| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
§
From the Laboratorio di Immunopatologia Renale,*
Dipartimento di Medicina Interna, and Dipartimento di Scienze
Biomediche ed Oncologia Umana,§
Università di Torino,
the Cattedra di Nefrologia,
Università
di Parma, and the Dipartimento di Scienze Cliniche e
Biologiche,
Università
dell'Insubria, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and
bacterial polysaccharides, induce the expression of IL-12 mRNA
and the synthesis of the protein by cultured mesangial cells.
Moreover, cultured mesangial cells were shown to bind IL-12 and
to express the human low-affinity IL-12 ß1-chain receptor. When
challenged with IL-12, mesangial cells produced PAF in a dose-
and time-dependent manner and superoxide anions. No production of tumor
necrosis factor- and IL-8 was observed. Moreover, we
demonstrate that IL-12 induced a delayed and sustained shape change of
mesangial cells that reached its maximum between 90 and 120 minutes of
incubation. The changes in cell shape occurred concomitantly with
cytoskeletal rearrangements and may be consistent with cell
contraction. As IL-12-dependent shape change of mesangial cells was
concomitant with the synthesis of PAF, which is known to
promote mesangial cell contraction, we investigated the role of
PAF using two chemically different PAF receptor antagonists. Both
antagonists inhibited almost completely the cell shape change induced
by IL-12, whereas they were ineffective on
angiotensin-II-induced cell shape change. In conclusion, our
results suggest that mesangial cells can either produce IL-12 or be
stimulated by this cytokine to synthesize PAF and to undergo shape
changes compatible with cell contraction.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, interleukin (IL)-1, and IL-6, may affect MC functions
by stimulating cell contraction, proliferation, or matrix
production.3
Recently, it has been shown that lipid
mediators may contribute to the biological activities exerted by
certain cytokines. In particular, it has been shown that PAF, a
phospholipid mediator of inflammation with a large spectrum of
biological activity,4-6
directly stimulates MC
contraction7
and that an endogenous production of PAF
mediates the contraction induced by TNF-8
and
endothelin-1.9
In several experimental models, PAF affects
glomerular filtration and permeability and contributes to glomerular
pathology.10
We have recently shown that the synthesis of
PAF induced by IL-12 contributes to the activation of human
neutrophils.11
IL-12 is an heterodimeric cytokine, composed
of a 40-kd and a 35-kd subunit, which displays a key role in the
initiation of both innate and antigen-specific pro-inflammatory
immunity.12-15
This cytokine is mostly produced by
phagocytic cells and B lymphocytes in response to lipopolysaccharide
(LPS) and other bacterial products.12,15
Recently, IL-12
has been also involved in the pathogenesis of autoimmune
diseases.16
In particular, a prominent IL-12-dependent Th1
response has been demonstrated in some experimental
glomerulonephritis.17
In MRL-Faslpr mice, which
develop a lupus nephritis, an enhanced expression of IL-12 within the
nephritic kidney has been shown.18
Cultured proximal
tubular cells derived from the MRL-Faslpr mouse kidney were
also capable of producing IL-12.18
However, the production
of IL-12 from glomerular cells has not been investigated. The aim of the present study was to evaluate whether MCs are capable of producing IL-12 and whether IL-12 may regulate some of the MC-related functions. In particular, we studied the ability of IL-12 to stimulate the production of PAF, superoxide anions (O2-), and cytokines and to induce changes of the shape of MCs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Polymyxin B, phospholipase A2, phospholipase A1, bovine serum albumin (BSA) fraction V (tested for not more than 1 ng of endotoxin per mg), Formyl-met-leu-phe (FMLP), sphingomyelin, and lyso-2-phosphatidylcholine (lyso-PC), fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG were purchased from Sigma Chemical Co. (St. Louis, MO). Collagenase from Clostridium histolyticum was from Boehringer (Mannheim, Germany); human factor VIII antiserum was from Nordic Immunology (Tilburg, The Netherlands); anti-smooth muscle cell myosin antibodies were from Immunotech (Marseille, France); and mouse monoclonal anti-cytokeratin antibodies, anti-collagen type IV antibodies, and anti-fibronectin were from Labometrics (Milano, Italy). IL-12 was a kind gift of G. Trincheri, Genetics Institute (Cambridge, MA). The anti-IL-12 neutralizing monoclonal antibody (MAb) C.8.6 and the anti-IL-12 non-neutralizing MAb C.11.515 were a kind gift from G. Trincheri. Anti-IL-12 receptor 12Rß.44 MAb19 was a kind gift from J. Ritz (Dana-Farber Cancer Institute, Boston, MA). All mouse anti-IL-12 MAbs and anti-IL-12 receptor MAbs were of the IgG1 isotype. The corresponding irrelevant isotypic control (mouse IgG1) was purchased from Cedarlane (Hornby, Ontario, Canada). Synthetic PAF (1-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) was obtained from Bachem Feinchemikalien (Bubendorf, Switzerland); the stock solution in chloroform was stored at -20°C until use. The chloroform was evaporated, and saline containing 0.25% BSA fraction V, low endotoxin (Sigma), was added immediately before use. WEB 2170 was obtained from Boehringer and CV 3988 from Takeda Chemical Industries (Kyoto, Japan).
TNF- and LPS from Escherichia coli (0111:B4) were
purchased from Sigma. The stock solution of LPS was prepared by
suspending 10 mg of LPS in 2 ml of 20 mmol/L EDTA and by sonicating
until clarification (three to five times a 20-second burst at maximal
intensity using a W375 sonicator with a number 419 microtip, Heat
Systems-Ultrasonics (Farmingdale, NY). Aliquots of LPS stocks (200
µl) were stored at -20°C and when thawed for use were sonicated
for 15 seconds using a microsonicator (Microson, Heat
Systems-Ultrasonics). LPS working dilutions were prepared in 10 mmol/L
Hepes saline formulated using 1 mol/L Hepes stock (Gibco Laboratories,
Grand Island, NY) and sterile, nonpyrogenic saline.
Culture of Human Mesangial Cells
Human glomeruli were isolated from surgical specimens of kidneys by the method described by Striker et al.20 The separated cortex was sliced and forced through a graded series of stainless steel meshes, and isolated encapsulated glomeruli were recovered. MCs were obtained from collagenase-treated, isolated glomeruli to remove the epithelial cell component.21 Washed glomerular remnants were plated at a density of ~300 glomeruli/cm2 in Dulbecco's modified Eagle's medium (DMEM) and 20% fetal calf serum tested for endotoxin levels less than 0.1 ng/ml (Sigma), 50 U/ml penicillin, and 50 µg/ml streptomycin; culture flasks were kept in a 95% air, 5% CO2 environment at 37°C. After three weeks in primary culture, MCs were harvested with 0.05% trypsin, 0.02% ethylene-diamine-tetracetate (EDTA). Subcultures were grown in the same medium. The MCs used were characterized by the following criteria:20 1) morphological appearance of stellate cells growing in interwoven bundles, 2) uniform fluorescence with FITC-phalloidin (F-PHD) specific for F-actin, 3) immunofluorescence staining for smooth muscle-type myosin, 4) immunofluorescence staining of extracellular matrix for type IV collagen and fibronectin using monospecific antisera, and 5) negative immunofluorescence staining for HLA-DR and leukocyte common antigen (CD-45) and human factor VIII antigens. In parallel experiments, cell viability was monitored by Trypan blue and ranged between 88% and 95%.
IL-12 Receptor Analysis
The presence of the IL-12 receptor on MCs was evaluated by cytofluorimetric analysis by assessing IL-12 binding to the putative receptor using the technique described in.22 MCs detached using 0.05 mol/L EDTA were first incubated with heat-inactivated human serum to block nonspecific sites. MCs (2 x 106) in 100 µl of staining buffer (PBS containing 2% heat-inactivated human serum and 0.1% sodium azide) were sequentially incubated with IL-12 (10 ng/ml) for 1 hour, followed by incubation with the anti-IL-12 MAb C.11.5 or the neutralizing anti-IL-12 MAb C.8.6 or the irrelevant isotypic control for 30 minutes and finally with FITC-conjugated goat anti-mouse IgG for 20 minutes. All incubations were performed in staining buffer at 4°C, and cells were washed twice between incubations. Phytohemagglutinin-activated peripheral blood mononuclear cells, prepared as described previously,22 were used as positive control.
The stained cells were analyzed on a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA).
Immunoprecipitation and Western Blot Analysis Studies
MCs (20 x 106) were extracted with cold detergent-insoluble matrix buffer (50 mmol/L Pipes, pH 6.8, 100 mmol/L NaCl, 5 mmol/L MgCl2, 300 mmol/L sucrose, 5 mmol/L EGTA, 2 mmol/L sodium orthovanadate) plus 1% Triton X-100 and a mixture of protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 0.15 U/ml aprotinin, 1 µg/ml pepstatin A) for 20 minutes at 4°C and centrifuged at 15,000 x g for 20 minutes. The clarified supernatant was precleaned for 1 hour with 50 µl of Sepharose-protein A (3 mg/sample). The protein concentration of MC lysates was determined by the Bradford's technique, and the protein content of the samples was normalized to 250 mg/sample by appropriate dilution with the cold DIM buffer. The samples were then incubated with 2 µg of 12Rß.44 MAb or the isotypic control and adsorbed by anti-mouse IgG coupled to Sepharose-protein A. Bound proteins were washed several times in DIM buffer and eluted in boiling Laemmli buffer. Thirty microliters of eluted proteins were subjected to 8% SDS-polyacrylamide gel electrophoresis. Lymphocytes (20 x 106) were extracted by sonication. Proteins were then transferred electrophoretically to nitrocellulose; the filters were incubated with blocking solution (10% low-fat milk in 20 mmol/L Tris/HCl, pH 7.6, and 17 mmol/L NaCl) for 1 hour. The anti-IL-12 receptor 12Rß.44 MAb (2 µg) was then added at the same solution, and the incubation was carried out overnight at room temperature. For detection, the filters were washed four times (15 minutes each wash) with PBS, 0.5% Tween 20 and reacted for 1 hour at room temperature with horseradish-peroxidase-conjugated protein A. The enzyme was removed by washing as above. The filters were reacted for 1 minute with a chemiluminescence reagent (ECL) and exposed to an autoradiography film for 1 to 15 minutes. To reprobe, nitrocellulose filters were first stripped of antibody by 62 mmol/L Tris/HCl, pH 6.7, 2% SDS, 100 mmol/L ß2-mercaptoethanol.
IL-12 mRNA Expression
IL-12 p40-specific mRNA was detected in total RNA extracted from MCs by guanidinium thyocyanate phenol-chloroform and precipitated with isopropanol. One microgram of RNA was treated with 6 U of RNAse-free DNAse for 1 hour at 37°C and then for 5 minutes at 94°C; complementary DNA was obtained by using random hexamer primers (Perkin-Elmer Cetus, Norwalk, CT). Reverse transcription was carried out at 42°C for 60 minutes; in addition to 1 µg of RNA, the reaction mixture (20 µl) contained 10 mmol/L Tris/HCl, pH 8.3, 50 mmol/L KCl, 5 mmol/L MgCl2, 1.0 mmol/L dNTPs, 20 U of ribonuclease inhibitor, and 50 U of Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer Cetus). cDNA was then subjected to 35 cycles of amplification by the polymerase chain reaction (PCR) in an automated DNA thermal cycler (Perkin-Elmer Cetus) by using human IL-12 p40 mRNA-specific primer pairs (R&D Systems, Abingdon, UK). The PCR reaction mixture (50 µl) contained 10 mmol/L Tris/HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 20 pmol of (+) and (-) primers, and 2 U of thermostable DNA polymerase (Perkin-Elmer Cetus). Times and temperatures for denaturation, annealing, and extension were 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C, respectively. Amplification product (559 bp) was analyzed in 2% agarose gels containing 0.5 µg/ml ethidium bromide.
Purification and Quantification of PAF
The production of PAF from MCs stimulated with IL-12 was studied. Cells were equilibrated for 15 minutes in Tris-buffered Tyrode's buffer containing 0.25% delipidized BSA (fraction V), as previously described,23,24 and incubated at 37°C for the indicated time with IL-12 at different concentrations. To assess the specificity of the reaction, IL-12 was preincubated for 10 minutes at 37°C with the neutralizing anti-IL12 MAb C.8.6 (10 µg/ml). Selected experiments were conducted in the presence of 5 µg/ml polymixin B for 30 minutes at 37°C to exclude LPS contamination. The supernatants and the cell pellets were extracted according to a modification of the Bligh and Dyer procedure,25 with formic acid added to lower the pH of the aqueous phase to 3.0. Each individual experiment was performed in duplicate.
PAF was quantified after extraction and purification by thin layer chromatography (silica gel plates 60 F254, Merck, Darmstadt, Germany) and high-pressure liquid chromatography (µPorasil column, Millipore Chromatographic Division, Waters, Milford, MA) by aggregation of washed rabbit platelets, as previously reported.23,24 The biologically active material extracted from cells and supernatants in different experiments was characterized by comparison with synthetic PAF according to the following criteria:23-27 1) induction of platelet aggregation by a pathway independent from both ADP and arachidonic acid/thromboxane-A2-mediated pathways, 2) specificity of platelet aggregation as inferred from the inhibitory effect of 5 µmol/L WEB 2170 or CV 3988, two different PAF receptor antagonists,28,29 3) thin-layer chromatography and high-pressure liquid chromatography behavior and physico-chemical characteristics, such as inactivation by strong bases and by phospholipase A2 treatment, but resistance to phospholipase A1, acids, weak bases, and 5 minutes of heating in boiling water.26,27
Cytokine Detection
The presence of IL-12 protein was measured in the supernatants
from MCs unstimulated or stimulated with LPS (100 ng/ml) or TNF-
(10 ng/ml) with an ELISA kit that specifically detects only the
heterodimeric form of the molecule (Genzyme, Cambridge, MA). The
quantitative determination of TNF- and IL-8 in the supernatant of
IL-12-stimulated MCs was performed by ELISA using specific kits
(Genzyme).
O2- Assay
Production of O2- was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C.30 MCs (2.5 x 106 cells) were incubated at 37°C in Tyrode's buffer (2.6 mmol/L KCl, 1 mmol/L MgCl2, 137 mmol/L NaCl, 6 mmol/L CaCl2, 0.1% glucose, 1 mmol/L Tris, pH 7.4) containing 80 µmol/L cytochrome C with or without superoxide dismutase (50 U/ml) and appropriately stimulated. Basal O2- production was assessed in the absence of stimulating factors. Supernatants were removed at specified times and centrifuged, and the absorbance was measured in a spectrophotometer at 550 nm. The extinction coefficient of ferricytochrome C at 550 nm was taken as 2.1 x 104 (mol/L)-1 cm-1. Protein content of MCs was measured according to the Lowry technique. O2- production was expressed as nmol/L cytochrome C reduced/mg of protein.30
Shape Change of MCs
MCs, seeded in small petri dishes (35-mm diameter) coated with dimethylpolyxiloxane at subconfluent density, in DMEM with 0.25% BSA, were kept in an attached, hermetically sealed plexiglass Nikon NP-2 incubator at 37°C. Cells were stimulated with IL-12 (20 ng/ml), AT-II (10-7 mol/L), and PAF (10 nmol/L). To evaluate the role of PAF in IL-12-dependent shape change, cells were incubated for 10 minutes with WEB 2170 (3 µmol/L) and CV 3988 (5 µmol/L) before stimulation. Cell shape change was studied over 2-hour period under a Nikon Diaphot inverted microscope with a 20x phase-contrast objective. Cell shape change was recorded using a JVC-1CCD video camera. Image analysis was performed with a MicroImage analysis system (Cast Imaging srl, Venice, Italy) and an IBM-compatible system equipped with a video card (Targa 2000, Truevision, Santa Clara, CA). Image analysis was performed by digital saving of image compared before stimulation and then at 5-minute intervals for 2 hours. The cell planar surface was calculated by the MicroImage software. The reduction of the planar cell surface >15% was used as a parameter of cell shape change compatible with a cell contraction. Both the number of contracted cells and the mean cell contraction were indicated. Between 10 and 25 cells were analyzed for each experimental condition and repeated at least four times. Values are given as mean ± SE.
Cytoskeleton Alterations
Actin microfilament alterations in IL-12-stimulated MCs was evaluated as binding of FITC-phalloidin. MCs seeded on glass coverslips coated with dimethylpolyxiloxane were stimulated for 2 hours with IL-12 (20 ng/ml), with IL-12 plus the neutralizing anti-IL-12 C.8.6 MAb, with IL-12 plus the PAF-receptor antagonist WEB 2170 (3 µmol/L), or with AT-II (10-7 mol/L) for 15 minutes. MCs were then fixed for 5 minutes at room temperature in 3% paraformaldehyde in PBS, pH 7.6, containing 2% sucrose. After rinsing in PBS, MCs were permeabilized by soaking the coverslips for 5 minutes in 20 nmol/L Hepes, pH 7.4, 300 nmol/L sucrose, 50 mmol/L MgCl2, and 0.5% Triton X-100. To stain actin microfilaments, direct staining using F-PHD (30 minutes at 37°C at the concentration of 2 µg/ml) was performed. F-PHD, which directly binds to F-actin, was used according to the method of Wulf et al.31
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We studied the expression by cultured MCs of the IL-12 p40 subunit
mRNA and the synthesis of the heterodimeric protein p70 in basal
conditions and after stimulation with LPS and TNF-. Figure 1
shows that the expression of IL-12
p40-specific mRNA in MCs was inducible on cell activation. MCs cultured
for 18 hours in basal conditions did not express detectable IL-12
p40-specific mRNA by reverse transcription PCR. In contrast, after
stimulation with LPS or TNF-, MCs expressed the IL-12 mRNA.
Moreover, MCs stimulated with LPS and TNF- synthesized and released
the IL-12 protein. The synthesis peaked at 24 hours to become
undetectable after 48 hours (Figure 2A)
.
MCs challenged with the vehicle alone as control did not synthesize
detectable amounts of IL-12. Figure 2B
shows the dose dependency of
IL-12 synthesis induced by TNF-. The inhibitory effect of
cycloheximide, which prevents protein synthesis, suggests that
the released IL-12 was newly synthesized (Figure 2B)
.
|
|
IL-12 binding to the putative receptor was evaluated by incubating
MCs with IL-12 followed by staining with the anti-IL-12 MAb C.11.5. As
shown in Figure 3
, MCs expressed
significant levels of IL-12 binding. MCs unchallenged with IL-12 or
incubated with the irrelevant isotypic control were not stained. IL-12
receptor detection specificity by this method was confirmed by the
diminished staining observed when the C.11.5 MAb was replaced with the
neutralizing anti-IL-12 C.8.6 MAb (Figure 3)
. Because the neutralizing
anti-IL-12 C.8.6 MAb recognizes the IL-12 receptor-binding epitope, the
reaction of IL-12 with its receptor prevents binding of neutralizing
anti-IL-12 MAb to cell-associated IL-12 as reported by Desai et
al.22
As positive control, IL-12 binding to the putative
receptor was detected on peripheral blood mononuclear cells activated
for 72 hours with phytohemagglutinin (data not shown).
|
. The expression of the ß1 receptor
in unstimulated lymphocytes was used as control.
|
IL-12 induced a time-dependent (Figure 5A)
and dose-dependent (Figure 5B)
production of PAF from MCs. Time course studies showed a peak of PAF
production 30 minutes after stimulation with IL-12 (Figure 5B)
. PAF
detected at that time was all cell associated, whereas 120 minutes
after stimulation PAF was detected as released in the supernatant. Cell
viability tested at the end of each experiment by Trypan blue dye
exclusion test was >90%. To evaluate the specificity of the effect
induced by IL-12, the cytokine preparation was incubated for 30 minutes
at 37°C with the neutralizing C.8.6 anti-IL12 MAb. Both
cell-associated and cell-released PAF production was almost completely
abrogated in the presence of the C.8.6 MAb, whereas the TNF--induced
PAF synthesis was not (Figure 5C)
. The synthesis of PAF induced by
IL-12 did not require protein synthesis, as treatment of MCs with
cycloheximide before stimulation with IL-12 did not prevent the
synthesis of PAF (Figure 5C)
.
|
We evaluated the production of TNF- and IL-8 by MCs stimulated
with various doses of IL-12 (5 to 20 ng/ml) for 8, 12, 24, and 48
hours. IL-12 failed to stimulate the production of TNF- or IL-8 by
MCs at all concentrations and times tested (data not shown).
IL-12 induced O2- production by MCs, evaluated
as reduction of cytochrome C. The production of
O2- peaked 1 minute after the addition of
IL-12 and was abrogated by preincubation of IL-12 with 10 µg/ml C.8.6
neutralizing anti-IL12 MAb (Figure 6)
.
|
Shape change of MCs, compatible with a cell contraction, was
evaluated as changes in planar surface area in response to different
stimuli. As shown in Table 1
, IL-12
induced a reduction of the cell planar surface of >15% in 84% of
MCs. Figure 7
is representative of MC
shape change observed after stimulation with IL-12. Preincubation with
neutralizing C.8.6 anti-IL-12 MAb prevented the IL-12-induced reduction
of the cell planar surface (Table 1)
. A similar reduction of the cell
planar surface was obtained with AT-II and synthetic PAF. However, the
kinetics of cell shape change were different depending on the stimuli
(Figure 8)
. Whereas the reduction of the
cell planar surface induced by IL-12 was delayed and sustained,
reaching its maximum between 90 and 120 minutes of incubation,
reduction of the cell planar surface induced by AT-II was rapid and
transient. The cell shape change induced by PAF was also rapid, but
sustained up to 60 minutes. As the kinetics of cell shape change
induced by IL-12 was concomitant with that of PAF production (Figure 5)
, we evaluated whether the production of PAF could mediate the shape
change of MCs induced by IL-12 using WEB 2170 and CV 3988, two
structurally different PAF-receptor antagonists.28,29
As
shown in Table 1
, the receptor antagonists significantly reduced cell
shape change induced by IL-12, as well as that induced by synthetic
PAF. In contrast, as previously reported,33
AT-II-induced
contraction was not prevented by the addition of the PAF-receptor
antagonists. The changes in cell shape of MCs were reversed by
replacement of the stimuli with fresh medium. No significant cell shape
change was observed in MCs stimulated with the vehicle alone.
|
|
|
Morphological alteration of IL-12-stimulated MCs were associated
with cytoskeleton changes. Resting MCs showed an elaborate array of
microfilament bundles of the stress fiber type after staining with
F-PHD, which binds specifically to F-actin (Figure 9A)
. Within 1 to 2 hours after addition
of IL-12, MCs lost their regular array of microfilament bundles, and
F-actin appeared to be predominantly associated with the cell
periphery; most stress fibers disappeared, and ruffles were often seen
(Figure 9B)
. Several MCs showed leading edges and a prominent tail
compatible with cell movement (Figure 9C)
. These changes of
cytoskeleton were similar to those induced by PAF8
and were
inhibited by WEB 2170 (Figure 9D)
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and that IL-12 interacts
with MCs and induces cell shape change and cytoskeletal reorganization
by a mechanism involving the synthesis of PAF. IL-12 is involved in
both the innate resistance mediated by phagocytic and NK cells and the
adaptive immune response mediated by T and B cells.12
IL-12
regulates the cytotoxic activity, the proliferation, and the cytokine
production of NK and T cells.13-15
IL-12 also regulates
the inflammatory cell recruitment in tissues, acting as a chemotactic
agent.34,35
In adaptive immunity, IL-12 induces a Th1-type
immune response that is particularly beneficial in infectious processes
involving intracellular pathogens and parasites.16
Furthermore, IL-12-dependent Th1 responses have been demonstrated to
cause or exacerbate autoimmune diseases.16
IL-12 has been
implicated in several experimental and human autoimmune diseases,
including autoimmune encephalitis in mice,36
diabetes in
NOD mice,37
and Wegener granulomatosis in
humans.38
Moreover, a prominent IL-12-dependent Th1
response has been implicated in some experimental
glomerulonephritis.17
In particular, the host's propensity
to develop a Th1-type response has been correlated with the
susceptibility and the severity of a crescentic glomerulonephritis in
mice and rat.39,40
The pathogenetic role of the Th1
response has been also demonstrated in a lupus-like glomerulonephritis
in mice.41
In this experimental model, the inhibition of
the Th1 response was capable of ameliorating the disease and of
down-regulating the appearance of Th1- mediated nephritogenic
IgG subclasses of antibodies. Recently, it has been demonstrated that
in MRL-Faslpr mice, which spontaneously develop a lupus
nephritis, IL-12 is up-regulated in the nephritic kidney.18
Moreover, cultured proximal tubular cells derived from
MRL-Faslpr mice produce IL-12. This observation is of
interest as only a few nonhematopoietic cells, such as
keratinocytes,42
have been previously shown to produce
IL-12. In the present study, we could show that, in vitro,
human MCs are capable of producing IL-12 in response to
pro-inflammatory stimuli such as TNF- and LPS. Additional studies
are needed to confirm whether MCs synthesize IL-12 in vivo
in experimental or human glomerular injury. Although MCs produce only
low quantities of IL-12 compared with LPS-activated
monocytes,15
a local production of IL-12 by MCs could
contribute to glomerular injury either by promoting recruitment of
inflammatory cells or by stimulating the development of a Th1 immune
response. Recently, the presence of Th1 cells and cytotoxic lymphocytes
has been shown in glomeruli of rats developing Heymann nephritis and
has been correlated with the onset of proteinuria.43
MCs were also shown to be directly stimulated by IL-12. The functional
high-affinity IL-12 receptor is composed of two ß-type cytokine
receptor subunits, each independently exhibiting a low-affinity binding
for IL-12.44
Whereas the ß1 subunit is expressed
in basal conditions on lymphocytes,45
the distribution of
the ß2 subunit is more restricted, and its expression appears to be
related to lymphocyte activation or differentiation.46,47
Herein, we demonstrate that cultured MCs bind IL-12 and express the
human low-affinity IL-12 ß1 chain receptor under basal conditions. A
similar expression of the low-affinity IL-12 receptor ß1 chain has
been observed in unstimulated T lymphocytes,45
in which
IL-12, even in the absence of the ß2 subunit, induces an efficient
production of interferon-
12
and in neutrophils, in which
IL-12 stimulates the synthesis of PAF.11
We observed that
IL-12 induces a dose-dependent synthesis of PAF also by MCs.
IL-12-induced synthesis of PAF by MCs started rapidly and was detected
both as associated to the cellular fraction and as released in the
supernatant. On phagocytic cells, IL-12 induces production of
interferon- and of other cytokines, such as granulocyte/macrophage
colony-stimulating factor, IL-8, and TNF-.12,48,49
As
MCs may synthesize PAF with a cytokine-dependent mechanism, we
evaluated whether IL-12 could induce cytokine synthesis by MCs. We
failed to detect synthesis of TNF- or IL-8 by IL-12-stimulated MCs.
Moreover, treatment of MCs with cycloheximide before stimulation with
IL-12 did not prevent the synthesis of PAF, suggesting a direct
stimulatory action of IL-12 on the synthesis of this mediator rather
then a cytokine-dependent synthesis. Previous studies have shown that
PAF exerts several effects on renal function.10
Beside a
vascular effect, PAF reduces the glomerular filtration by inducing the
contraction of mesangial cells.7
The contraction of
mesangium is in fact correlated with a decrease of the filtration area
and may therefore affect the coefficient of filtration.1-3
Herein, we demonstrate that IL-12 can also induce shape change of MCs
compatible with cell contraction. The cell shape change induced by
IL-12 was delayed and sustained, reaching its maximum between 90 and
120 minutes of incubation. The changes in cell shape occurred
concomitantly with cytoskeletal rearrangements; in particular, the
fluorescence of F-actin decreased and its distribution appeared more
associated with the cell periphery. These alterations were similar to
those induced by PAF and by TNF-, which has been shown to act via
the synthesis of PAF.8
Moreover, the kinetic of
IL-12-dependent MC shape change was concomitant with that of PAF
production. A role for PAF in IL-12-dependent cell shape change was
investigated using two different PAF receptor antagonists. Both
antagonists almost completely inhibited the cell shape change induced
by IL-12 although they were ineffective on AT-II-induced cell shape
change, as previously reported by Neuwirth et al.33
In conclusion, these results suggest that cultured MCs produce IL-12, possess the IL-12 low-affinity ß1 receptor, and can be directly stimulated by this cytokine to produce PAF and to change their shape with a cell retraction. IL-12 produced by MCs could, therefore, have an autocrine effect by regulating MC functions and a paracrine effect by activating inflammatory cells and stimulating a Th1 response within the glomeruli.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC); by CNR, Target project Biotechnology; by Istituto Superiore di Sanità, target project Artificial Organs and Organ Transplantation; and MURST 40% to G. Camussi. B. Bussolati is a postdoctoral student in Nephrology, University of Parma, Italy.
Accepted for publication November 17, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression. Eur J Immunol 1997, 27:647-652[Medline]
1-proteinase inhibitor or 1-antichymotrypsin and is stimulated by proteinases. J Exp Med 1988, 168:1293-1306 production by NK cells stimulatory factor (NKSF): characterization of the responder cells and synergy with other inducers. J Exp Med 1991, 173:869-879This article has been cited by other articles:
 |
|
 |
|
  P. Dentelli, A. Rosso, A. Zeoli, R. Gambino, L. Pegoraro, G. Pagano, R. Falcioni, and M. F. Brizzi Oxidative Stress-mediated Mesangial Cell Proliferation Requires RAC-1/Reactive Oxygen Species Production and beta4 Integrin Expression J. Biol. Chem., September 7, 2007; 282(36): 26101 - 26110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Herrera, S. Bruno, S. Buttiglieri, C. Tetta, S. Gatti, M. C. Deregibus, B. Bussolati, and G. Camussi Isolation and Characterization of a Stem Cell Population from Adult Human Liver Stem Cells, December 1, 2006; 24(12): 2840 - 2850. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Brown, H. R. Lock, S. H. Sacks, and M. G. Robson TLR2 Stimulation of Intrinsic Renal Cells in the Induction of Immune-Mediated Glomerulonephritis J. Immunol., August 1, 2006; 177(3): 1925 - 1931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Bussolati, S. Bruno, C. Grange, S. Buttiglieri, M. C. Deregibus, D. Cantino, and G. Camussi Isolation of Renal Progenitor Cells from Adult Human Kidney Am. J. Pathol., February 1, 2005; 166(2): 545 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Bussolati, G. Peri, G. Salvidio, D. Verzola, A. Mantovani, and G. Camussi The Long Pentraxin Ptx3 Is Synthesized in IgA Glomerulonephritis and Activates Mesangial Cells J. Immunol., February 1, 2003; 170(3): 1466 - 1472. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. I. Khan, P. A. Blennerhassett, Y. Deng, J. Gauldie, B. A. Vallance, and S. M. Collins IL-12 gene transfer alters gut physiology and host immunity in nematode-infected mice Am J Physiol Gastrointest Liver Physiol, July 1, 2001; 281(1): G102 - G110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. TIMOSHANKO, A. R. KITCHING, S. R. HOLDSWORTH, and P. G. TIPPING Interleukin-12 from Intrinsic Cells Is an Effector of Renal Injury in Crescentic Glomerulonephritis J. Am. Soc. Nephrol., March 1, 2001; 12(3): 464 - 471. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or of TNF-
) as control. Data
represent the mean ± SD of three individual experiments.
or
stimulated with 20 ng/ml IL-12. PAF was detected as cell-associated
(
) and as released in
the supernatant (
).
Values are given as mean ± SE of five individual experiments.
