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Anti-inflammatory lymphokine mRNA expression in antibody-induced glomerulonephritis
Kidney International, VoL 49 (1996), pp. 117—126
Anti-inflammatory lymphokine mRNA expression in antibody-induced glomerulonephritis E'wi G. LAKKIS, FADY K. BADDOURA, EDDIE N. CRUET, KANNAN R. PAREKH, MEGUMU FUKUNAGA, and KAREN A. MUNGER Renal Division, Emoty University School of Medicine and Veterans Administration Medical Center, Atlanta, Georgia, and Pathology and Laboratoty Medicine, Veterans Administration Medical Center, Buffalo, New Yorlç, USA
Anti-inflammatory lymphokine mRNA expression in antibody-induced glomerulonephritis. T helper subset 2 (Th2) lymphocytes produce inter-
leukin 4 (IL-4) and IL-b, which exert anti-inflammatory actions on monocytes and macrophages. Thi lymphocytes, on the other hand, secrete interferon-y (IFNy) which promotes tissue inflammation. The functional dichotomy between Thl and Th2 lymphocyte subsets suggests that these cells play a regulatory role in inflammatory disease. The participation of
Th subpopulations and their lymphokine products in experimental gb-
merubonephritis (GN) has not been previously evaluated. In this study, we examined renal expression of Thi and Th2-type lymphokines in the first 48 hours of passive anti-glomerular basement membrane (anti-GBM) GN in the rat. Using the reverse transcriptase-polymerase chain reaction (RTPCR) method, apparent increase in expression of both Thi-type (IL-2 and IFNy) and Th2-type (IL-4 and IL-b) lymphokine mRNA was observed in gbomerular-enriched renal tissue obtained from nephritic rats. Induction
cytokine products of activated macrophages (IL-i, IL-6, and IL-8) are implicated in the pathogenesis of immune-complex mediated glomerular injury [10]. In contrast, Th2 lymphocytes secrete IL-4,
IL-b, and IL-13 which suppress production of inflammatory mediators by macrophages/monocytes [11, 12]. The anti-inflammatory effect of Th2-type lymphokines on macrophages in vitro is characterized by inhibition of IL-i, IL-6, IL-8, and TNFcr production while inducing IL-i receptor antagonist (IL-iRA) synthesis [12—14]. In addition, IL-4 and IL-13 induce de novo expression of
monocyte 15-lipoxygenase (15-LO), an enzyme which catalyzes the formation of anti-inflammatory eicosanoids from arachidonic acid [15—17]. Cross-regulation between Th lymphocytes has also of monocyte-derived IL-la and IL-i receptor antagonist (IL-IRA) been demonstrated: IFN'y inhibits proliferation of Th2 cells while mRNA expression was also detected shortly after initiation of GN. IL-4 and IL-b suppress production of Thi lymphokines [18]. In Evidence for influx of mononuclear cells including T lymphocytes into the vitro and in vivo data suggest that the outcome of an immune or an kidney was noted during the same time period as cytokine mRNA inflammatory response is influenced by the net balance of Thi and expression. Utilizing a monoclonal anti-rat IL-4 antibody, we also detected interleukin 4-producing cells in the renal cortex 24 hours following Th2 lymphokines [11]. Although T lymphocytes have been implicated in the pathogeninduction of GN. These experiments demonstrate for the first time
anti-inflammatory lymphokine (IL-4 and IL-b) mRNA expression and IL-4 protein production in the kidney during antibody-mediated GN. We hypothesize that Th lymphocyte subsets modulate glomerular inflammation by producing lymphokines with opposing actions.
esis of human and experimental GN, their role is not clearly Table 1. PCR primer-pairs and oligonucleotide probes utilized in this study
The participation of T lymphocytes in human and in experimental glomerulonephritis (GN) is inferred from several histological studies [1, 21. Small numbers of T cells have been detected in
Cytokine
Rat IFNy
normal rat kidney using monoclonal antibodies to cell surface markers [3, 4]. Further influx of T lymphocytes, predominantly CD4 +, is observed following induction of accelerated anti-gb- Rat IL-2 merular basement membrane (anti-GBM) GN [4—6]. CD4+ T helper cells (Th) segregate into at least two subpopulations based on the pattern of bymphokine production and the type of immune responses they promote [7—9]. Thi lymphocytes secrete interbeukin-2 (IL-2), interferon-gamma (IFN7), lymphotoxin, and tumor necrosis factor-alpha (TNFa), which promote delayed-type hyper-
Oligonucleotide sequence
Rat IL-4
Prediced size of amplified segment bp
5'-atgagtgctacacgccgcgtcttgg-3' A 5' -gagttcattgacagctttgtgctgg-3' P 5 '-gtcatcgaatcgcacctgatcactaacttc-3'
405
S
409
S
5'-catgtacagcatgcagctcgcatcc-3'
A 5' -ccaccacagttgctggctcatcat-3'
P 5 '-cacgtttaaattttacttgcccaagcaggcc-3' S
5'-tgatgggtctcagccccaccttgc-3'
378
A 5 '-ctttcagtgttgtgagcgtggactc-3' P 5' -cttacggcaacaaggaacaccacggagaac-3' Rat IL-b
S
5'-ttcccagtcagccagacccacatgc-3'
530
A 5 '-tttccaaggagttgctcccgttagc-3'
sensitivity and macrophage activation [7]. IFNY, TNFa, and Rat IL-la
P 5' -cagctgcgacgctgtcatcgatttctcccc-3' S
5'-agagggagtcaactcattggcgc-3'
800
A 5' -ggaaatctatcatggagggcagtcc-3' Received for publication April 4, 1994 and in revised form August 2, 1995 Accepted for publication August 3, 1995
P 5 '-accatccaacccagatcagcacctcacagc-3' Mouse
IL-iRA
© 1996 by the International Society of Nephrobogy
S
5'-caagccttcagaatctgggat-3'
A 5'-gtcttcctggaagtagaa-3'
Abbreviations are: S, sense; A, anti-sense; P, probe.
117
422
118
Lakkis et a!: Anti-inflammatory lymphokines in GN
Table 2. Enzymatic substrates and monoclonal antibodies used in this study and their specificities in the rat. Substrate! antibody Naphthol AS-D chioroacetate MRC HIS-17 (W3!13) MRC OX-22
15 -
CD designation —
anti-CD43
anti-CD45RC
ED1
—
ED2 MRC OX-81
— —
Reference
Specificity
Neutrophils Mast cells
[27]
Pan-T cells Myeloid cells" T helper 1 cells T cytotoxic cells Natural killer cells B cells
[38]
Monocytes Macrophages Dendritic cells
[43, 441
Tissue macrophages Monoclonal mouse Anti-rat interleukin-4
[43, 44]
[39, 40]
DW Masona
a CD, cluster of differentiation b
Variable expression of CD43 antigen MRC, University of Oxford, England (personal communication)
0
2
24
48
Time, hours Fig. 1. Induction of proteinuria following NTS injection. Urine samples were obtained from rat urinary bladders at time of sacrifice. UpJUcr is the spot urinary protein to creatinine ratio. Symbols are: (1) NTS; (D) NRS.
*Significantly greater than baseline (P <
0.05;
Dunnet multiple
comparison).
A 1353
B
Time, hours
k#O 2 162448
Time, hours
0 2 162448
1078
872 603
IFNy
310 276 234
IL-2
I L-4
IL-b
Fig. 2. Analysis of renal cortical lymphokine expression by RT-PCR following induction of anti-GBM GN: (A) experimental group (rats injected with NTS), (B) control group (rats injected with NRS). Reverse transcribed RNA samples were subjected to 40 PCR cycles in each case. Amplification products were electrophoresed on 2% agarose gels and visualized with ethidium bromide. Marker= 0X174 RF DNA!HaeIII fragment ladder.
defined [1, 2, 4]. The functional dichotomy between Thi and Th2 GN has not been previously evaluated. In this report, we studied lymphocyte subpopulations suggests that these cells could play a renal expression of Thi and Th2-type lymphokine mRNA during regulatory role in glomerular inflammation. The participation of the first 48 hours of passive anti-GBM GN in the rat. In addition, Th subpopulations and their lymphokine products in experimental mononuclear cell infiltration of the kidney was visualized and
119
Lakkis et al: Anti-inflammatoty lymphokines in GN
Time, hours 0 2 16 24 48
A GAPDH
0 2162448
Time, hours 0 2 16 24 48
Ctrl
Trme. hours 0
Time. hoL
IL
I L-4
CtrI
CtrI
B Time,hours 0 2 16 24
Time,hours 0 2 162448 IL-b
I L-4
Fig. 3. Slot-blot and Southern blot analysis of renal cortical lymphokine expression following induction of anti-GBM GN: (A) Slot-blot analysis of RT-PCR products from three different rat groups injected with NTS and one group injected with control NRS using peroxidase-labeled oligonucleotide probes (Table 1). (B) Southern
blot analysis of rat IL-4 and IL-b RT-PCR products using peroxidase-labeled eDNA probes. Bands shown correspond in size to those visualized by ethidium bromide staining (Fig. 2A).
quantitated using monoclonal antibodies to T lymphocyte and macrophage surface antigens. To substantiate the mRNA studies, in situ production of IL-4 protein by infiltrating cells was investigated using a monoclonal anti-rat IL-4 antibody. Methods Induction of glomerulonephritis
Male Sprague-Dawley rats (200 to 250 g; Charles River, MA,
USA) were injected in the femoral vein with 150 d of rabbit
examination of renal tissue and by measuring protein to creatinine ratio (Up/Ur) in urine samples obtained from the bladder at time of sacrifice. Protein concentration was determined by the Bio-Rad Protein Assay system (Bio-Rad Laboratories, Inc., USA) based on the Bradford method, and creatinine concentration was measured
using an assay kit (Sigma) based on formation of Janovski complex following addition of picric acid. To validate the nephritogenicity of NTS used in these experiments, two groups of rats (N = 5 each) were anesthetized with
mactin (100 mg/kg i.p.), and the ureters exposed via a midline incision. The ureters were catheterized with PE-lO tubing, and were injected with an equal volume of heat-inactivated, non- following a 45-minute equilibration period, baseline (pre-injecimmune, normal rabbit serum (NRS) (Sigma, St. Louis, MO, tion) urine was collected for 30 minutes. Rats were then injected USA). Rats were sacrificed at 0, 2, 16, 24 and 48 hours post- intravenously with 150 ml of either NTS or NRS, and two hours injection (N = 4 per time point). Kidneys were flushed with 20 ml later another 30-minute urine collection was obtained. Animals ice-cold Hanks balanced salt solution (HBSS) and removed. were sacrificed immediately at the end of urine collection, and Excised kidneys were immediately used for glomerular enrich- renal tissue was removed for histochemical staining of neutroment except for small sections dedicated for immunohistochemi- phils (methodology described later). All urine samples were cal studies. Disease induction was confirmed by histochemical stored at —20°C prior to protein determination. Intergroup anti-rat GBM nephrotoxic serum (NTS) (provided by Dr. Kamal F. Badr, Emory University, Atlanta, GA, USA). Control animals
120
Lakkis et at: Anti-infiammatoiy lymphokines in GN
Time, hours Control group
Experimental group L
0
2 16 24 48 2 16 24 48 C
L_.J
603 - -
310 --
S
p
41
—————————
-I
Fig. 4. GAPDH PCR amplification following reverse transcription of renal cortical RNA samples (A) and without reverse transcription (B). All samples were amplified (40 cycles) simultaneously. Control group, rats injected with NTS; experimental group, rats injected with NRS; L, 0X174 RF DNAIHaeIII fragment ladder; C, control RT-PCR in the absence of RNA.
statistical analysis was performed using the Dunnet multiple comparison test. Enrichment for rat glomeruli
Renal cortical tissue was enriched for glomeruli by graded sieving as previously described [19]. Briefly, kidneys were placed in ice-cold PBS, the renal capsule was removed, and the outer and
[211. Amplified products were electrophoresed on 2% SeaKem LE agarose (American Bioanalytical, MA, USA) and stained with ethidium bromide. DNA band corresponding to the target cytokine was identified by its predicted size (Table 1). Southern and slot-blot analysis
Products of IL-4 and IL-b RT-PCR reactions were electrophomid cortices dissected and minced with a razor blade, Minced resed on 2% agarose gels, transferred to Hybond-N+ nylon cortices were then passed stepwise through stainless steel sieves membranes (Amersham, IL, USA) by capillary Southern blotting (120 mm to 63 mm). The fraction, retained on top of the 63 mm [22], and covalently linked by UV-irradiation using a Stratalinker sieve, was washed in ice-cold PBS, examined by phase microscopy, UV cross linker (Stratagene, CA, USA). Rat IL-4 eDNA was and used immediately for RNA extraction. Glomeruli constituted purified from the plasmid pRIL-4.C10 (gift of Dr. A.J. McKnight, greater than 90% of this fraction. MRC, Oxford, UK) [23] following digestion with HindIIl (New England Biolabs, MA, USA). Rat IL-b was purified from plasmid RNA extraction and RT-PCR analysis pILl 0-30 (gift of Dr. James A. Baker, Cornell University, Ithaca, Total cellular RNA was purified from glomerular-enriched NY, USA) [241 following digestion with Hindlil and BamHI. cortical tissue by the Chomczynski and Sacchi method [201 using cDNAs were denatured and labeled with horseradish peroxidase RNAzoI B reagent (Biotecx Laboratories, Houston, TX, USA). using Enhanced Chemiluminescence (ECL) direct nucleic acid Cortical RNA from four animals/time point was pooled, and 2 sg labeling and detection system (Amersham). Membranes were was reverse transcribed using an oligo-dT primer and Moloney hybridized overnight with the labeled eDNA probes in 0.5 M NaCI murine leukemia virus reverse transcriptase (Perkin Elmer, CT, solution at 42°C, washed in 0.5 X SSC at 42°C, and detection USA). Forty cycles of PCR amplification were performed in a reagents were added as described by the manufacturer (AmerDNA Thermal Cycler 480 (Perkin-Elmer) using oligonucleotide sham). Autoradiography was performed at room temperature for primers specific for rat cytokines (Table 1). Primer pairs were one minute. designed using PCRPLAN software (pcgene, Geneva, SwitzerIL-2, IFN7, IL-4, IL-b, and IL-la RT-PCR products from land) based on the published eDNA sequences of the target three replica animal experiments were also captured on Hycytokines. Oligonucleotides were synthesized on an Applied Bio- bond-N+ nylon membrane using a slot-blot apparatus (Hoeffer systems DNA synthesizer by Dr. James Murtagh, Jr. (Emory Scientific Instruments, San Francisco, CA, USA). Hybridization University and VAMC, Atlanta, GA, USA). PCR parameters and detection was performed as described above, except that were optimized for each cytokine to achieve maximum specificity
target-specific oligonucleotide probes (Table 1) were used instead
121
Lakkis et al: Anti-inflammatoty lymphokines in GN
Time, hours
(Pharmingen Inc., CA, USA), and monocytes/macrophages (EDt
0 2 16 24 48
1353
1078
872 —.. 603
IL-i cx
310 —
and ED2) (Harlan/Serotec, IN, USA) using an immunoenzymatic kit (DAKO Corp., CA, USA) according to published procedure [26]. Monoclonal antibodies and enzymatic substrates used in this study and their specificities are summarized in Table 2. Briefly, demercurized and rehydrated B5-fixed sections were digested at RT in 0.1 mg/ml Proteinase K in 0.6 M Tris HC1/0,1%CaC12 for 30
minutes (CD43 and CD45RC) and in 0.1% Trypsin in 0.05 M Tris HC1/0.1% CaC12, pH 7.6 for three minutes (ED1, ED2, 0X81). Non-specific antibody binding sites were blocked with prediluted
276 234
IL-iRA
goat serum for 30 minutes. Sections were then incubated with primary mouse anti-rat antibody diluted 1:100 in Tris-salineTriton (TST) for one hour. Polyclonal anti-mouse link antibodies were applied for 30 minutes followed by alkaline-phosphatase anti-alkaline phosphatase (APAAP) immune complex for 30 minutes. Sections were developed in the dark with fast red chromagen
for 30 minutes. The entire procedure was carried out at room temperature, and all washes were performed in TST. Appropriate
-' a a a a
GAPDH
titers of primary antibodies were predetermined by staining similarly processed normal rat spleen sections. For neutrophil detection, formalin-fixed sections were stained cytochemically with the esterase substrate Naphthol AS-D Chloroacetate (CAE;
Sigma) according to a previously described method [27]. All sections were counterstained with hematoxylin. Positive cells
Time, hours 0
within 200 randomly selected glomeruli per animal were enumerated by light microscopy. Intraglomerular infiltration was quanti-
2 16 24 48
tated by calculating the average number of positive cells per glomerulus. Intensity of the cortical interstitial infiltrate was
exp.1
determined by counting all positive cells in the interstitial area of cortex containing 100 glomeruli. Unpaired Student's t-test was used for statistical comparisons (confidence interval set at 95%). IL-i a
exp.2
exp.3
Results Induction of glomerulonephritis
'
Nephritogenicity of anti-GBM nephrotoxic serum (NTS) used in this study was confirmed by measuring urinary protein excre-
Fig. 5. IL-la and IL-IRA mRNA induction in the renal cortex following NTS injection. Reverse transcribed RNA samples were subjected to 40 PCR cycles in each case. Amplification products were electrophoresed on 2% agarose gels and visualized with ethidium bromide (A). Slot-blot analysis of IL-la mRNA from three different animal experiments (B).
of cDNA probes [25] and autoradiography was performed at room temperature for 10 minutes. Each oligonucleotide was designed such that it hybridizes to a mid-region of the target cDNA and not to PCR amplification primers. Densitometric analysis was performed using a Molecular Dynamics densitometer (Sun Valley, CA, USA).
tion rate and magnitude of glomerular neutrophil infiltration prior to and after injection of NTS. Proteinuria increased from 0.011
0.003 mg/mm (mean
SD) pre-NTS to 0.188
0.050
mg/mm two hours following NTS administration (N = 5, P < 0.05). In contrast, proteinuria did not increase in rats injected with NRS: 0.010 0.004 mg/mm pre-injection and 0.012 0.006 two hours post-injection (N = 2). NTS administration also resulted in
a significant increase in the number of infiltrating neutrophils from 0.10 0.07 neutrophil/glomerulus in healthy rats to 3.3 1.7 neutrophils/glomerulus in rats sacrificed two and a half hours following NTS injection (N = 5, P < 0.05). Induction of glomerular injury in the rats utilized in gene expression and immunohistochemical studies was demonstrated by measuring urinary pro-
tein (Un) and creatinine concentrations (Ur) and calculating
Tissue samples from kidneys excised at 0, 2, 16, 24, and 48 hours following injection of NTS (N = 4/group/time point) or NRS (N = 2/group/time point) were formalin and/or B5-fixed and
Up/U ratios at time of sacrifice. UP/UCr increased significantly two hours following injection of NTS and declined afterwards (Fig. 1). Up/Ucr ratios did not change significantly in rats that received NRS (Fig. 1).
paraffin-embedded for accurate localization of target cell populations. Deparaffinized tissue sections mounted on silanized slides were immunostained with monoclonal antibodies against rat IL-4
Detection of Thi and Th2-type lymphokine mRNA in glomerularenriched cortical tissue
Histochemical studies
(0X81, provided by Dr. D.W. Mason, MRC, University of
Figure 2A summarizes the time course of renal Thi and Th2
Oxford, UK), CD43 (MRC HIS-17), CD45RC (MRC OX-22)
lymphokine mRNA expression during the first 48 hours of passive
4
A
B
10
* 8 a)
a,
0C 0)
E
0 +
C,,
6
4
0
*
0 2 0
0
0 8 16 24 32 40 48 56
0 8 16 24 32 40 48 56
Time, hours
Time, hours D
C
1.00 0 E 0
Fig. 6. Temporal patterns of leukocytic infiltration into glomendi. (A) CAE+ cells (LII, neutrophils); (B) CD43+ cells (E, T lymphocytes); (C) CD45RC+ cells (Thi lymphocytes) [(LI) CD45RC (NTS), (I) CD45RC (NRS)1; (D) ED1 + cells (monocytes/
0.75
0) a)
+
0
0.5
U,
00
0.25 0
0 8 16 24 32 40 48 56
0 8 1624 32404856
Time, hours
Time, hours
macrophages) [(LI) ED-i (NTS), ('I') ED-i (NRS)j. The NTS group is the rats injected with nephrotoxic serum, and the NRS group rats are injected with normal rabbit serum. All data points represent mean SEM. *significantly greater than baseline (time 0) (P < 0.05; unpaired t-test). **Significantly greater than time-matched NRS control (P < 0.05; unpaired t-test).
anti-GBM GN. A weak RT-PCR amplification signal was detected by ethidium bromide staining in healthy animals (time 0)
70
for all lymphokines studied. The intensity of the ethidium-
60
*
bromide stained bands, however, increased two hours following induction of disease in all cases. An apparent increase in staining intensity was also present at later time points but appeared to be transient in case of IL-4. Unlike the experimental group, amplification of RNA isolated from renal cortices of control animals injected with NRS did not result in a pattern of increasing staining intensity (Fig. 2B). These findings were confirmed by slot-blotting of RT-PCR products and hybridization to target-specific labeled oligonucleotide probes in three separate experimental animal
**
50
-(I,40
p0 ... 20
groups and one representative control group (Fig. 3). In each replica experiment, NTS administration resulted in an apparent
10
increase in cytokine mRNA levels above baseline (0 time point) or time-matched NRS controls (Fig. 3A). Semiquantitative densitometric measurements performed on slot-blot analyses showed on
0 0
8
16
24 32 Time, hours
40
48
56
Fig. 7. Temporal patterns of leukocytic infiltration into cortical interstitial
areas. Symbols are: (LI) CAE-NTS; (I) CD45RC-NTS; (0) ED2-NTS; (A) CD45RC-NRS. CAE+ cells (neutrophils); CD45RC+ cells (Thi
the average a 4.4-fold and 6.8-fold increase in IFNY and IL-2 mRNA expression over baseline in nephritic animals. When compared to control animals injected with NRS, IL-4 and IL-b mRNA levels increased by an average of 3.4-fold and 12-fold, respectively. Following agarose gel electrophoresis, the identities
of IL-4 and IL-b RT-PCR products were also confirmed by Southern transfer and hybridization to labeled cDNA probes (Fig.
lymphocytes); ED2+ cells (tissue macrophages); NTS group, rats injected
3B). Although IL-4 and IL-lU mRNA were barely detected in
glomeruli.
normal renal cortical tissue (time 0) when oligonucleotide probes were used (Fig. 3A), baseline expression could be demonstrated in cDNA-probed Southern blots (Fig. 3B). Figure 4 demonstrates that GAPDH amplification product was uniformly absent when reverse transcription was excluded, thus
with nephrotoxic serum; NRS group, rats injected with normal rabbit serum. All data points represent mean SEM. *Signiflcantly greater than baseline (time 0) (P < 0.05; unpaired t-test). 00Signiflcantly greater than time-matched NRS control (P < 0.05; unpaired t-test). Interstitial area in this study was defined as area of cortical interstitium containing 100
.1(1
-s
V.. —
S
I. •
p
3.
t-d.
c
.
Mm -
.—.
"r ,.
.lt9 "a.
r2.: 'I-. 4 4 so t4 •a.. •. S
r
Fig. 8. Histochemical analysis of paraffin-embedded renal sections from glomerulonephritic rats (positively stained cells appears bright red). (Magnification
x400). (A) CAE+ cells (neutrophils) at two hours following NTS injection; (B) CD43+ cells (T lymphocytes) at 24 hours; (C) CD45RC+ cells (Thi) lymphocytes at 24 hours; (D) ED1 + cells (monocytes/macrophages) at 24 hours; (E) ED2+ interstitial cells (tissue macrophages) at 48 hours; (F) IL-4-producing (OX-81 +) cells in control spleen section; (G) periglomerular IL 4-producing (OX-81+) cells at 24 hours.
124
Lakkis et al: Anti -infiammatoty lymphokines in GN
confirming the absence of DNA contamination in all RNA agents and to self-antigens implicated in autoimmune disease [11, 181. In addition, some of the cytokines secreted by Thi and
samples analyzed.
Th2 lymphocytes exert opposite effects on production of inIL-la and IL-iRA mRNA in glomerular-enriched cortical tissue flammatory mediators in vitro and are likely candidates for Because production of macrophage-derived cytokines is regu- modulating inflammatory responses in vivo [11—14]. In this lated by T cell lymphokines [13], we investigated IL-la and report we provided evidence for renal expression of antiIL-iRA mRNA expression during passive anti-GBM GN. Figure inflammatory Th2-type (IL-4 and IL-b) lymphokine mRNA 5 demonstrates the induction of both IL-la and IL-iRA mRNA and of IL-4 protein during the heterologous phase of antibodyin glomerular-enriched renal tissue as early as two hours following induced experimental GN. NTS administration. An apparent increase in IL-la mRNA Because a small number of T lymphocytes infiltrate the kidney expression is confirmed by slot-blot analysis using a target-specific
in passive anti-GBM GN, we took advantage of the high sensitivity
oligonucleotide probe (Fig. 5B). IL-la and IL-iRA mRNA of reverse transcriptase-polymerase chain reaction (RT-PCR) to expression in glomerular-enriched renal tissue from control rats injected with NRS was not detected (data not shown). Characterization of glomerular and interstitial cellular infiltrates
To determine the cellular correlate of cytokine mRNA expression in the kidney, immunohistochemical studies were performed
study temporal expression of lymphokine genes [25, 28—30]. The specifcity of the method was insured by designing primers specific for each rat cytokine analyzed and by optimizing PCR conditions
as described in the literature [21]. Identities of the amplified products were confirmed by Southern and/or slot blot analysis using target-specific cDNA and oligonucleotide probes. The slot-
on fixed renal tissue to quantitate intraglomerular and cortical blot format also allowed rapid semiquantitation of cytokine interstitial leukocytic infiltrates. The results are summarized in mRNA expression as previously described [251. In addition, we Figures 6 and 7. Representative micrographs are shown in Figures
ruled out DNA contamination of RNA samples by demonstrating
8A—E. Three separate patterns of cellular infiltration were observed during the first 48 hours of passive anti-GBM GN. A transient neutrophil influx, identified by CAE staining, appeared in both glomerular and cortical interstitial areas two hours after
that GAPDH PCR product can be visualized only if reverse transcription is performed prior to amplification. Using this
with ED1 monoclonal antibody, also rose significantly between 16
[31]. Like IL-4 and IL-b, IL-13 is mainly produced by Th2
and 24 hours (Fig. 6C). Interstitial ED1+ cells increased after induction of GN and were too numerous to count (Fig. 8D). A gradually increasing number of ED2 + tissue macrophages in the cortical interstitium was observed at 24 and 48 hours (Fig. 7). Intraglomerular ED2+ cells were not detected during the time frame of this study. In contrast to NTS, NRS administration did not increase the number of glomerular CD45RC+ and ED1 +
lymphocytes and possesses anti-inflammatory properties in vitro and in vivo [32]. We also demonstrated induction of IL-la and IL-IRA mRNA in glomerular-enriched cortical tissue following anti-GBM antibody administration. These cytokines are secreted by activated monocytes/macrophages and possibly mesangial cells and their production is regulated by Thi and Th2 lymphokines. IFNY, for example, is a potent macrophage activator which induces production of IL-i and other inflammatory mediators [33]. IL-i in turn contributes to the pathogenesis of glomerular immune injury [34, 35]. On the other hand, several anti-inflammatory actions of IL-4,
methodology, we demonstrated an apparent increase in expression of both Thi and Th2-type lymphokine mRNA in the renal injection of NTS (Figs. 6A and 7). A mononuclear cellular cortex during the first 48 hours of passive anti-GBM GN. Renal infiltrate, which stained positively with anti-CD43 or anti- tissue obtained from animals that received non-nephrotoxic seCD45RC, followed the neutrophil influx and peaked in number at rum, on the other hand, did not show an increase in lymphokine 24 hours (Figs. 6 B,C). CD45RC+ cell infiltration of the cortical mRNA expression. Detection of IL-4 and IL-b mRNA in the interstitium persisted longer than its intraglomerular counterpart kidney following induction of anti-GBM GN is consistent with our (Fig. 7). The number of intraglomerular monocytes, identified prior demonstration of IL-13 gene expression in the same model
cells over that found in healthy animals (Figs. 6 C, D). Moreover,
an interstitial CD45RC+ infiltrate was not induced by NRS (Fig. 7).
In situ production of rat IL-4 by infiltrating mononuclear cells
Utilizing a monoclonal mouse anti-rat IL-4 antibody (0X81), IL-4-producing mononuclear cells were observed in the renal interstitium following induction of GN. Figure 8F demonstrates the periglomerular location of 0X81 + cells in a renal section obtained 24 hours after NTS injection. Normal rat spleen tissue served as a positive control for immunohistochemical detection of IL-4 protein (Fig. 8G). Renal tissue obtained at other experimental time points lacked OX-81 + cells. OX-81 staining was also absent in the kidneys of control rats injected with NRS. Discussion
In contrast to the plethora of information available on the
including suppression of IL-i production and upregulation of IL-iRA synthesis, have been demonstrated in vitro and in vivo [13,
36, 371. Whether anti-inflammatory cytokines produced during passive anti-GBM GN modulate IL-i and IL-iRA production by infiltrating monocytes remains to be determined. Of significance to the aim of this study is that RT-PCR data were substantiated by immunohistochemical demonstration of rat IL-4 protein within periglomerular mononuclear cells. IL-4-producing (OX8 1+) cells were observed transiently 24 hours following induction of GN, which correlates with the apparent maximal expression of IL-4 mRNA at 16 hours. Because conventional paraffin immunohistochemistry lacks high sensitivity, it is possible
that IL-4 protein was missed in renal sections obtained at other experimental time points. how inflammation is regulated during the course of glomDemonstration of lymphokine mRNA is also corroborated by erulonephritis. CD4+ T-lymphocyte subsets, Thi and Th2, play immunohistochemical findings in paraffin-embedded sections. A a central role in regulating immune responses to infectious sparse mononuclear cell infiltrate was observed within glomeruli pathogenesis of glomerular immune injury, less is known about
125
Lakkis et al: Anti-inflammatory lymphokines in GN
and in the interstitium during the first 48 hours of disease. The cells stained positively for CD43 and peaked in number between 16 and 24 hours following induction of GN. Anti-CD43 monoclonat antibody serves as a pan-T cell marker [38] and has been utilized to detect T lymphocytes in renal tissue [3, 5]. Previous studies have shown a similar temporal pattern of T cell infiltration in accelerated anti-GBM GN [4, 51. Our results, however, showed a surprisingly high number of intraglomerular and interstitial CD43+ cells. This cannot be attributed to staining of neutrophils because they have a distinct temporal pattern of inflitrating the kidney. It is possible that anti-CD43 staining is also detecting
Acknowledgments This work was supported by a Young Investigator award from the National Kidney Foundation (FGL) and by a VA Merit Review Grant (FGL). Portions of this work were presented at the American Society of Nephrology 26th annual meeting (1993) and appeared in abstract form (JAm Soc Nephrol 4: 613, 1993). We thank Dr. Kamal F. Badr, Emory University, Atlanta, Georgia, USA, for his excellent advice. Reprint requests to Fadi G. Lakkis, MD., VAMC - Atlanta, Research 151, 1670 Clairmont Road, Decatur, Georgia 30033, USA.
References
monocytes or that the CD43 antigen is induced on resident mesangial cells and other mesenchymal cells in the interstitium during the same time frame as T cell influx. The latter speculation requires more testing by evaluating anti-CD43 staining in resting and activated cultured mesangial cells.
We also observed positive staining for CD45RC (OX-22) antigen in the mononuclear infiltrate. Although the number of CD45RC+ cells detected was small, the temporal pattern of infiltration was parallel to that of CD43+ cells. Monoclonal
antibodies to CD45RC (OX-22) serve as a marker for Thi lymphocytes in the rat [39, 40]. Based on the immunostaining profile of the mononuclear cells, the data suggest that T lymphocytes including Thi cells infiltrate the kidney and peak in number around 24 hours following induction of passive anti-GBM GN. Our results, however, are not definitive because of the lack of specificity of anti-CD43 and anti-CD45RC for T lymphocytes (Table 2). Further phenotypic characterization of these cells is limited by the inability to detect more specific T lymphocyte markers (CD3, CD4, and CD8) in paraffin sections using currently
available anti-rat antibodies and by the absence of a specific marker for Th2 lymphocytes. Frozen section immunohistochem-
istry is suboptimal for accurate localization of a rare cellular
1. MAIN 1W, NIKOLIC-PATERSON DJ, ATKINS RC: T cells and macrophages and their role in renal injury. Semin Nephrol 12:395—407, 1992
2. RoviN BH, SCJIREINER GF: Cell-mediated immunity in glomerular disease. Annu Rev Med 42:25—33, 1991 3. EDDY AA, CRARY GS, MICHAEL AF: Identification of lymphohemopoietic cells in the kidneys of normal rats. Am J Pathol 124:335—342, 1986
4. LAN HY, PATERSON DJ, ATKINS RC: Initiation and evolution of interstitial leukocytic infiltration in experimental glomerulonephritis. Kidney mt 40:425—433, 1991 5. TIPPING PG, NEALE TJ, HOLDSWORTH SR: T lymphocyte participation
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6. BOYCE NW, TIPPING PG, HOLDSWORTH SR: Lymphokine (MIF) production by glomerular T-lymphocytes in experimental glomerulonephritis. Kidney mt 30:673—677, 1986 7. MOSMANN TR, COFFMAN RL: TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145—173, 1989 8. PAPP I, WIEDER KJ, SABLINSKI T, O'CONNELL P1, MILFORD EL, STROM TB, KUPIEC-WEGLINSKI JW: Evidence for functional heterogeneity of rat CD4+ T-cells in vivo. Differential expression of IL-2 and IL-4 mRNA in recipients of cardiac allografts. J Immunol 148:1308— 1314, 1992 9. DEL PRETE GF, DE CARLI M, MASTROMAURO C, BIAGLOTFI R, MACCHIA D, FALAGIANI P, RiccI M, ROMAGNANI S: Purified protein
Our data does not prove that IL-4 and IL-lU mRNA is
derivate of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T-cells with stable and
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been shown to produce IL-4 while macrophages can produce IL-b [33, 41]. Monocytes and macrophages which infiltrate the renal cortex 24 to 48 hours following NTS injection could account for persistent IL-lU mRNA expression observed in this study.
The role of Th lymphocyte subpopulations in modulating inflammation in vivo is inferred from several animal studies [8, 11,
opposite (type I T helper and type 2 T helper) profile of cytokine production. J Clin Invest 88:346—350, 1991 Nephrol 12:428—440, 1992
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Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human m000cytes. Comparison with IL-4 and modulation
40, 42]. In experimental allergic encephalitis, predominance of by IFNy or IL-lU. J Immunol 151:6370—6381, 1993 Thi lymphokines was found to be associated with increased 13. WONG HL, COSTA GL, LOTZE MT, WAI-IL SM: Interleukin (IL) 4 disease activity while expansion of Th2 cells and their lymphodifferentially regulates monocyte IL-i family gene expression and synthesis in vitro and in viva. J Exp Med 177:775—781, 1993 kines correlated with decreased inflammation and spontaneous 14. FIORENTINO DF, ZLOTNIK A, MOSMANN TR, HOWARD MH, O'GARRA recovery [40]. Similarly, spontaneous remission of collagen-inA: IL-lO inhibits cytokine production by activated macrophages. J duced arthritis in mice correlates with increased serum IgE level Immunol 147:3815—22, 1991 which is a marker of IL-4 activity [42]. This report provides 15. NASSAR GM, MORROW JD, ROBERTS I II, LAKKIS, FG, BADR KF: Induction of 15-lipoxygenase by interleukin-13 in human blood monoevidence for renal expression of both anti-inflammatory and cytes. J Biol Chem 269:27631—27634 pro-inflammatory lymphokines in antibody-induced GN. It is 16. BADR KF: 15-lipoxygenase products as leukotriene antagonists: Therpossible that the role of T lymphocytes in experimental GN is not apeutic potential in glomerulonephritis. Kidney mt 44(Suppl 42): S101—S108, 1992 necessarily limited to mediating tissue injury. Instead, we hypothesize that Th-lymphocyte subsets modulate glomerular inflamma- 17. KATOH T, LAKKIS FG, MAKITA N, BADE KF: Co-regulated expression of glomerular 12/15-lipoxygenase and interleukin-4 mRNAs in rat tion by producing lymphokines with opposing actions. The net nephrotoxic nephritis. Kidney Int 46:341—349, 1994 balance between pro-inflammatory (Thi-type) and anti-inflam- 18. FITCH FW, McKI5Ic MD, LANCKI DW, GAJEWSKI TF: Differential matory (Th2-type) lymphokines could then determine the outregulation of murine T-lymphocyte subsets. Annu Rev Immunol 11:29—48, 1993 come of the inflammatory process.
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T: Mechanism of action of angiotensin II on isolated rat glomeruli. Kidney mt 36:985—991, 1989 20. CHOMCZYNSKI P, SACCHI N: Single-step method of RNA isolation by
cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today 15:19—26, 1994 33. ABBAS AK, LICHTMAN AH, POBER iS: Cellular and Molecular Immu-
nology (2nd ed). Philadelphia, WB Saunders, 1994, pp 239—260
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interleukin 1 gene is expressed by rat mesangial cells and is augmented in immune complex glomerulonephritis. J Immunol 138:3207—3212, 1987 35. TOMOSUGI NI, CASHMAN Si, HAY H: Modulation of antibody-mediated glomerular injury in viva by bacterial lipopolysaccharide, tumor necrosis factor, and IL-i. J Immunol 142:3083—3090, 1989 36. ALLEN JB, WONG HL, COSTA GL, BIENKOWSKI Mi, WAHL SM: Suppression of monocyte function and differential regulation of IL-i and IL-lra by IL-4 contribute to resolution of experimental arthritis. Jlmmunol 151:4344—4351, 1993
21. HOSTA L, FLICK P: Enhancement of specificity and yield in PCR, in How to Optimize PCR Reactions. Editorial Comments (United States Biochemical Corp. publication), Winter 1991—1992, vol 18, pp 1—5 22. BROWN T: Analysis of DNA sequences by blotting and hybridization, in Current Protocols in Molecular Biology, edited by AUSUBEL FM, BRENT R, KINGSTON RE, MOORE DD, SEIDMAN JG, SMITH JA, STRUHL K, New York, Greene Publishing Associates and WileyInterscience, 1989, p 2.9.1 23. MCKNIGHT AJ, BARCLAY AN, MASON DW: Molecular cloning of rat
interleukin-4 cDNA and analysis of the cytokine repertoire of subsets of CD4+ T cells. EurJlmmunol 21:1187—1194, 1991 24. GOODMAN RE, OBLAK J, BELL RG: Synthesis and characterization of
rat interleukin-lO (IL-b) cDNA clones from the RNA of cultured 0X8+ 0X22+ thoracic duct T cells. Biochem Biophys Res Commun 189:1—7, 1992
25. MURPHY E, HIENY 5, SHER A, O'GARRA A: Detection of in vivo
expression of interleukin-lO using a semi-quantitative polymerase
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26. CORDELL JL: Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). JHistochem Cyochem 32:219, 1984 27. YAM LT, LI CY, CROSBY WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol 55:283, 1971 28. DALLMAN MJ, LARSEN CP, MORRIS PJ: Cytokine gene transcription in
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reaction. J Exp Med 174:493—496, 1991 29. TAKEUCHI T, LOWRY RP, KONIECZNY B: Heart allografts in murine systems: The differential activation of Th2-like effector cells in peripheral tolerance. Transplantation 53:1281—1294, 1992 30. NORONHA IL, KRUGER C, ANDRASSY K, RITZ E, WALDHERR R: In situ
production of TNFa, IL-i and IL-2R in ANCA-positive glomerulonephritis. Kidney mt 43:682—692, 1993 31. LAKKIS FG, CRUET EN: Cloning of rat interleukin-13 (IL-13) cDNA and analysis of IL-13 gene expression in experimental glomerulonephritis. Biochem Biophys Res Commun 197:612—618, 1993
32. ZURAWSKI G, DE VRIES JE: Interleukin 13, an interleukin 4-like
37. MULLIGAN MS, JONES ML, VAPORCIYAN AA, HOWARD MC, WARD
PA: Protective effects of IL-4 and IL-lO against immune complexinduced lung injury. J Immunol 151:5666—5674, 1993 38. KROESE FGM, WUBBENA AS, JOLING P, NIEUWENHUIS P: T lympho-
cytes in rat lymphoid follicles are a subset of T helper cells. Adv Exp Med Biol 186:443—449, 1985 39. SPICKET GP, BRANDON MR, MASON DW, WILLIAMS AF, WOOLLETr
GR: MRC OX-22, a monoclonal antibody that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocyte-common antigen. J Exp Med 158:795—810, 1983 40. KI-IOURY Si, HANCOCK WW, WEINER HL: Oral tolerance to myelin basic protein and natural recovery from experimental allergic enceph-
alitis are associated with downregulation of inflammatory cytokines and differential upregulation of transforming growth factor beta, IL-4, and prostaglandin E expression in the brain. J Exp Med 176:1355— 1364, 1992
41. MOORE KW, O'GARRA A, DE WAAL MALEFYT R, VIEIRA P, MOSMAN
TR: Interleukin-lO. Annu Rev Immunol 11:165—190, 1993 42. MARCELLErrI JF, Oiw JI, KATZ DH: Collagen-induced arthritis in
mice. Relationship of collagen-specific and total IgE synthesis to disease. J Immunol 147:4185—4191, 1991 43. DIJKSTRA CD, DoP EA, JOLING P, KRAAL G: The heterogeneity of
mononuclear phagocytes in lymphoid organs: Distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2, and ED3. Immunology 54:589—599, 1985 44. BOYCE NW, HOLDSWORTH SR, DIJKSTRA CD, ATKINS RC: Quantita-
tion of intraglomerular mononuclear phagocytes in experimental glomerulonephritis in the rat using specific monoclonal antibodies. Pathology 19:290—293, 1987
Anti-inflammatory lymphokine mRNA expression in antibody-induced glomerulonephritis E'wi G. LAKKIS, FADY K. BADDOURA, EDDIE N. CRUET, KANNAN R. PAREKH, MEGUMU FUKUNAGA, and KAREN A. MUNGER Renal Division, Emoty University School of Medicine and Veterans Administration Medical Center, Atlanta, Georgia, and Pathology and Laboratoty Medicine, Veterans Administration Medical Center, Buffalo, New Yorlç, USA
Anti-inflammatory lymphokine mRNA expression in antibody-induced glomerulonephritis. T helper subset 2 (Th2) lymphocytes produce inter-
leukin 4 (IL-4) and IL-b, which exert anti-inflammatory actions on monocytes and macrophages. Thi lymphocytes, on the other hand, secrete interferon-y (IFNy) which promotes tissue inflammation. The functional dichotomy between Thl and Th2 lymphocyte subsets suggests that these cells play a regulatory role in inflammatory disease. The participation of
Th subpopulations and their lymphokine products in experimental gb-
merubonephritis (GN) has not been previously evaluated. In this study, we examined renal expression of Thi and Th2-type lymphokines in the first 48 hours of passive anti-glomerular basement membrane (anti-GBM) GN in the rat. Using the reverse transcriptase-polymerase chain reaction (RTPCR) method, apparent increase in expression of both Thi-type (IL-2 and IFNy) and Th2-type (IL-4 and IL-b) lymphokine mRNA was observed in gbomerular-enriched renal tissue obtained from nephritic rats. Induction
cytokine products of activated macrophages (IL-i, IL-6, and IL-8) are implicated in the pathogenesis of immune-complex mediated glomerular injury [10]. In contrast, Th2 lymphocytes secrete IL-4,
IL-b, and IL-13 which suppress production of inflammatory mediators by macrophages/monocytes [11, 12]. The anti-inflammatory effect of Th2-type lymphokines on macrophages in vitro is characterized by inhibition of IL-i, IL-6, IL-8, and TNFcr production while inducing IL-i receptor antagonist (IL-iRA) synthesis [12—14]. In addition, IL-4 and IL-13 induce de novo expression of
monocyte 15-lipoxygenase (15-LO), an enzyme which catalyzes the formation of anti-inflammatory eicosanoids from arachidonic acid [15—17]. Cross-regulation between Th lymphocytes has also of monocyte-derived IL-la and IL-i receptor antagonist (IL-IRA) been demonstrated: IFN'y inhibits proliferation of Th2 cells while mRNA expression was also detected shortly after initiation of GN. IL-4 and IL-b suppress production of Thi lymphokines [18]. In Evidence for influx of mononuclear cells including T lymphocytes into the vitro and in vivo data suggest that the outcome of an immune or an kidney was noted during the same time period as cytokine mRNA inflammatory response is influenced by the net balance of Thi and expression. Utilizing a monoclonal anti-rat IL-4 antibody, we also detected interleukin 4-producing cells in the renal cortex 24 hours following Th2 lymphokines [11]. Although T lymphocytes have been implicated in the pathogeninduction of GN. These experiments demonstrate for the first time
anti-inflammatory lymphokine (IL-4 and IL-b) mRNA expression and IL-4 protein production in the kidney during antibody-mediated GN. We hypothesize that Th lymphocyte subsets modulate glomerular inflammation by producing lymphokines with opposing actions.
esis of human and experimental GN, their role is not clearly Table 1. PCR primer-pairs and oligonucleotide probes utilized in this study
The participation of T lymphocytes in human and in experimental glomerulonephritis (GN) is inferred from several histological studies [1, 21. Small numbers of T cells have been detected in
Cytokine
Rat IFNy
normal rat kidney using monoclonal antibodies to cell surface markers [3, 4]. Further influx of T lymphocytes, predominantly CD4 +, is observed following induction of accelerated anti-gb- Rat IL-2 merular basement membrane (anti-GBM) GN [4—6]. CD4+ T helper cells (Th) segregate into at least two subpopulations based on the pattern of bymphokine production and the type of immune responses they promote [7—9]. Thi lymphocytes secrete interbeukin-2 (IL-2), interferon-gamma (IFN7), lymphotoxin, and tumor necrosis factor-alpha (TNFa), which promote delayed-type hyper-
Oligonucleotide sequence
Rat IL-4
Prediced size of amplified segment bp
5'-atgagtgctacacgccgcgtcttgg-3' A 5' -gagttcattgacagctttgtgctgg-3' P 5 '-gtcatcgaatcgcacctgatcactaacttc-3'
405
S
409
S
5'-catgtacagcatgcagctcgcatcc-3'
A 5' -ccaccacagttgctggctcatcat-3'
P 5 '-cacgtttaaattttacttgcccaagcaggcc-3' S
5'-tgatgggtctcagccccaccttgc-3'
378
A 5 '-ctttcagtgttgtgagcgtggactc-3' P 5' -cttacggcaacaaggaacaccacggagaac-3' Rat IL-b
S
5'-ttcccagtcagccagacccacatgc-3'
530
A 5 '-tttccaaggagttgctcccgttagc-3'
sensitivity and macrophage activation [7]. IFNY, TNFa, and Rat IL-la
P 5' -cagctgcgacgctgtcatcgatttctcccc-3' S
5'-agagggagtcaactcattggcgc-3'
800
A 5' -ggaaatctatcatggagggcagtcc-3' Received for publication April 4, 1994 and in revised form August 2, 1995 Accepted for publication August 3, 1995
P 5 '-accatccaacccagatcagcacctcacagc-3' Mouse
IL-iRA
© 1996 by the International Society of Nephrobogy
S
5'-caagccttcagaatctgggat-3'
A 5'-gtcttcctggaagtagaa-3'
Abbreviations are: S, sense; A, anti-sense; P, probe.
117
422
118
Lakkis et a!: Anti-inflammatory lymphokines in GN
Table 2. Enzymatic substrates and monoclonal antibodies used in this study and their specificities in the rat. Substrate! antibody Naphthol AS-D chioroacetate MRC HIS-17 (W3!13) MRC OX-22
15 -
CD designation —
anti-CD43
anti-CD45RC
ED1
—
ED2 MRC OX-81
— —
Reference
Specificity
Neutrophils Mast cells
[27]
Pan-T cells Myeloid cells" T helper 1 cells T cytotoxic cells Natural killer cells B cells
[38]
Monocytes Macrophages Dendritic cells
[43, 441
Tissue macrophages Monoclonal mouse Anti-rat interleukin-4
[43, 44]
[39, 40]
DW Masona
a CD, cluster of differentiation b
Variable expression of CD43 antigen MRC, University of Oxford, England (personal communication)
0
2
24
48
Time, hours Fig. 1. Induction of proteinuria following NTS injection. Urine samples were obtained from rat urinary bladders at time of sacrifice. UpJUcr is the spot urinary protein to creatinine ratio. Symbols are: (1) NTS; (D) NRS.
*Significantly greater than baseline (P <
0.05;
Dunnet multiple
comparison).
A 1353
B
Time, hours
k#O 2 162448
Time, hours
0 2 162448
1078
872 603
IFNy
310 276 234
IL-2
I L-4
IL-b
Fig. 2. Analysis of renal cortical lymphokine expression by RT-PCR following induction of anti-GBM GN: (A) experimental group (rats injected with NTS), (B) control group (rats injected with NRS). Reverse transcribed RNA samples were subjected to 40 PCR cycles in each case. Amplification products were electrophoresed on 2% agarose gels and visualized with ethidium bromide. Marker= 0X174 RF DNA!HaeIII fragment ladder.
defined [1, 2, 4]. The functional dichotomy between Thi and Th2 GN has not been previously evaluated. In this report, we studied lymphocyte subpopulations suggests that these cells could play a renal expression of Thi and Th2-type lymphokine mRNA during regulatory role in glomerular inflammation. The participation of the first 48 hours of passive anti-GBM GN in the rat. In addition, Th subpopulations and their lymphokine products in experimental mononuclear cell infiltration of the kidney was visualized and
119
Lakkis et al: Anti-inflammatoty lymphokines in GN
Time, hours 0 2 16 24 48
A GAPDH
0 2162448
Time, hours 0 2 16 24 48
Ctrl
Trme. hours 0
Time. hoL
IL
I L-4
CtrI
CtrI
B Time,hours 0 2 16 24
Time,hours 0 2 162448 IL-b
I L-4
Fig. 3. Slot-blot and Southern blot analysis of renal cortical lymphokine expression following induction of anti-GBM GN: (A) Slot-blot analysis of RT-PCR products from three different rat groups injected with NTS and one group injected with control NRS using peroxidase-labeled oligonucleotide probes (Table 1). (B) Southern
blot analysis of rat IL-4 and IL-b RT-PCR products using peroxidase-labeled eDNA probes. Bands shown correspond in size to those visualized by ethidium bromide staining (Fig. 2A).
quantitated using monoclonal antibodies to T lymphocyte and macrophage surface antigens. To substantiate the mRNA studies, in situ production of IL-4 protein by infiltrating cells was investigated using a monoclonal anti-rat IL-4 antibody. Methods Induction of glomerulonephritis
Male Sprague-Dawley rats (200 to 250 g; Charles River, MA,
USA) were injected in the femoral vein with 150 d of rabbit
examination of renal tissue and by measuring protein to creatinine ratio (Up/Ur) in urine samples obtained from the bladder at time of sacrifice. Protein concentration was determined by the Bio-Rad Protein Assay system (Bio-Rad Laboratories, Inc., USA) based on the Bradford method, and creatinine concentration was measured
using an assay kit (Sigma) based on formation of Janovski complex following addition of picric acid. To validate the nephritogenicity of NTS used in these experiments, two groups of rats (N = 5 each) were anesthetized with
mactin (100 mg/kg i.p.), and the ureters exposed via a midline incision. The ureters were catheterized with PE-lO tubing, and were injected with an equal volume of heat-inactivated, non- following a 45-minute equilibration period, baseline (pre-injecimmune, normal rabbit serum (NRS) (Sigma, St. Louis, MO, tion) urine was collected for 30 minutes. Rats were then injected USA). Rats were sacrificed at 0, 2, 16, 24 and 48 hours post- intravenously with 150 ml of either NTS or NRS, and two hours injection (N = 4 per time point). Kidneys were flushed with 20 ml later another 30-minute urine collection was obtained. Animals ice-cold Hanks balanced salt solution (HBSS) and removed. were sacrificed immediately at the end of urine collection, and Excised kidneys were immediately used for glomerular enrich- renal tissue was removed for histochemical staining of neutroment except for small sections dedicated for immunohistochemi- phils (methodology described later). All urine samples were cal studies. Disease induction was confirmed by histochemical stored at —20°C prior to protein determination. Intergroup anti-rat GBM nephrotoxic serum (NTS) (provided by Dr. Kamal F. Badr, Emory University, Atlanta, GA, USA). Control animals
120
Lakkis et at: Anti-infiammatoiy lymphokines in GN
Time, hours Control group
Experimental group L
0
2 16 24 48 2 16 24 48 C
L_.J
603 - -
310 --
S
p
41
—————————
-I
Fig. 4. GAPDH PCR amplification following reverse transcription of renal cortical RNA samples (A) and without reverse transcription (B). All samples were amplified (40 cycles) simultaneously. Control group, rats injected with NTS; experimental group, rats injected with NRS; L, 0X174 RF DNAIHaeIII fragment ladder; C, control RT-PCR in the absence of RNA.
statistical analysis was performed using the Dunnet multiple comparison test. Enrichment for rat glomeruli
Renal cortical tissue was enriched for glomeruli by graded sieving as previously described [19]. Briefly, kidneys were placed in ice-cold PBS, the renal capsule was removed, and the outer and
[211. Amplified products were electrophoresed on 2% SeaKem LE agarose (American Bioanalytical, MA, USA) and stained with ethidium bromide. DNA band corresponding to the target cytokine was identified by its predicted size (Table 1). Southern and slot-blot analysis
Products of IL-4 and IL-b RT-PCR reactions were electrophomid cortices dissected and minced with a razor blade, Minced resed on 2% agarose gels, transferred to Hybond-N+ nylon cortices were then passed stepwise through stainless steel sieves membranes (Amersham, IL, USA) by capillary Southern blotting (120 mm to 63 mm). The fraction, retained on top of the 63 mm [22], and covalently linked by UV-irradiation using a Stratalinker sieve, was washed in ice-cold PBS, examined by phase microscopy, UV cross linker (Stratagene, CA, USA). Rat IL-4 eDNA was and used immediately for RNA extraction. Glomeruli constituted purified from the plasmid pRIL-4.C10 (gift of Dr. A.J. McKnight, greater than 90% of this fraction. MRC, Oxford, UK) [23] following digestion with HindIIl (New England Biolabs, MA, USA). Rat IL-b was purified from plasmid RNA extraction and RT-PCR analysis pILl 0-30 (gift of Dr. James A. Baker, Cornell University, Ithaca, Total cellular RNA was purified from glomerular-enriched NY, USA) [241 following digestion with Hindlil and BamHI. cortical tissue by the Chomczynski and Sacchi method [201 using cDNAs were denatured and labeled with horseradish peroxidase RNAzoI B reagent (Biotecx Laboratories, Houston, TX, USA). using Enhanced Chemiluminescence (ECL) direct nucleic acid Cortical RNA from four animals/time point was pooled, and 2 sg labeling and detection system (Amersham). Membranes were was reverse transcribed using an oligo-dT primer and Moloney hybridized overnight with the labeled eDNA probes in 0.5 M NaCI murine leukemia virus reverse transcriptase (Perkin Elmer, CT, solution at 42°C, washed in 0.5 X SSC at 42°C, and detection USA). Forty cycles of PCR amplification were performed in a reagents were added as described by the manufacturer (AmerDNA Thermal Cycler 480 (Perkin-Elmer) using oligonucleotide sham). Autoradiography was performed at room temperature for primers specific for rat cytokines (Table 1). Primer pairs were one minute. designed using PCRPLAN software (pcgene, Geneva, SwitzerIL-2, IFN7, IL-4, IL-b, and IL-la RT-PCR products from land) based on the published eDNA sequences of the target three replica animal experiments were also captured on Hycytokines. Oligonucleotides were synthesized on an Applied Bio- bond-N+ nylon membrane using a slot-blot apparatus (Hoeffer systems DNA synthesizer by Dr. James Murtagh, Jr. (Emory Scientific Instruments, San Francisco, CA, USA). Hybridization University and VAMC, Atlanta, GA, USA). PCR parameters and detection was performed as described above, except that were optimized for each cytokine to achieve maximum specificity
target-specific oligonucleotide probes (Table 1) were used instead
121
Lakkis et al: Anti-inflammatoty lymphokines in GN
Time, hours
(Pharmingen Inc., CA, USA), and monocytes/macrophages (EDt
0 2 16 24 48
1353
1078
872 —.. 603
IL-i cx
310 —
and ED2) (Harlan/Serotec, IN, USA) using an immunoenzymatic kit (DAKO Corp., CA, USA) according to published procedure [26]. Monoclonal antibodies and enzymatic substrates used in this study and their specificities are summarized in Table 2. Briefly, demercurized and rehydrated B5-fixed sections were digested at RT in 0.1 mg/ml Proteinase K in 0.6 M Tris HC1/0,1%CaC12 for 30
minutes (CD43 and CD45RC) and in 0.1% Trypsin in 0.05 M Tris HC1/0.1% CaC12, pH 7.6 for three minutes (ED1, ED2, 0X81). Non-specific antibody binding sites were blocked with prediluted
276 234
IL-iRA
goat serum for 30 minutes. Sections were then incubated with primary mouse anti-rat antibody diluted 1:100 in Tris-salineTriton (TST) for one hour. Polyclonal anti-mouse link antibodies were applied for 30 minutes followed by alkaline-phosphatase anti-alkaline phosphatase (APAAP) immune complex for 30 minutes. Sections were developed in the dark with fast red chromagen
for 30 minutes. The entire procedure was carried out at room temperature, and all washes were performed in TST. Appropriate
-' a a a a
GAPDH
titers of primary antibodies were predetermined by staining similarly processed normal rat spleen sections. For neutrophil detection, formalin-fixed sections were stained cytochemically with the esterase substrate Naphthol AS-D Chloroacetate (CAE;
Sigma) according to a previously described method [27]. All sections were counterstained with hematoxylin. Positive cells
Time, hours 0
within 200 randomly selected glomeruli per animal were enumerated by light microscopy. Intraglomerular infiltration was quanti-
2 16 24 48
tated by calculating the average number of positive cells per glomerulus. Intensity of the cortical interstitial infiltrate was
exp.1
determined by counting all positive cells in the interstitial area of cortex containing 100 glomeruli. Unpaired Student's t-test was used for statistical comparisons (confidence interval set at 95%). IL-i a
exp.2
exp.3
Results Induction of glomerulonephritis
'
Nephritogenicity of anti-GBM nephrotoxic serum (NTS) used in this study was confirmed by measuring urinary protein excre-
Fig. 5. IL-la and IL-IRA mRNA induction in the renal cortex following NTS injection. Reverse transcribed RNA samples were subjected to 40 PCR cycles in each case. Amplification products were electrophoresed on 2% agarose gels and visualized with ethidium bromide (A). Slot-blot analysis of IL-la mRNA from three different animal experiments (B).
of cDNA probes [25] and autoradiography was performed at room temperature for 10 minutes. Each oligonucleotide was designed such that it hybridizes to a mid-region of the target cDNA and not to PCR amplification primers. Densitometric analysis was performed using a Molecular Dynamics densitometer (Sun Valley, CA, USA).
tion rate and magnitude of glomerular neutrophil infiltration prior to and after injection of NTS. Proteinuria increased from 0.011
0.003 mg/mm (mean
SD) pre-NTS to 0.188
0.050
mg/mm two hours following NTS administration (N = 5, P < 0.05). In contrast, proteinuria did not increase in rats injected with NRS: 0.010 0.004 mg/mm pre-injection and 0.012 0.006 two hours post-injection (N = 2). NTS administration also resulted in
a significant increase in the number of infiltrating neutrophils from 0.10 0.07 neutrophil/glomerulus in healthy rats to 3.3 1.7 neutrophils/glomerulus in rats sacrificed two and a half hours following NTS injection (N = 5, P < 0.05). Induction of glomerular injury in the rats utilized in gene expression and immunohistochemical studies was demonstrated by measuring urinary pro-
tein (Un) and creatinine concentrations (Ur) and calculating
Tissue samples from kidneys excised at 0, 2, 16, 24, and 48 hours following injection of NTS (N = 4/group/time point) or NRS (N = 2/group/time point) were formalin and/or B5-fixed and
Up/U ratios at time of sacrifice. UP/UCr increased significantly two hours following injection of NTS and declined afterwards (Fig. 1). Up/Ucr ratios did not change significantly in rats that received NRS (Fig. 1).
paraffin-embedded for accurate localization of target cell populations. Deparaffinized tissue sections mounted on silanized slides were immunostained with monoclonal antibodies against rat IL-4
Detection of Thi and Th2-type lymphokine mRNA in glomerularenriched cortical tissue
Histochemical studies
(0X81, provided by Dr. D.W. Mason, MRC, University of
Figure 2A summarizes the time course of renal Thi and Th2
Oxford, UK), CD43 (MRC HIS-17), CD45RC (MRC OX-22)
lymphokine mRNA expression during the first 48 hours of passive
4
A
B
10
* 8 a)
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0C 0)
E
0 +
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6
4
0
*
0 2 0
0
0 8 16 24 32 40 48 56
0 8 16 24 32 40 48 56
Time, hours
Time, hours D
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Fig. 6. Temporal patterns of leukocytic infiltration into glomendi. (A) CAE+ cells (LII, neutrophils); (B) CD43+ cells (E, T lymphocytes); (C) CD45RC+ cells (Thi lymphocytes) [(LI) CD45RC (NTS), (I) CD45RC (NRS)1; (D) ED1 + cells (monocytes/
0.75
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00
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0 8 16 24 32 40 48 56
0 8 1624 32404856
Time, hours
Time, hours
macrophages) [(LI) ED-i (NTS), ('I') ED-i (NRS)j. The NTS group is the rats injected with nephrotoxic serum, and the NRS group rats are injected with normal rabbit serum. All data points represent mean SEM. *significantly greater than baseline (time 0) (P < 0.05; unpaired t-test). **Significantly greater than time-matched NRS control (P < 0.05; unpaired t-test).
anti-GBM GN. A weak RT-PCR amplification signal was detected by ethidium bromide staining in healthy animals (time 0)
70
for all lymphokines studied. The intensity of the ethidium-
60
*
bromide stained bands, however, increased two hours following induction of disease in all cases. An apparent increase in staining intensity was also present at later time points but appeared to be transient in case of IL-4. Unlike the experimental group, amplification of RNA isolated from renal cortices of control animals injected with NRS did not result in a pattern of increasing staining intensity (Fig. 2B). These findings were confirmed by slot-blotting of RT-PCR products and hybridization to target-specific labeled oligonucleotide probes in three separate experimental animal
**
50
-(I,40
p0 ... 20
groups and one representative control group (Fig. 3). In each replica experiment, NTS administration resulted in an apparent
10
increase in cytokine mRNA levels above baseline (0 time point) or time-matched NRS controls (Fig. 3A). Semiquantitative densitometric measurements performed on slot-blot analyses showed on
0 0
8
16
24 32 Time, hours
40
48
56
Fig. 7. Temporal patterns of leukocytic infiltration into cortical interstitial
areas. Symbols are: (LI) CAE-NTS; (I) CD45RC-NTS; (0) ED2-NTS; (A) CD45RC-NRS. CAE+ cells (neutrophils); CD45RC+ cells (Thi
the average a 4.4-fold and 6.8-fold increase in IFNY and IL-2 mRNA expression over baseline in nephritic animals. When compared to control animals injected with NRS, IL-4 and IL-b mRNA levels increased by an average of 3.4-fold and 12-fold, respectively. Following agarose gel electrophoresis, the identities
of IL-4 and IL-b RT-PCR products were also confirmed by Southern transfer and hybridization to labeled cDNA probes (Fig.
lymphocytes); ED2+ cells (tissue macrophages); NTS group, rats injected
3B). Although IL-4 and IL-lU mRNA were barely detected in
glomeruli.
normal renal cortical tissue (time 0) when oligonucleotide probes were used (Fig. 3A), baseline expression could be demonstrated in cDNA-probed Southern blots (Fig. 3B). Figure 4 demonstrates that GAPDH amplification product was uniformly absent when reverse transcription was excluded, thus
with nephrotoxic serum; NRS group, rats injected with normal rabbit serum. All data points represent mean SEM. *Signiflcantly greater than baseline (time 0) (P < 0.05; unpaired t-test). 00Signiflcantly greater than time-matched NRS control (P < 0.05; unpaired t-test). Interstitial area in this study was defined as area of cortical interstitium containing 100
.1(1
-s
V.. —
S
I. •
p
3.
t-d.
c
.
Mm -
.—.
"r ,.
.lt9 "a.
r2.: 'I-. 4 4 so t4 •a.. •. S
r
Fig. 8. Histochemical analysis of paraffin-embedded renal sections from glomerulonephritic rats (positively stained cells appears bright red). (Magnification
x400). (A) CAE+ cells (neutrophils) at two hours following NTS injection; (B) CD43+ cells (T lymphocytes) at 24 hours; (C) CD45RC+ cells (Thi) lymphocytes at 24 hours; (D) ED1 + cells (monocytes/macrophages) at 24 hours; (E) ED2+ interstitial cells (tissue macrophages) at 48 hours; (F) IL-4-producing (OX-81 +) cells in control spleen section; (G) periglomerular IL 4-producing (OX-81+) cells at 24 hours.
124
Lakkis et al: Anti -infiammatoty lymphokines in GN
confirming the absence of DNA contamination in all RNA agents and to self-antigens implicated in autoimmune disease [11, 181. In addition, some of the cytokines secreted by Thi and
samples analyzed.
Th2 lymphocytes exert opposite effects on production of inIL-la and IL-iRA mRNA in glomerular-enriched cortical tissue flammatory mediators in vitro and are likely candidates for Because production of macrophage-derived cytokines is regu- modulating inflammatory responses in vivo [11—14]. In this lated by T cell lymphokines [13], we investigated IL-la and report we provided evidence for renal expression of antiIL-iRA mRNA expression during passive anti-GBM GN. Figure inflammatory Th2-type (IL-4 and IL-b) lymphokine mRNA 5 demonstrates the induction of both IL-la and IL-iRA mRNA and of IL-4 protein during the heterologous phase of antibodyin glomerular-enriched renal tissue as early as two hours following induced experimental GN. NTS administration. An apparent increase in IL-la mRNA Because a small number of T lymphocytes infiltrate the kidney expression is confirmed by slot-blot analysis using a target-specific
in passive anti-GBM GN, we took advantage of the high sensitivity
oligonucleotide probe (Fig. 5B). IL-la and IL-iRA mRNA of reverse transcriptase-polymerase chain reaction (RT-PCR) to expression in glomerular-enriched renal tissue from control rats injected with NRS was not detected (data not shown). Characterization of glomerular and interstitial cellular infiltrates
To determine the cellular correlate of cytokine mRNA expression in the kidney, immunohistochemical studies were performed
study temporal expression of lymphokine genes [25, 28—30]. The specifcity of the method was insured by designing primers specific for each rat cytokine analyzed and by optimizing PCR conditions
as described in the literature [21]. Identities of the amplified products were confirmed by Southern and/or slot blot analysis using target-specific cDNA and oligonucleotide probes. The slot-
on fixed renal tissue to quantitate intraglomerular and cortical blot format also allowed rapid semiquantitation of cytokine interstitial leukocytic infiltrates. The results are summarized in mRNA expression as previously described [251. In addition, we Figures 6 and 7. Representative micrographs are shown in Figures
ruled out DNA contamination of RNA samples by demonstrating
8A—E. Three separate patterns of cellular infiltration were observed during the first 48 hours of passive anti-GBM GN. A transient neutrophil influx, identified by CAE staining, appeared in both glomerular and cortical interstitial areas two hours after
that GAPDH PCR product can be visualized only if reverse transcription is performed prior to amplification. Using this
with ED1 monoclonal antibody, also rose significantly between 16
[31]. Like IL-4 and IL-b, IL-13 is mainly produced by Th2
and 24 hours (Fig. 6C). Interstitial ED1+ cells increased after induction of GN and were too numerous to count (Fig. 8D). A gradually increasing number of ED2 + tissue macrophages in the cortical interstitium was observed at 24 and 48 hours (Fig. 7). Intraglomerular ED2+ cells were not detected during the time frame of this study. In contrast to NTS, NRS administration did not increase the number of glomerular CD45RC+ and ED1 +
lymphocytes and possesses anti-inflammatory properties in vitro and in vivo [32]. We also demonstrated induction of IL-la and IL-IRA mRNA in glomerular-enriched cortical tissue following anti-GBM antibody administration. These cytokines are secreted by activated monocytes/macrophages and possibly mesangial cells and their production is regulated by Thi and Th2 lymphokines. IFNY, for example, is a potent macrophage activator which induces production of IL-i and other inflammatory mediators [33]. IL-i in turn contributes to the pathogenesis of glomerular immune injury [34, 35]. On the other hand, several anti-inflammatory actions of IL-4,
methodology, we demonstrated an apparent increase in expression of both Thi and Th2-type lymphokine mRNA in the renal injection of NTS (Figs. 6A and 7). A mononuclear cellular cortex during the first 48 hours of passive anti-GBM GN. Renal infiltrate, which stained positively with anti-CD43 or anti- tissue obtained from animals that received non-nephrotoxic seCD45RC, followed the neutrophil influx and peaked in number at rum, on the other hand, did not show an increase in lymphokine 24 hours (Figs. 6 B,C). CD45RC+ cell infiltration of the cortical mRNA expression. Detection of IL-4 and IL-b mRNA in the interstitium persisted longer than its intraglomerular counterpart kidney following induction of anti-GBM GN is consistent with our (Fig. 7). The number of intraglomerular monocytes, identified prior demonstration of IL-13 gene expression in the same model
cells over that found in healthy animals (Figs. 6 C, D). Moreover,
an interstitial CD45RC+ infiltrate was not induced by NRS (Fig. 7).
In situ production of rat IL-4 by infiltrating mononuclear cells
Utilizing a monoclonal mouse anti-rat IL-4 antibody (0X81), IL-4-producing mononuclear cells were observed in the renal interstitium following induction of GN. Figure 8F demonstrates the periglomerular location of 0X81 + cells in a renal section obtained 24 hours after NTS injection. Normal rat spleen tissue served as a positive control for immunohistochemical detection of IL-4 protein (Fig. 8G). Renal tissue obtained at other experimental time points lacked OX-81 + cells. OX-81 staining was also absent in the kidneys of control rats injected with NRS. Discussion
In contrast to the plethora of information available on the
including suppression of IL-i production and upregulation of IL-iRA synthesis, have been demonstrated in vitro and in vivo [13,
36, 371. Whether anti-inflammatory cytokines produced during passive anti-GBM GN modulate IL-i and IL-iRA production by infiltrating monocytes remains to be determined. Of significance to the aim of this study is that RT-PCR data were substantiated by immunohistochemical demonstration of rat IL-4 protein within periglomerular mononuclear cells. IL-4-producing (OX8 1+) cells were observed transiently 24 hours following induction of GN, which correlates with the apparent maximal expression of IL-4 mRNA at 16 hours. Because conventional paraffin immunohistochemistry lacks high sensitivity, it is possible
that IL-4 protein was missed in renal sections obtained at other experimental time points. how inflammation is regulated during the course of glomDemonstration of lymphokine mRNA is also corroborated by erulonephritis. CD4+ T-lymphocyte subsets, Thi and Th2, play immunohistochemical findings in paraffin-embedded sections. A a central role in regulating immune responses to infectious sparse mononuclear cell infiltrate was observed within glomeruli pathogenesis of glomerular immune injury, less is known about
125
Lakkis et al: Anti-inflammatory lymphokines in GN
and in the interstitium during the first 48 hours of disease. The cells stained positively for CD43 and peaked in number between 16 and 24 hours following induction of GN. Anti-CD43 monoclonat antibody serves as a pan-T cell marker [38] and has been utilized to detect T lymphocytes in renal tissue [3, 5]. Previous studies have shown a similar temporal pattern of T cell infiltration in accelerated anti-GBM GN [4, 51. Our results, however, showed a surprisingly high number of intraglomerular and interstitial CD43+ cells. This cannot be attributed to staining of neutrophils because they have a distinct temporal pattern of inflitrating the kidney. It is possible that anti-CD43 staining is also detecting
Acknowledgments This work was supported by a Young Investigator award from the National Kidney Foundation (FGL) and by a VA Merit Review Grant (FGL). Portions of this work were presented at the American Society of Nephrology 26th annual meeting (1993) and appeared in abstract form (JAm Soc Nephrol 4: 613, 1993). We thank Dr. Kamal F. Badr, Emory University, Atlanta, Georgia, USA, for his excellent advice. Reprint requests to Fadi G. Lakkis, MD., VAMC - Atlanta, Research 151, 1670 Clairmont Road, Decatur, Georgia 30033, USA.
References
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