. IFN-
␥
levels
were normalized to

-actin and expressed as fg IFN-
␥
/pg

-actin
mRNA. PCR products were confirmed by melt-curve analysis.
Assessment of Humoral Immune Reactants in Glomeruli
Immunofluorescence was performed on 4-
m cryostat-cut tissue.
Glomerular mouse Ig was evaluated using FITC-conjugated sheep anti-
mouse Ig (Silenus) and C3 using FITC-conjugated goat anti-mouse C3
(Cappel, Durham, NC). For both mouse Ig and C3, a single dilution of
1:100 was scored (0 to 3⫹, based on fluorescence intensity). Sections in
which only some glomeruli were positive were graded as 0.5. In addi-
tion, quantitative evaluation of the extent of glomerular antibody dep-
osition was made by capturing images of at least 10 randomly selected
glomeruli (high power) from each mouse and analyzing mean fluores-
cence intensity in each glomerular tuft by tracing each tuft, after re-
moving background values (e.g., light emanating from stained section
without tissue) for each slide (NIH Image) as previously published
(12,30). Values are expressed as arbitrary units of fluorescence per pixel.
Renal Function and Urinary Protein Excretion
Urinary creatinine concentrations were measured by the alkaline
picric acid. Serum creatinine concentrations were measured by an
enzymatic creatininase assay. Urinary protein excretions were deter-
mined by the Bradford method on 24-h urine collections from mice
before disease and from each mouse over the final 24 h of the experi-
ment. Urinary protein excretion was expressed by a 24-h value and as
a urinary protein to urinary creatinine ratio.
Results
IL-12p40 Is the Key Th1-Defining Cytokine Chain in
Antigen-Specific Cell-Mediated Immune Responses
Systemic immune responses to the nephritogenic antigen
(SG) were assessed in the presence and the absence of endog-
enous IL-12p40, IL-18, or both IL-12p40 and IL-18 (Figure 2).
IL-12p40
⫺/⫺
mice developed significantly diminished dermal
DTH, and a similar trend was evident in IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice. However, deficiency of IL-18 alone did not result in
diminished dermal DTH. Total serum antigen-specific antibody
titers were marginally and nonsignificantly decreased in both
groups that lacked IL-18 (Figure 2B). The Th2-associated IgG1
subclass was unchanged (Figure 2C). There was a trend to
decreased IgG3 in both IL-18 –deficient groups of mice (Figure
2D). Antigen-stimulated splenocytes from IL-12p40
⫺/⫺
mice
did not produce detectable amounts of IFN-
␥
(Figure 3A), TNF
(Figure 3C), or GM-CSF (Figure 3B), confirming the key role for
IL-12p40 in the development of Th1 responses. However, there
was no effect on any of these three key proinflammatory cyto-
kines in the absence of IL-18, showing that IL-18 is not required
for Th1 cytokine production by immune cells. In IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice, cytokine production was low, except in the
case of TNF, in which levels seemed paradoxically elevated
in IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice. Deletion of IL-12p40 sup
-
pressed IL-4 and IL-5 production (Figure 3, D and E; as
previously observed [16,34]). Levels of IL-4 and IL-5 were
increased in the absence of IL-18. IL-1

was measured by
ELISA, but only one mouse produced measurable levels of
IL-1

(sensitivity 3.9 pg/ml).
IL-12p40 Is the Key Cytokine Chain in Glomerular Crescent
Formation
Ten days after challenge with sheep anti-mouse GBM glob-
ulin, sensitized WT C57BL/6 mice developed severe prolifera-
tive and crescentic GN, with significant tubulointerstitial injury
(Figures 4, A and E, and 5). Glomerular and tubulointerstitial
disease was markedly attenuated in the absence of IL-12p40
(Figure 4, B and F). In the absence of IL-18, histologic renal
injury was still severe but qualitatively marginally less so than
that seen in WT mice (Figure 4, C and G). IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice developed disease that was less severe than in WT mice
but more severe than in IL-12p40
⫺/⫺
mice (Figure 4, D and H).
Analysis of glomerular crescent formation in two separate ex-
periments (Figure 5, A and B) showed decreased crescent for-
mation in IL-12p40
⫺/⫺
mice but no significant decrease in
either IL-18
⫺/⫺
or combined IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice. Sig
-
nificant reductions in the proportions of glomeruli severely
affected by crescent formation, proliferative changes, or necro-
sis were evident in all three groups of genetically deficient mice
(Figure 5, C and D).
J Am Soc Nephrol 16: 2023–2033, 2005 Endogenous IL-12p40 and IL-18 in GN 2025
Both IL-12p40 and IL-18 Contribute to Leukocyte
Accumulation in Inflammatory Renal Disease
In glomeruli of mice with GN, CD4⫹ cell (Figure 6A) and
CD11b(Mac-1)
⫹
cell (Figure 6C) accumulation was reduced in
all groups of genetically deficient mice, with the reduction in
infiltrate greatest in mice deficient in IL-12p40 (including IL-
12p40
⫺/⫺
IL-18
⫺/⫺
–deficient mice). Changes in CD8
⫹
cell in
-
filtration (Figure 6B) were significant in IL-12p40
⫺/⫺
mice but
not in IL-18
⫺/⫺
or IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice. Similar findings
were present in the interstitium (Figure 6, D through F), al-
though the reduction in CD8
⫹
cells in combined IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice was significant, and reductions in Mac-1
⫹
cells
seemed more prominent in IL-18
⫺/⫺
mice.
IL-18 Expression in Kidneys of Mice with Anti-GBM GN
Given the reduced leukocytic infiltrates in IL-18
⫺/⫺
mice
despite the lack of a key role for systemic/lymphoid IL-18 to
influence Th1 responses, local IL-18 production was examined.
IL-18 mRNA was detected in renal tissue from WT C57BL/6
mice with GN (4.1 ⫾ 1.2 AU) by RNAse protection assay. IL-18
mRNA was present to a similar level in IL-12p40
⫺/⫺
mice with
GN (4.2 ⫾ 0.3 AU) but was not detected in IL-18 and combined
IL-12p40
⫺/⫺
IL-18
⫺/⫺
–deficient mice. Immunohistochemistry,
using a rabbit anti-mouse IL-18 antibody, revealed local accu-
mulation of IL-18 (Figure 7). Staining was abrogated by substi-
tuting normal rabbit Ig for the primary antibody (Figure 7B), by
preabsorbing the primary antibody with excess rmIL-18 (Figure
7C), or by staining tissue from an IL-18
⫺/⫺
mouse with GN
(data not shown).
Intrarenal Chemokine Production and Adhesion Molecule
Expression in Mice with GN
To determine whether endogenous IL-18 alters renal chemo-
kine and adhesion molecule expression, we measured intrare-
nal chemokine mRNA expression by RNAse protection assay
(Table 1). There were no significant effects on mRNA expres-
sion of the T cell–related chemokines CXCL10 (IP-10), XCL1
(lymphotactin), CCL5 (RANTES), or CCL1 (TCA-3). However,
endogenous IL-18 does regulate macrophage and neutrophil
chemoattractants. Compared with WT C57BL/6 mice with GN,
IL-18
⫺/⫺
mice had significantly reduced CCL3 (MIP-1
␣
) and
Figure 2. Dermal delayed type hypersensitivity (DTH; A), serum antigen (sheep globulin)-specific antibody responses (B), with the
antigen-specific IgG subclasses IgG1 (C) and IgG3 (D) in mice with anti– glomerular basement membrane (GBM) glomerulone-
phritis (GN), showing reduced dermal DTH particularly in IL-12p40
⫺/⫺
mice but not in IL-18
⫺/⫺
mice, no statistically significant
alterations in total antigen-specific Ig, IgG1, or IgG3, although IL-18
⫺/⫺
and IL-12p40
⫺/⫺
IL-18
⫺/⫺
groups had a trend to reduced
IgG3. *P ⬍ 0.05 versus WT C57BL/6 mice (ANOVA). Results are expressed as the mean ⫾ SEM. Numbers of mice studied are WT
C57BL/6 (n ⫽ 4), IL-12p40
⫺/⫺
(n ⫽ 5), IL-18
⫺/⫺
(n ⫽ 4), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 7) in A and WT C57BL/6 (n ⫽ 11 to 20),
IL-12p40
⫺/⫺
(n ⫽ 5), IL-18
⫺/⫺
(n ⫽ 10 to 16), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 12 to 13) in B through D.
2026 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2023–2033, 2005
CCL4 (MIP-1

). In IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice, reductions in
both CCL3 and CCL4 were seen as for IL-18
⫺/⫺
mice, and the
reduction in CXCL1 (MIP-2) was significant. In both IL-18
⫺/⫺
and IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice, expression of CCL2 (MCP-1)
mRNA fell to approximately two thirds of that in WT mice with
GN, but this reduction did not reach statistical significance.
Semiquantitative assessment of immunofluorescent staining for
the adhesion molecules P-selectin and ICAM-1 revealed no
significant differences in the expression of either molecule (Ta-
ble 2), although as previously found (16), a trend to decreased
ICAM-1 in IL-12p40 –deficient mice (in either IL-12p40
⫺/⫺
or
IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice) was noted. The minimal interstitial
expression of P-selectin was not formally assessed.
Intrarenal Cytokine Production in Mice with GN
Kidneys of WT C57BL/6 mice with GN expressed mRNA for
IL-1

, TNF, MIF, IFN-
␥
, and LT-
␣
(Figure 8). Genetically defi-
cient mice expressed less intrarenal IL-1

and TNF mRNA (not
reaching significance in IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice for IL-1

mRNA and IL-12p40
⫺/⫺
mice for TNF mRNA). Upregulation
of TNF in IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice was not seen in the
kidney. In contrast to the findings in antigen-stimulated spleno-
cytes (Figure 3A), renal IFN-
␥
mRNA, measured by real-time
PCR, was reduced in IL-18
⫺/⫺
mice but not in IL-12p40
⫺/⫺
mice. LT-
␣
mRNA levels were similar in all groups of mice.
Detectable levels of mRNA for the anti-inflammatory cytokine
IL-10 were found only in mice that lacked IL-12p40 (IL-
12p40
⫺/⫺
and IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice), suggesting that IL-
12p40 but not IL-18 suppresses this protective cytokine in in-
flammatory renal disease. Intrarenal MIF mRNA expression
was unchanged. IL-4, IL-12p40, IL-13, and lymphotoxin-

mRNA were not detected by RNAse protection assay in any
group of mice with GN.
Effects of IL-12p40 and IL-18 on Humoral Mediators of
Renal Injury
Autologous antibody and C3 were detected in glomeruli by
immunofluorescence (Table 3) and assessed by both semiquan-
titative scoring of fluorescence intensity (0 to 3⫹; WT C57BL/6
n ⫽ 18, IL-12p40
⫺/⫺
n ⫽ 5, IL-18
⫺/⫺
n ⫽ 16, and IL-12p40
⫺/⫺
IL-18
⫺/⫺
n ⫽ 13). There was little difference in the deposition
of Ig in glomeruli by this method (WT C57BL/6 1.5 ⫾ 0.2,
IL-12p40
⫺/⫺
1.6 ⫾ 0.4, IL-18
⫺/⫺
1.2 ⫾ 0.1, IL-12p40
⫺/⫺
IL-
18
⫺/⫺
1.2 ⫾ 0.1), although C3 deposition seemed diminished in
the IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice (WT C57BL/6 2.1 ⫾ 0.2, IL-
12p40
⫺/⫺
1.6 ⫾ 0.3, IL-18
⫺/⫺
1.6 ⫾ 0.2, IL-12p40
⫺/⫺
IL-18
⫺/⫺
1.2 ⫾ 0.1; P ⬍ 0.01 versus WT C57BL/6 mice). Quantitative
analyses of fluorescence intensity performed from tissues in an
additional experiment (Table 3) demonstrated increased mouse
Ig in glomeruli in both IL-18
⫺/⫺
and combined IL-12p40
⫺/⫺
IL-
18
⫺/⫺
mice and no alterations in C3 deposition.
Markers of Progressive Renal Disease: Intrarenal TGF-

and
␣
1(I) Procollagen mRNA
As markers of renal fibrotic potential in mice with GN, renal
mRNA expression of the anti-inflammatory but profibrotic cy-
tokine TGF-

1 and mRNA expression for
␣
1(I) procollagen
were measured (Figure 9). Intrarenal TGF-

1 mRNA expres-
sion was reduced in IL-12p40
⫺/⫺
and IL-18
⫺/⫺
mice (trend in
IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice). All groups of genetically deficient
mice with GN had reduced expression of intrarenal
␣
1(I) pro-
collagen mRNA expression compared with WT C57BL/6 mice
with GN. TGF-

3 mRNA was unchanged in all groups.
Functional Indices of Injury in Mice with GN
WT C57BL/6 mice with crescentic GN developed significant
proteinuria and moderate renal impairment (Table 4). Al-
though values did not reach statistical significance, both serum
creatinine values and urinary protein excretion values reflected
the lesser severity of disease in IL-12p40
⫺/⫺
mice, which had
Figure 3. Cytokine production by antigen-stimulated spleno-
cytes from mice developing GN. Cytokine production was
measured in culture supernatants (4 ⫻ 10
6
cells/ml) at 72 h by
ELISA. IFN-
␥
(〈), GM-CSF (B), TNF (C), IL-4 (D), and IL-5 (E)
were undetectable in splenocyte cultures from IL-12p40
⫺/⫺
mice with GN, but in IL-18
⫺/⫺
mice, cytokine production was
either similar (IFN-
␥
, GM-CSF, and TNF) or increased (IL-4 and
IL-5). Results are expressed as the mean ⫾ SEM. Numbers of
mice studied for are WT C57BL/6 (n ⫽ 11 to 14), IL-12p40
⫺/⫺
(n ⫽ 5), IL-18
⫺/⫺
(n ⫽ 8 to 16), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽
8 to 13).
J Am Soc Nephrol 16: 2023–2033, 2005 Endogenous IL-12p40 and IL-18 in GN 2027
serum creatinine values not significantly different from mice
without GN.
Discussion
Glomerular crescent formation indicates severe and rapidly
progressive GN. The presence of effector T cells and macro-
phages in patients with this lesion, together with studies in
experimental models of GN, suggests that CD4
⫹
cell-directed,
macrophage-mediated effector responses, particularly Th1 re-
sponses, are important in the genesis of glomerular crescent
formation. We have comprehensively characterized severe glo-
merular injury in this model. At an effector level, it is depen-
dent on effector CD4
⫹
cells (6,11,14), independent of autolo
-
gous antibodies (14), complement (11), and CD8
⫹
cells (35). At
a molecular level, IL-12p40, IFN-
␥
, GM-CSF, TNF, and IL-1

are pathogenetic (7,29,36–38), whereas endogenous IL-4 and
IL-10 are protective (8,9).
The current studies used mice that were genetically deficient
in IL-12p40, the common chain of both IL-12 and the more
recently described cytokine IL-23, and/or IL-18 to define the
relative roles of these molecules in severe immune renal injury.
The key findings from these studies are (1) IL-12p40 is the key
Figure 4. Histologic features of injury in WT C57BL/6, IL-12p40
⫺/⫺
, IL-18
⫺/⫺
, and IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice with GN. Severe
proliferative and crescentic GN with significant tubulointerstitial injury was present in WT C57BL/6 mice with GN (A and E).
Injury was significantly attenuated in IL-12p40
⫺/⫺
mice (B and F) and marginally less severe in IL-18
⫺/⫺
mice (C and G).
Combined IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice (D and H) showed an intermediate phenotype between IL-12p40
⫺/⫺
and IL-18
⫺/⫺
groups.
Figure 5. Analysis of glomerular injury in mice with accelerated autologous-phase anti-GBM GN at 10 d. Significant crescent
formation seen in C57BL/6 mice was attenuated in the absence of IL-12p40 but not reduced in the absence of IL-18. Crescent
formation was intermediate in extent in the absence of both IL-12p40 and IL-18. Severe histologic changes were most prominently
reduced in IL-12p40
⫺/⫺
mice but also less common in IL-18
⫺/⫺
and combined IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice. A and C, and B and
D show analyses of separate experiments. *P ⬍ 0.01 versus IL-18
⫺/⫺
;**P ⬍ 0.01 versus WT C57BL/6; ***P ⬍ 0.001 versus WT
C57BL/6 (ANOVA). Results are expressed as the mean ⫾ SEM. Numbers of mice studied are WT C57BL/6 (n ⫽ 20), IL-12p40
⫺/⫺
(n ⫽ 5), IL-18
⫺/⫺
(n ⫽ 16), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 13) for A and C and WT C57BL/6 (n ⫽ 7), IL-12p40
⫺/⫺
(n ⫽ 6),
IL-18
⫺/⫺
(n ⫽ 5), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 7) for B and D.
2028 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2023–2033, 2005
cytokine chain in defining Th1 nephritogenic immune respons-
es; (2) whereas IL-18 is not required for systemic Th1 responses
that generate Th1 effectors and crescent formation, it plays a
role in local inflammation, the severity of glomerular injury,
and the production of chemoattractants; (3) deficiency of both
of these molecules does not offer additional protection; and (4)
reducing inflammation by deleting either of these molecules
reduces surrogate markers of progressive renal disease.
This study confirms our (7,16,17) and others’ (18) previous
work that has shown a key role for IL-12p40 in crescentic GN.
IL-12p40
⫺/⫺
mice did not make strong Th1 responses, and
glomerular crescent formation, severe glomerular changes, and
leukocytic infiltration were significantly inhibited. In addition
to IL-12p40’s effects on the systemic immune responses, local
production of TNF mRNA was reduced in the absence of
IL-12p40. Local expression of IL-10 mRNA, which has been
shown to be protective in this lesion (9), was increased in the
IL-12p40
⫺/⫺
mice. There was only minor, if any, effect on
chemoattractants and adhesion molecules, suggesting that ef-
fector CD4
⫹
cells that are generated in the absence of IL-12p40
lack the appropriate receptors (e.g., CXCR3) to localize to glo-
meruli. Whereas systemic IFN-
␥
was reduced in IL-12p40
⫺/⫺
mice, local IFN-
␥
mRNA production was unchanged, suggest-
ing that intrinsic renal cell– derived IFN-
␥
may not be induced
by IL-12p40 but by IL-18. The potential differential roles of
IL-12 and IL-23 in immune renal disease remain to be explored,
although current data in EAE suggest both systemic and local
roles for IL-23 in inflammatory disease (20).
Figure 6. Leukocyte accumulation in mice with accelerated autologous-phase anti-GBM GN at 10 d. (A through C) Values for
glomeruli, expressed as cells per glomerular cross section (c/gcs). (D through F) Values for the tubulointerstitium expressed as
cells per high-power field (c/hpf). *P ⬍ 0.05 versus WT; **P ⬍ 0.01 versus WT; ***P ⬍ 0.001 versus WT (ANOVA). Results are
expressed as the mean ⫾ SEM. Numbers of mice studied are WT C57BL/6 (n ⫽ 7), IL-12p40
⫺/⫺
(n ⫽ 6), IL-18
⫺/⫺
(n ⫽ 5), and
IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 7).
Figure 7. Expression of IL-18 protein in experimental crescentic GN. Mice with crescentic anti-GBM GN showed local expression
of IL-18 (A; brown reaction product). Negative controls included the substitution of nonimmune rabbit Ig for the primary antibody
(B) and preabsorbing the primary antibody with excess rmIL-18 (C). High-power views using diaminobenzidine (brown reaction
product) and hematoxylin counterstain (blue).
J Am Soc Nephrol 16: 2023–2033, 2005 Endogenous IL-12p40 and IL-18 in GN 2029
Acute glomerular injury was only modestly attenuated in the
absence of IL-18. Although glomerular crescent formation was
not significantly reduced in its absence, the proportion of glo-
meruli that were severely affected was diminished. The current
studies dissect this local role of IL-18 in experimental GN.
Systemically, in this response, IL-18 is not required for the
generation of a Th1 response, in contrast to IL-12p40. Antigen-
stimulated IFN-
␥
, TNF, and GM-CSF production by spleno-
cytes was not reduced in the absence of IL-18, although IL-4
and IL-5 production (as Th2 markers) was increased. Further-
more, IL-18
⫺/⫺
mice made normal dermal DTH responses. The
presence of normal antigen-specific systemic Th1 responses
means that in IL-18
⫺/⫺
mice (unlike IL-12p40
⫺/⫺
mice), CD4
⫹
cells localizing to glomeruli are likely to possess a Th1 pheno-
type. The localization of these injurious leukocytes requires the
expression of the relevant adhesion molecules and chemokines.
IL-18 was expressed locally in the kidneys of mice with GN,
suggesting that it may upregulate local recruitment. In its ab-
sence, chemokine mRNA expression (particularly MIP-1
␣
and
MIP-1

) was reduced. Consequently, leukocyte recruitment
was attenuated but not abrogated. Although less histologic
injury (excluding crescent formation) and proinflammatory cy-
tokine mRNA (TNF, IL-1

, and IFN-
␥
) was observed, injury
remained substantial. There are three likely explanations for
this finding: (1) Leukocytes, particularly CD4
⫹
cells, infiltrating
glomeruli in IL-18
⫺/⫺
mice possessed a Th1 proinflammatory
phenotype (in contrast to cells from IL-12p40
⫺/⫺
mice); (2)
numbers of Th1 cells in glomeruli IL-18
⫺/⫺
mice exceeded a
threshold number of glomerular leukocytes required to trigger
significant crescent formation (observed previously in this
model [10]); and (3) unlike IL-12p40
⫺/⫺
mice, local mRNA
expression of the protective cytokine IL-10 was not upregu-
lated. The current results, highlighting a potential role for local
IL-18 production in amplifying leukocyte-mediated immune
renal injury, are consistent with other studies showing that
IL-18 is produced locally in renal inflammation (23–25) and that
its production regulates leukocyte chemoattractants (22,39).
IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice did not show additional protec
-
tion from injury beyond that demonstrated in IL-12p40
⫺/⫺
mice. In IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice, key indicators of injury,
such as glomerular crescent formation and leukocytic infiltra-
tion, were not less than in IL-12p40
⫺/⫺
mice. If anything,
values for IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice were intermediate (less
injury than in IL-18
⫺/⫺
mice but more than in IL-12p40
⫺/⫺
mice), similar to findings in an antigen-induced model of ar-
thritis (40). The reasons for this lack of effect are not clear.
Whereas IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice usually exhibited the phe
-
notype consistent with the single gene–deficient mouse that
showed the lesser inflammation, splenocytes from IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice seemed to produce more TNF than IL-12p40
⫺/⫺
mice (supported by one previously published study [41]). How-
ever, renal TNF mRNA expression was not increased in IL-
12p40
⫺/⫺
IL-18
⫺/⫺
mice. Although combined deficiency of
three key Th1 cytokines could have switched glomerular injury
to a Th2 predominant pattern in IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice,
this is unlikely as (1) Increased antigen-specific IgG1 levels
Table 1. Intrarenal chemokine mRNA levels in mice with glomerulonephritis
a
WT C57BL/6 IL-12p40
⫺/⫺
IL-18
⫺/⫺
IL-12p40
⫺/⫺
/IL-18
⫺/⫺
CXCL10 (IP-10) 75.8 ⫾ 12.0 83.0 ⫾ 17.4 93.4 ⫾ 31.3 56.8 ⫾ 10.6
XCL1 (lymphotactin) 2.03 ⫾ 0.44 2.88 ⫾ 1.22 1.20 ⫾ 0.32 1.16 ⫾ 0.37
CCL5 (RANTES) 86.4 ⫾ 13.0 54.6 ⫾ 9.9 80.4 ⫾ 22.5 53.6 ⫾ 13.1
CCL1 (TCA-3) 7.89 ⫾ 0.99 7.27 ⫾ 1.04 7.99 ⫾ 2.15 5.61 ⫾ 1.16
CCL2 (MCP-1) 100.3 ⫾ 11.2 78.9 ⫾ 16.5 66.0 ⫾ 19.4 63.1 ⫾ 8.8
CCL3 (MIP-1
␣
) 8.06 ⫾ 1.43 6.22 ⫾ 0.40 2.91 ⫾ 0.48
c,d
3.96 ⫾ 0.56
b
CCL4 (MIP-1

) 11.6 ⫾ 1.35 7.53 ⫾ 1.66 4.12 ⫾ 1.03
d
5.98 ⫾ 0.96
b
CXCL1 (MIP-2) 17.0 ⫾ 3.4 10.5 ⫾ 3.9 7.73 ⫾ 2.22 5.27 ⫾ 0.68
b
a
Results are expressed as the means ⫾ SEM (arbitrary units). WT, wild-type mice. Numbers of mice studied are WT
C57BL/6 (n ⫽ 4), IL-12p40
⫺/⫺
(n ⫽ 4), IL-18
⫺/⫺
(n ⫽ 5), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 5).
b
P ⬍ 0.05 versus WT C57BL/6.
c
P ⬍ 0.05 versus IL-12p40
⫺/⫺
.
d
P ⬍ 0.01 versus WT C57BL/6 (ANOVA).
Table 2. P-selectin and ICAM-1 expression in mice with glomerulonephritis
a
WT C57BL/6 IL-12p40
⫺/⫺
IL-18
⫺/⫺
IL-12p40
⫺/⫺
/IL-18
⫺/⫺
Glomerular P-selectin 2.00 ⫾ 0.19 1.77 ⫾ 0.20 1.99 ⫾ 0.15 2.11 ⫾ 0.15
Glomerular ICAM-1 1.99 ⫾ 0.18 1.36 ⫾ 0.23 1.74 ⫾ 0.18 1.50 ⫾ 0.21
Interstitial ICAM-1 2.57 ⫾ 0.12 2.06 ⫾ 0.27 2.40 ⫾ 0.09 2.15 ⫾ 0.17
a
Results are expressed as the mean ⫾ SEM of the semiquantitative score of immunofluorescence intensity (0 to 3⫹).
Numbers of mice studied are WT C57BL/6 (n ⫽ 7), IL-12p40
⫺/⫺
(n ⫽ 6), IL-18
⫺/⫺
(n ⫽ 5), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 7).
ICAM-1, intercellular adhesion molecule-1.
2030 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 2023–2033, 2005
were not observed, (2) systemic IL-4 or IL-5 production was not
increased, (3) local increases in IL-4/IL-13 mRNA were not ob-
served, and (4) an increased neutrophil infiltrate (that can occur in
deviant Th2 immune responses) was not seen. The finding of a
modest increase of mouse IgG in glomeruli (at least by one
method of measurement) suggests a component of increased hu-
moral injury, perhaps relating to higher antigen-induced produc-
tion of IL-4 and IL-5. It has been shown that severe injury in this
model is independent of autologous antibody (6,14), meaning that
in this context, humoral immunity is unlikely to be the key deter-
minant of glomerular injury.
Figure 8. Intrarenal cytokine mRNA in crescentic GN. Genetically deficient mice had lower levels of IL-1

and TNF (values not
reaching significance for IL-1

in IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice and for TNF in IL-12p40
⫺/⫺
mice). IL-18
⫺/⫺
mice had lower levels
of intrarenal IFN-
␥
, suggesting that IL-18 promotes secretion of this cytokine locally. MIF and LT-
␣
levels were unchanged. IL-10
mRNA could be detected only in mice that were deficient in IL-12p40 (IL-12p40
⫺/⫺
and IL-1240
⫺/⫺
IL-18
⫺/⫺
mice). *P ⬍ 0.05
versus WT C57BL/6 mice; **P ⬍ 0.01 versus WT C57BL/6 mice (ANOVA). Results are expressed as the mean ⫾ SEM (arbitrary
units). Numbers of mice studied are WT C57BL/6 (n ⫽ 4 to 7), IL-12p40
⫺/⫺
(n ⫽ 4 to 6), IL-18
⫺/⫺
(n ⫽ 5 to 6), and IL-12p40
⫺/⫺
IL-
18
⫺/⫺
(n ⫽ 5to6).
Figure 9. Intrarenal expression of TGF-

1, TGF-

3, and type 1
procollagen (
␣
1 chain) mRNA in mice with GN. Cytokine-defi-
cient mice exhibited reduced expression of TGF-

1 and
␣
1(I)
procollagen mRNA, except for the IL-12p40
⫺/⫺
IL-18
⫺/⫺
group,
in which TGF-

1 mRNA expression did not reach statistical sig-
nificance. TFG-

3 mRNA expression was similar in all groups of
mice. *P ⬍ 0.05 versus WT C57BL/6 mice; **P ⬍ 0.01 versus WT
C57BL/6 mice (ANOVA). Results are expressed as the mean ⫾
SEM (arbitrary units). Numbers of mice studied are WT C57BL/6
mice (n ⫽ 5), IL-12p40
⫺/⫺
(n ⫽ 5), IL-18
⫺/⫺
(n ⫽ 5), and IL-
12p40
⫺/⫺
IL-18
⫺/⫺
mice (n ⫽ 5).
Table 3. Ig and complement (C3) deposition in mice
developing GN
a
Glomerular Ig
(AU)
Glomerular C3
(AU)
WT C57BL/6 68 ⫾ 3.4 50 ⫾ 3.2
IL-12p40
⫺/⫺
91 ⫾ 5.9 61 ⫾ 5.2
IL-18
⫺/⫺
103 ⫾ 4.8
c
59 ⫾ 6.6
IL-12p40
⫺/⫺
IL-18
⫺/⫺
96 ⫾ 9.7
b
53 ⫾ 3.9
a
Results are expressed as the mean ⫾ SEM of the
quantification of Ig deposition by image capture and NIH
image analysis of fluorescence from glomeruli (antibody
dilution 1:100) for each animal. Numbers of mice studied are
WT C57BL/6 (n ⫽ 7), IL-12p40
⫺/⫺
(n ⫽ 6), IL-18
⫺/⫺
(n ⫽ 5),
and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 7). GN, glomerulonephritis.
b
P ⬍ 0.05 versus WT C57BL/6 mice (ANOVA).
c
P ⬍ 0.05 versus WT C57BL/6 mice (ANOVA).
J Am Soc Nephrol 16: 2023–2033, 2005 Endogenous IL-12p40 and IL-18 in GN 2031
Progression of inflammatory renal disease is characterized
not only by ongoing inflammation but also by the production of
profibrotic cytokines, such as TGF-

1 and matrix proteins, for
example type I collagens. Renal TGF-

1 and
␣
1(I) procollagen
mRNA was reduced in all groups (TGF-

1 in IL-12p40
⫺/⫺
IL-
18
⫺/⫺
mice not reaching significance). It has been suggested
that Th2 responses are profibrotic (42), but the current studies
demonstrate that in renal inflammation, deletion of key Th1
cytokines does not drive immune responses toward pathoge-
netic Th2 responses or seem to induce a local profibrotic state.
Rather, the dampening of renal inflammation in mice deficient
in IL-12p40 and/or IL-18 is associated with decreased produc-
tion of mRNA for prototypic markers of renal fibrosis, suggest-
ing that in this lesion, dampening ongoing inflammation will
lead to reduced progressive renal fibrosis.
In summary, these studies demonstrate that IL-18 plays local
proinflammatory roles in immune renal inflammation, whereas
IL-12p40 is the key cytokine chain in nephritogenic Th1 re-
sponses that lead to crescentic GN.
Acknowledgments
These studies were supported by Project and Programme grants from
the National Health and Medical Research Council of Australia.
Parts of this work was previously published in abstract form (Clin
Invest Med 27: 58B; Nephrology 9[Suppl 1]: A5–A6; J Am Soc Nephrol 15:
40A–41A, 2004).
We acknowledge the technical assistance of J. Sharkey and A. Wright.
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Table 4. Renal function and urinary protein excretion in C57BL/6 WT, IL-12p40
⫺/⫺
, IL-18
⫺/⫺
, and
IL-12p40
⫺/⫺
IL-18
⫺/⫺
mice with GN
a
Serum Creatinine
(
mol/L)
Urinary Protein
(mg/24 h)
Urinary Protein:Creatinine
(mg/
mol)
No GN 11.1 ⫾ 0.8 1.2 ⫾ 0.8 ND
WT C57BL/6 GN 19.4 ⫾ 1.2
b
7.9 ⫾ 1.1 3.0 ⫾ 0.6
IL-12p40
⫺/⫺
GN
16.1 ⫾ 1.5 4.8 ⫾ 1.0 1.3 ⫾ 0.2
IL-18
⫺/⫺
GN
18.0 ⫾ 1.8
c
5.6 ⫾ 1.0 3.1 ⫾ 0.6
IL-12p40
⫺/⫺
/IL-18
⫺/⫺
GN
18.5 ⫾ 0.9
c
5.1 ⫾ 0.4 1.9 ⫾ 0.2
a
Results are expressed as the mean ⫾ SEM. Numbers of mice studied for serum creatinine are No GN (n ⫽ 8) WT C57BL/6
(n ⫽ 20), IL-12p40
⫺/⫺
(n ⫽ 4), IL-18
⫺/⫺
(n ⫽ 16), and IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 13) and for proteinuria are No GN (n ⫽ 13)
WT C57BL/6 (n ⫽ 19), IL-12p40
⫺/⫺
(n ⫽ 5), and IL-18
⫺/⫺
(n ⫽ 16), IL-12p40
⫺/⫺
IL-18
⫺/⫺
(n ⫽ 13).
b
P ⬍ 0.01 versus No GN.
c
P ⬍ 0.05 versus No GN (ANOVA).
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J Am Soc Nephrol 16: 2023–2033, 2005 Endogenous IL-12p40 and IL-18 in GN 2033
