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Activated (IL-2R+) intraglomerular mononuclear cells in crescentic glomerulonephritis
Kidney International, Vol. 39 (1991), pp. 793—798
RAPID COMMUNICATION
Activated (IL-2R+) intraglomerular mononuclear cells in crescentic glomerulonephritis HAI-LING Li, WAYNE W. HANCOCK, JOHN P. DOWLING, and ROBERT C. ATKINS Departments of Nephrology and Anatomical Pathology, Prince Henry's Hospital, Melbourne, Victoria, Australia
Activated (IL-2R+) intraglomerular mononuclear cells in crescentic glomerulouephritis. We recently reported evidence for the involvement
of local cellular immune activation in the immunopathogenesis of human IgA nephropathy, particularly in cases of IgA disease featuring crescent formation. In the current study, using monoclonal antibodies, we investigated whether mononuclear cells bearing receptors for interleukin 2 (IL-2R+ MNC) were present within glomeruli or associated crescents in biopsies from patients with crescentic glomerulonephntis (>60% crescents, N = 19), IgA disease with crescents (N = 9), or other types of proliferative glomerulonephritis with crescents (10 to 44%, N = 6), compared with normal control kidneys (N = 10). Biopsies were further classified into those showing active (cells, fibrin) (N = 15) or inactive (sclerosed) crescents (N = 19), to determine whether IL-2R+ MNC were particularly associated with active crescent formation. Few leucocytes were found within glomerular tufts of normal kidneys (2.4
the correlations observed between the presence of activated intraglomerular tuft MNC and decreased parameters of renal function, these results suggest that crescent formation involves recruitment of macrophages to the glomerulus by sensitised T cells, analogous to events occurring in a DTH response, and are consistent with a direct role for cellular immunity in the immunopathogenesis and deteriorating renal function characteristic of human crescentic glomerulonephritis.
Extensive analysis of experimental models of glomerulonephritis has resulted in considerable insights into the mechanisms of immunologically induced glomerular injury. Individual
models based on antibody-mediated injury, immune complex deposition or T cell-dependent glomerular damage are well biopsies from patients with active crescentic glomerulonephritis, total described. However, few studies have concerned identification 1.7 (P < 0.01 intraglomerular tuft leucocytes were increased to 14.0 vs. normal kidneys), largely due to increased numbers of intraglomer- of similar mechanisms in human glomerulonephritis, particuular monocytes (10.4 1.1, P <0.01) and T cells (3.7 0.6, P <0.01). larly T cell-dependent mechanisms (Type IV hypersensitivity Biopsies with active crescents also contained significantly increased reactions). We recently reported that mononuclear cells bearing numbers of intraglomerular tuft IL-2R+ MNC (4.0 0.7, 29% of total intraglomerular leucocytes), and there was a strong correlation between receptors for interleukiri 2 (IL-2R+ MNC) were present in significantly increased numbers in biopsies from patients with the numbers of intraglomerular IL-2R+ MNC and T cells (P < 0.001). Moreover, in this active crescentic group, increases in the numbers of IgA nephropathy with crescents, compared to patients with IgA intraglomerular IL-2R+ MNC (P < 0.05), macrophages (P < 0.01) and nephropathy without associated crescent formation or normal T cells (P < 0.01) were correlated with raised serum creatinine levels, and the numbers of both IL-2R+ MNC (P < 0.05) and macrophages controls [II. Moreover, the numbers of IL-2R+ MNC correlated with raised serum creatinine and decreased creatinine (P< 0.01) correlated with decreased creatinine clearance. Patients with inactive crescentic glomerulonephritis also showed increased levels of clearance, suggesting that local cellular immune activation is tuft MNC compared to normal kidneys, but significantly less intraglo- involved in the immunopathogenesis of IgA disease, particumerular MNC than biopsies from patients with active crescentic dis- larly in cases with crescent formation. ease. Thus, in inactive crescentic disease there was a four-fold increase The current study examined whether IL-2R+ MNC, T cells in glomerular MNC infiltration (total leucocytes 10.0 1.4, P < 0.01), due to macrophages (6.6 1.0, P < 0.01 compared to normals or active and macrophages were present within glomeruli and/or associcrescentic glomerulonephritis) and T cells (2.9 0.3, P < 0.01), and ated crescents in a larger series of biopsies from patients with including IL-2R+ MNC (2.2 0.3, P < 0.01 compared to normals or more common forms of crescentic glomerulonephritis, and active crescentic disease). The cellular composition of actual crescents was also determined. Leucocytes contributed more than 60% of the more importantly, whether the presence of such activated MNC total cells within active crescents, due primarily to the presence of was associated with deteriorating renal function. 0.7 cells/glomerular cross-section; mean
SEM). By contrast, in
macrophages (43.1% 5.1%) and T cells (7.1% 2.1%); 8% of these leucocytes within crescents were IL-2R+. Glomeruli with active cres-
Methods
cents contained extensive ruptures of Bowman's capsules. Inactive crescents, which generally displayed intact Bowman's capsules, presumeably as a result of fresh synthesis of basement membrane components, contained fewer leucocytes (19.1% 2.2%), including reduced 1.9%) and T cells (0 to 3, ranges). numbers of macrophages (7.2% Given the close regulation of IL-2R expression on activated MNC, and
Tissues
Renal biopsies from 19 patients with crescentic glomerulone-
Received for publication August 21, 1990 and in revised form December II, 1990 Accepted for publication December 12, 1990
phritis (> 60% crescents), were compared with biopsies from nine patients with IgA nephropathy with crescents, and six patients with other forms of proliferative glomerulonephritis and focal crescent formation (10 to 44%) (Table 1). Biopsies with predominantly fleshy, cellular crescents and associated fibrin deposition were further classified as active crescents (N = 15), whereas those with largely organized, fibrous or fibrocel-
© 1991 by the International Society of Nephrology
lular crescents and largely lacking fibrin deposits were classified
793
794
Li et a!: IL-2R+ MNC in human crescentic ON Table 1. Histologic classification of renal biopsies
No. of
Active Inactive biopsies Crescents crescentsa crescentsa
Diagnosis
Immune complex GN IgA nephropathy Focal proliferative ON Idiopathic GN Anti-GBM disease Pyelonephritis Membranoproliferative GN Totals
14 9 4 3 2
%
70—100%
8—71 % 35—44 % 70—100% 60—100%
1
30%
1
10%
34
6 8 2
8
1 1
2 2 2 0 0
19
15
1
0
a As defined in Methods
Renal function Serum creatinine, endogenous 24-hour creatinine clearance, and glomerular filtration rate were determined in each patient at the time of biopsy.
Statistical analysis Data were analyzed on a personal computer using programs from the CSS system (Statsoft, Tulsa, Oklahoma, USA). The numbers of labelled cells in different groups were compared using the two-tailed Student's t-test. The percentages of la-
belled cells within glomeruli or crescents in biopsies from different groups were compared using a two-tailed Wilcoxon Rank sum test, and correlations determined using Spearman's
test. Results
as inactive crescents (N 19) [2, 3]. Normal control tissues Intraglomerular MNC consisted of portions of cadaver nephrectomies not used for or kidneys removed because of localtransplantation (N = 7) Few leucocytes were present in normal renal tissue (2.4 0.7 ized tumors (N = 3). All biopsies were routinely assessed by cells/glomerular cross section); these consisted of monocytes light microscopy, immunofluorescence and electron micros- (1.4 0.4) and lesser number of T cells (0.3, range 0.1 to 0.4) copy, as well as by immunoperoxidase labelling using anti- (Table 2). No IL-2R+ cells were detected. In contrast, biopsies leucocyte and other monoclonal antibodies (mAbs). Specimens from patients with active crescentic glomerulonephritis showed for immunoperoxidase studies were fixed in PLP at 4°C, washed increased intraglomerular leucocytes (14.0 1.7, P < 0,01), in PBS containing 7% sucrose, snap frozen and stored at —80°C including macrophages (10.4 1.1, P < 0.01), T cells (3.7 until cryostat sectioning, as described previously [41. 0.6, P < 0.01) of both CD4+ and CD8+ phenotypes (P < 0.01),
Monoclonal antibodies
Infiltrating leucocytes were identified using the following mAbs: all leucocytes—PHMI (CD45); all T cells—Leu 4 and OKT3 (CD3); helper/inducer T cell subset—Leu 3A and OKT4 (CD4); cytotoxic/suppressor T cell subset—OKT8 (CD8); macrophages-FMC32 (CD14); IL-2R+ MNC—Tac-l (CD25); B cells—B 1 (CD2O), and NK cells—Leu 7 (CD57). Details of
and IL-2R+ MNC (4.0 0.7, P < 0.01; Table 2). Moreover, though the nature of IL-2R+ MNC was not directly established
in this study, there were strong correlations between the numbers of intraglomerular IL-2R+ MNC and both T cells (r = 0.76, P < 0.001, Fig. 1) and macrophages (r = 0.75, P < 0.001,
Fig. 2). Furthermore, the numbers of infiltrating macrophages and T cells were also highly correlated (r = 0.77, P < 0.001, Fig. 3). B cells and NK cells were rarely identified within glomeruli these mAbs and their use were presented previously [5]. of any group (< 0.1 cells/glomerular cross section). Biopsies from patients with inactive crescentic glomeruloneBiopsies were also labelled using a mAb to glomerular parietal phritis also showed increased numbers of intraglomerular leuand visceral epithelial cells (PHM5) 12, 61. Immunoperoxidase labelling
cocytes, though to a lesser extent than in active crescentic disease (Table 2). Thus, glomeruli in patients with inactive crescentic disease showed increased total leucocytes (10.0
Cryostat sections (4 m) were labelled using a four-layer 1.4, P < 0.01 vs. normal), due to increases in macrophages (6.6 immunoperoxidase (PAP) technique (4).
Evaluation of immunostained sections
Renal biopsies were examined for the presence of labelled cells within glomerular tufts and any associated glomerular crescents; biopsies contained 3 to 20 glomeruli. Labelled cells within each glomerulus were counted and results were expressed as the number of cells per glomerular cross section. Labelled cells within glomerular crescents were expressed as a percentage of total crescent cells. Cell counts were performed three times to reduce counting errors, and quantitative analysis of infiltrating MNC was performed independently and without knowledge of clinical or histopathologic data. Data were also expressed in terms of whether most crescentic glomeruli (> 75%) in a given biopsy retained intact Bowman's capsules, or whether such capsules were predominantly intact [7]; this was assessed by examination of silver-stained paraffin sections, prepared at three different levels from each biopsy, and by immunoperoxidase labelling with PHM5 mAb [6].
1.0, P < 0.01 compared to both normal and active crescentic disease) and T cells (2.9 0.3, P <0.01 vs. normal) of both CD4+ and CD8+ phenotypes, as well as IL-2R+ MNC (2.2
0.3, P < 0.01 compared to normal or active disease). The numbers of intraglomerular total leucocytes, macrophages, T cells and IL-2R+ MNC present in biopsies classified primarily as crescentic glomcrulonephritis, or proliferative glomerulonephritis with associated crescent formation, were significantly
increased compared with our previously studied groups of patients with IgA nephropathy with crescents (Fig. 4)111. Cells in glomerular crescents Leucocytes contributed some 60% of cells within crescents of patients with active crescentic disease (N 15) (Table 3).
Macrophages were the main cell type identified, contributing 5.1% of cells within active crescents, and in some 43.1% glomeruli up to 90% of crescent cells were macrophages. T cells were also present (7.1% 2.1%), including both CD4+ (4.9%
0.7%) and CD8+ (2.5% 0.5%) phenotypes, and IL-2R+ MNC
795
Li et a!: IL-2R+ MNC in human crescentic GN Table 2. Intraglomerular tuft MNC in biopsies from patients with crescentic glomerulonephritis (N =
Normal kidneys
10
Inactive crescentse Active crescentse
'
Total WBC
No.
Group
2.4a
10.0" 14.0"
19 15
T cells
Monocytes 1.4 0.4
0.7 1.4 1.7
a Expressed as cells/glomerular cross section (mean
6.6C
1.0
104c.d
1.1
34)
versus normal kidneys (N= 10)
CD8+
CD4+ cells
cells
IL-2R+ cells 0.0
03b
0.1
0.1
(0.1—0.4)
(0.1—0.2)
(0.1—0.2) l.2C 0.2
2.2"
0.3
0.3
4.0""
0.7
2.9" 3.7"
0.3 0.6
1.6" 27c.d
0.5 0.4
1.8"
SEM)
Ranges are
shown when numbers were very small P < 0.01, between normal and crescentic glomerulonephritis d P < 0.05, between inactive and active crescentic glomerulonephritis as defined in Methods
25 C
0
r = 0.755
C)
U,
0
0
9
20 -
P<0.0001
r0.77
P<0.0001
0
0
CC
15 C)
E
0
.
6
0
D)
.
C)
0
.
...
3
0 0
.
z 0
t10_
.. . .
8 E
5-
S
.. .
E
S
•..
..-• •
S
.
.• •
S
2
4
6
8
10
No. of L-2P + MNC/glomerular cross section Fig. 1. Correlation between the number of intraglomerular T cells and
IL-2R+ MNC in active crescentic disease, determined by Spearman's
test.
10 4 6 8 2 0 No. of L-2R + MNC/glomerular cross section Fig. 2. Correlation between the number of intro glomerular macrophages and JL-2R+ MNC in active crescent disease, determined by Spearman's test.
1.4%). No crescent cells expressed B cell or NK cell (P < 0.05), macrophages (P < 0.01) and IL-2R+ MNC (P < markers. 0.05) were correlated with decreased creatinine clearance. Nineteen patients with older, scierosed crescents were stud- However, none of these cells were correlated with urinary ied (Table 3). In contrast to biopsies with active, cellular protein or RBC excretion. crescents, the proportion of leucocytes in scierosed crescents (5.0%
was reduced to 19.1%
(P <
consisting of macrophages (7.2% 1.9%, P <0.01) and only very few T cells 2.2%
0.01),
Discussion
(range of 0 to 3%, P < 0.01) or IL-2R+ MNC (range of 0 to 2%,
This study investigated the contribution of leucocyte infiltration to the glomerular hypercellularity, accumulation of cells in
P < 0.01). Morphologically, most crescent cells at this stage
Bowman's space, and associated decreased renal function
appeared to be fibroblastoid or epithelioid in nature. The
characteristic of the various forms of human crescentic glomerulonephritis [31. We found that essentially all biopsies from patients with crescentic glomerulonephritis, regardless of etiology, but particularly those with active disease, showed signifi-
influence of the integrity of Bowman's capsule on the composition of crescent cells was also considered (Table 4). For each leucocyte population, the numbers of leucocytes within glomeruli with ruptured capsules were significantly greater (P < 0.01) than in those with intact capsules. Correlation between intro glomerular leucocytes and decreased renal function
cantly increased numbers of intraglomerular tuft leucocytes, consisting predominantly of macrophages and lesser numbers of T cells. Moreover, the findings that in active disease over 25% of these tuft leucocytes showed evidence of immune activation (Table 2), as indicated by expression of IL-2R, and that the presence of these cells correlated with decreased renal function (Table 5), were consistent with the developing concept
Intraglomerular MNC infiltration was strongly associated with decreased renal function (Table 5). Thus, the numbers of that MNC activation is important to the immunopathogenesis of total leucocytes (P < 0.01), monocytes (P < 0.01), T cells (P < human crescentic glomerulonephritis. 0.01) and IL-2R+ MNC (P < 0.05) were each correlated with Since our initial report [8], using glomerular culture, of increased serum creatinine, and the numbers of total leucocytes macrophage infiltration of glomeruli from patients with crescen-
Li et a!: IL-2R+ MNC in human crescentic GN
796
12 C
0
r= 0.784
a,
CO
U,
C
0
P< 0.0001
C,
C, U,
9
U,
0
CO
0
C-)
C, ci, ci,
E
6
.
0 DC
a)
F-
0 0
3
.
z 0
E
0
Di S
. .
.
C-)
U)
.
0 U)
0
5
10
15
20
No. of macrophages/glomerular cross section
3. Correlation between the numbers of intraglomerular macrophages and T cells, determined by Spearman's test. Fig.
Fig. 4. Numbers of intraglomerular total leucocytes and cell subsets present in biopsies classified as IgA nephropathy with crescents (IgA), other proliferative GN with crescents (GN), or crescentic GN (>60%
crescents, CGN). CGN and GN biopsies had significantly greater numbers of intraglomerular total leucocytes (U), macrophages ( cells (NJ) and IL-2R+ MNC (U) than IgA biopsies (P < 0.05).
), T
tic glomerulonephritis, a number of groups have confirmed that expression is controlled by cytokine(s) produced locally by leucocytes contribute to the glomerular hypercellularity usually activated helper T cells [19], the current results suggest a found in this group of diseases [reviewed in 31. Other groups possible source of cytokine(s) for such macrophage activation. Following activation, both IL-2R+ T cells and macrophages [9—111 have also reported that, based on the use of mAbs, small numbers of T cells accompany the more prominent influx of shed IL-2R into surrounding fluids, and measurement of soluble macrophages into the glomerulus during crescentic glomerulo- IL-2R levels is currently used for monitoring of a number of nephritis. However, the new and more important finding of the diseases with an immunologic basis [121. Yorioka et al [201 have current study was the demonstration that many of these MNC recently reported that serum IL-2R levels were significantly expressed IL-2R, a receptor whose induction and transient elevated in 55 patients with noncrescentic glomerulonephritis, expression play a key role in T cell activation [121. including IgA nephropathy, membranous glomerulonephritis In fact, a variety of leucocytes are now known to be capable and mesangial proliferative glomerulonephritis, compared to
healthy controls. Soluble IL-2R levels correlated with serum creatininc and decreased creatinine clearance [201. The current results, combined with our previous report of IL-2R+ MNC within glomeruli of patients with crescentic IgA disease [1], indicate that the presence of increased serum IL-2R levels may with induction of IL-2R, and production of a panopoly of reflect, at least in part, local immune activation and IL-2R biologically active cytokines, but notably IL-2, required for induction on MNC within damaged glomeruli. The basis for the correlations between increased numbers of clonal expansion of activated T cells [121, and interferonintraglomerular macrophages, T cells and IL-2R+ MNC and gamma, a potent activator of macrophages [13]. Macrophages can themselves be activated by IL-2 or interferon-gamma to increased serum creatinine and decreased creatinine clearance transiently express IL-2R [14, 151, and indeed, IL-2R+ macro- in these patients is unknown. However, it is of interest that phages are found in a variety of human diseases involving T glomerular cell cultures from patients with crescentic glomerucell-dependent immune responses, including allograft rejection lonephritis contain significant levels of IL-i [211, and that the [16] and sarcoidosis [17]. Hence, in the absence of double binding of IL-2 to IL-2R+ human macrophages is a potent labelling studies, it was unclear whether intraglomerular IL- inducer of IL-i [22] and TNF [23] production. It is possible that 2R+ MNC detected in the current study were actually activated intraglomerular production of IL-i and TNF (or additional T cells, macrophages or a mixture of both cell types; statisti- cytokines) may be responsible, at least in part, for the decally, the presence of both cell types was strongly correlated creased renal function, consistent with the effects o. these with IL-2R+ MNC, as well as with each other, and the cytokines on glomerular mesangial [241 and endothelial cells of expressing IL-2R. However, with relevance to the present study, IL-2R are expressed upon activation of both T cells and macrophages, but are absent from resting cells. T cells recognize antigens located on the surfaces of Class Il-bearing macrophages, and in the presence of IL-i, undergo rapid activation
morphologic features of IL-2R+ MNC in cryostat sections were indistinct. In addition, we have previously reported that intraglomerular macrophages in biopsies from patients with crescen-
tic glomeruionephritis expressed the procoagulant molecule,
[25—271 in vitro and in vivo.
Conversely, the decreased renal function may simply result from disruption of normal glomerular filtration due to accumulation and proliferation of cells in Bowman's space. We found
tissue factor, and that such procoagulant expression was that some 60% of crescent cells during active disease were closely associated with the fibrin deposition generally found in leucocytes, consistent with our previous findings [21, but that in these patients [181. Since induction of macrophage tissue factor addition to a large proportion of macrophages, these cells
797
Li et al: IL-2R+ MNC in human crescentic GN Table 3. Leucocytes within crescents of biopsies from patients with active or inactive crescentic glomerulonephritisa Group Active
(N=
Total WBC 60.5
50"
19.1
2.2
43.1
CD4+ cells
T cells
Monocytes 5.1"
7.1
2.1"
4.9
CD8+ cells
05b
2.5
0.7"
IL-2R+ cells 14b
5.0
15)
Inactive
7.2
1.9
0—2
0—3"
0—1
0—2
(N= 19) a Expressed as % of total crescent cells (mean SaM), and determined by immunoperoxidase labelling with mAbs. b p < 0.01, between active and inactive crescents
Ranges are shown when numbers very small
Table 4. Leucocytes within crescents of glomeruli with ruptured versus intact Bowman's capsules (BC) Group
No.
Total WBC
Monocytes
T cells
Ruptured BC Intact BC
20
635b 5.8
451b 55
75b 2.2
14
15.9
2.9
6.9
1.7
0—3"
a Leucocytes determined by mAb labelling and expressed as % total crescent cells (mean silver-stained paraffin sections and/or by immunoperoxidase labelling with PHM5 mAb. "P < 0.01, between groups with predominantly intact vs. ruptured BC "Ranges are shown when numbers were very small
CD4+ cells
48b
0.9
0—1.5
CD8+ cells
27b 0.5 0-0.8
IL-2R+ cells 55b
1.3
0—2
5EM); integrity of BC determined by evaluation of
Table 5. Correlations between in traglomerular MNC and renal function i n crescentic glomeronephritis (N = 34) Renal function Total CD4+ CD8+ WBC T cells cells cells parameter Monocytes r = 0.69 r = 0.68 r = 0.69 r = 0.62 r = 0.60 Serum creatinine P < 0.01 P < 0.01 P < 0.01 P < 0.05 P < 0.05 r = —0.68 r = —0.80 Creatinine clearance NS NS NS P < 0.05 P < 0.01 Urine protein excretion NS NS NS NS NS Urine RBC excretion NS NS NS NS NS
IL-2R+ cells
r = 0.61 P < 0.05
r=
—0.63
P < 0.05 NS NS
included significantly increased numbers of T cells and IL-2R+ rophages, as observed here, but increased proportions of epiMNC, compared to inactive disease. Moreover, in agreement thelial cells [3]. with the results of Boucher et al [71, such increases were usually In summary, these studies indicate that activated MNC are accompanied by tears in Bowman's capsule (Table 4). Hence, present within glomeruli and Bowman's space during active
whether these leucocytes originate from within damaged tuft human crescentic disease, and correlate with decreased renal capillaries, or migrate into Bowman's space through breaks in function, suggesting the importance of cellular immune activathe capsule, is unknown and warrants careful and serial analysis tion to the evolution of crescentic glomerulonephritis. Further studies are necessary to identify the precise nature of these using experimental models. Current concepts of crescent formation suggest that egress of IL-2R+ MNC, and to determine, initially using experimental MNC into Bowman's space may follow intense inflammation models, whether targeting of this critical subpopulation of within the tuft with release of proteolytic enzymes, superoxide activated MNC is relevant to the therapy of crescentic glomeranions, and cytokines which damage the glomerular capillary ulonephritis, analogous to the successful abrogation of transwalls [31. Hence, continuance of an active immune response plant rejection achieved through the administration of antiwithin Bowman's space, as reflected by the content of IL-2R+ IL-2R mAbs [16]. MNC in active disease, may contribute to this damage, for Acknowledgments example, by promoting macrophage production of IL-l and Parts of this work were presented at the 26th Annual Meeting of the TNF, and procoagulant activity. The latter suggestion is consistent with knowledge that crescent macrophages express Australasian Society of Nephrology, Sydney, February 1990, and at the tissue factor [18, 28], and the almost universal finding of fibrin XIth International Congress of Nephrology, Toyko, July 1990. deposition within Bowman's space during active disease [3]. Reprint requests to Professor Robert C. Atkins, M.D., Department of Moreover, -thrombin was recently reported to induce glomeru- Nephrology, Prince Henry's Hospital, St. Kilda Road, Melbourne, lar epithelial cell proliferation in vitro [29], suggesting a possible link between the initial active phase of crescentic disease, when T cell-induced macrophage activation and procoagulant expres-
sion may occur, and a later phase when crescents become organized and contain significantly decreased numbers of mac-
Victoria 3004, Australia.
References I. Li HL, HANCOCK WW, HOOKE DH, DOWLINO JP: Mononuclear
cell activation and decreased renal function in IgA nephropathy with crescents. Kidney mt 37: 1552—1556, 1990
Li et a!: IL-2R+ MNC in human crescentic GN
798
2. HANCOCK WW, ATKINS RC: Cellular composition of crescents in human rapidly progressive glomerulonephritis defined using monoclonal antibodies. Am J Nephrol 4:177—181, 1984 3. ATKINS RC, THOMSON NM: Rapidly progressive glomerulonephri-
tis, in Diseases of the Kidney, edited by SCHRIER RW, GOTTSCHALK CW, Boston, Little, Brown and Company, 1988, pp. 1903—1927
4. HANCOCK WW, ATKINS RC: Immunohistological studies with monoclonal antibodies, in Immunochemical Techniques: Hybridoma Technology and Monoclonal Antibodies. Methods in Enzymology (vol 121), edited by LANGONE JJ, VAN VUNAKIS H, Orlando,
C, HOGGN, TILNEY NL: Identification of IL-2R+ T cells and macrophages within rejecting rat cardiac allografts, and comparison of the effects of treatment with anti-IL-2R monoclonal antibody or cyclosporin. J Immunol 138:164—170, 1987 17. HANCOCK WW, K0BzIK L, COLBY AJ, O'HARA CJ, COOPER AG, GODLESKI JJ: Detection of lymphokines and lymphokine receptors in pulmonary sarcoidosis: Immunohistologic evidence that inflammatory macrophages express IL-2 receptors. AmfPathol 123:1—8, FR, DIJKSTRA
1986 18. HANCOCK WW, ATKINS RC: Activation of coagulation pathways
and fibrin deposition in human glomerulonephritis. Semin Nephro/
Academic Press, 1986, pp. 828—848
5. HANCOCK WW, GEE D, DE MOERLOOSE P, RICKLES FR, EWAN VA, ATKINS RC: Immunohistological analysis of serial biopsies
taken during ongoing human renal allograft rejection: Changing profile of infiltrating cells and involvement of the coagulation system. Transplantation 39:430—438, 1985 6. HANCOCK WW, ATKINS RC: Monoclonal antibodies to human glomerular cells: A marker for glomerular epithelial cells. Nephron 33:83—90, 1983 7. BOUCHER A, DROZ D, ADAFER E, NOEL LH: Relationship between
the integrity of Bowman's capsule and the composition of cellular crescents in human crescentic glomerulonephritis. Lab Invest 56:
5:67—77,
19. HELIN
cell-instructed monocyte procoagulant response is rapid and is elicited by HLA-DR. J Exp Med 158:962—967, 1983 20. YORIOKA N, HIRABAYASHI A, KANAHARA K, TAKEMASA A,
ODA
H, JOARDER ZH, YAMAKIDO M: Serum soluble interleukin-2 receptor in patients with glomerulonephritis. Am J Nephro! 10:181—185, 1990 21. MATSUMOTO K, DOWLING J, ATKINS RC: Production of interleukin
in glomerular cell cultures from patients with rapidly progressive crescentic glomerulonephritis. Am J Nephro! 8:463—470, 1988 1
Studies of the human and rat macrophage receptor for interleukin-2 (IL-2R): Induction and demonstra-
22. PLEAU ME, HANCOCK WW:
526—533, 1987 8.
1985
H, EDGINGTON TS: Allogeneic induction of the human T
ATKINS RC, HOLDSWORTH SR. GLASGOW EF, MATTHEWS FE:
tion that IL-2 boosts interleukin-l production by IL-2R+ macro-
The macrophage in human rapidly progressive glomerulonephritis. Lancet ii:830—83l, 1976
phages. Transplant Proc 21:140—141, 1989 23. HERRMANN F, GEBAUER G, LINDEMANN A, BRACH M, MERTELSMANN R: Interleukin-2 and interferon-gamma recruit different sub-
I, SI L, MADAN E, WHITESIDE T: Mononuclear cell in human renal disease. Enumeration in tissue sections with monoclonal antibodies, Clin Immunol Immunopathol 3:362—373,
9. STACHURA
sets of human peripheral blood and monocytes to secrete interleukin-I beta and tumour necrosis factor-alpha. Clin Exp Immunol
subsets
1984
F, CAMERON JS, HARTLEY B, COELHO GH, REUBEN R: Intraglomerular T cells and monocytes in nephritis: Study with
77:97—100, 1989
LOVETT DH, RYAN JL, STERZL B: Stimulation of
rat mesangial cell proliferation by macrophage interleukin 1. J Immunol 131:2830—
10. NOLASCO
24.
monoclonal antibodies. Kidney mt 31:1160—1166, 1987 11. BOLTON WK, INNES DJ, STURGILL BC, KAISER DL: T cells and
2835, 1983 25. BERTANI T, ABBATE M, ZOJA C, CORNA D, PERICO N, GHEZZI P, REMUZZI G: Tumor necrosis factor induces glomerular damage in the rabbit. Am J Pathol 134:419—430, 1989 26. HANCOCK WW, COTRAN RS: Induction of activation antigens on
macrophages in rapidly progressive glomerulonephritis: Clinicopathological correlations. Kidney mt 32:869—876, 1987 12. SMITH KA: Interleukin 2: Inception, impact and implications. Science 240:1169—1 176, 1988
13. ADAMS DO, HAMILTON TA: The cell biology of macrophage activation. Annu Rev Immunol 2:283—3 18, 1984 14. HANCOCK WW, MULLER WA, COTRAN RS: Interleukin-2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis, and are inducible by lymphokine treatment of normal human
human glomerular endothelium by interleukin 1 (IL-i), interferongamma (IFN-y), and endotoxin (LPS). (abstract) Kidney mt 3 1:322, 1987
27. HANCOCK WW: IL-l and TNF depress glomerular endothelial thrombomodulin expression in vitro and in vivo. (abstract) Kidney mt 38:557, 1990
lung macrophages, blood monocytes and monocyte cell lines. J 28. HANCOCK WW, RICKLES FR, EWANVA, ATKINS RC: lmmunohisImmunol 138:185—191, 1987 tological studies with AI-3, a monoclonal antibody to activated human monocytes and macrophages. J Immuno! 136:2416—2420, 15. HANCOCK WW, PLEAU ME, KOBZIK L: Recombinant granulocytemacrophage colony-stimulating factor down-regulates expression of IL-2 receptors on human mononuclear phagocytes by induction of prostaglandin E. J Immunol 140:3021—3025, 1988 16. HANCOCK WW, LORD RH, COLBY AJ, DIAMANTSTEIN T, RICKLES
1986 29.
HE CJ, RONDEAU E, LACAVE R, DELARNE F, SRAER JD: Thrombin
stimulates proliferation decreases fibrinolytic activity of human glomerular epithelial cells. (abstract) Kidney liii 37:195, 1990
RAPID COMMUNICATION
Activated (IL-2R+) intraglomerular mononuclear cells in crescentic glomerulonephritis HAI-LING Li, WAYNE W. HANCOCK, JOHN P. DOWLING, and ROBERT C. ATKINS Departments of Nephrology and Anatomical Pathology, Prince Henry's Hospital, Melbourne, Victoria, Australia
Activated (IL-2R+) intraglomerular mononuclear cells in crescentic glomerulouephritis. We recently reported evidence for the involvement
of local cellular immune activation in the immunopathogenesis of human IgA nephropathy, particularly in cases of IgA disease featuring crescent formation. In the current study, using monoclonal antibodies, we investigated whether mononuclear cells bearing receptors for interleukin 2 (IL-2R+ MNC) were present within glomeruli or associated crescents in biopsies from patients with crescentic glomerulonephntis (>60% crescents, N = 19), IgA disease with crescents (N = 9), or other types of proliferative glomerulonephritis with crescents (10 to 44%, N = 6), compared with normal control kidneys (N = 10). Biopsies were further classified into those showing active (cells, fibrin) (N = 15) or inactive (sclerosed) crescents (N = 19), to determine whether IL-2R+ MNC were particularly associated with active crescent formation. Few leucocytes were found within glomerular tufts of normal kidneys (2.4
the correlations observed between the presence of activated intraglomerular tuft MNC and decreased parameters of renal function, these results suggest that crescent formation involves recruitment of macrophages to the glomerulus by sensitised T cells, analogous to events occurring in a DTH response, and are consistent with a direct role for cellular immunity in the immunopathogenesis and deteriorating renal function characteristic of human crescentic glomerulonephritis.
Extensive analysis of experimental models of glomerulonephritis has resulted in considerable insights into the mechanisms of immunologically induced glomerular injury. Individual
models based on antibody-mediated injury, immune complex deposition or T cell-dependent glomerular damage are well biopsies from patients with active crescentic glomerulonephritis, total described. However, few studies have concerned identification 1.7 (P < 0.01 intraglomerular tuft leucocytes were increased to 14.0 vs. normal kidneys), largely due to increased numbers of intraglomer- of similar mechanisms in human glomerulonephritis, particuular monocytes (10.4 1.1, P <0.01) and T cells (3.7 0.6, P <0.01). larly T cell-dependent mechanisms (Type IV hypersensitivity Biopsies with active crescents also contained significantly increased reactions). We recently reported that mononuclear cells bearing numbers of intraglomerular tuft IL-2R+ MNC (4.0 0.7, 29% of total intraglomerular leucocytes), and there was a strong correlation between receptors for interleukiri 2 (IL-2R+ MNC) were present in significantly increased numbers in biopsies from patients with the numbers of intraglomerular IL-2R+ MNC and T cells (P < 0.001). Moreover, in this active crescentic group, increases in the numbers of IgA nephropathy with crescents, compared to patients with IgA intraglomerular IL-2R+ MNC (P < 0.05), macrophages (P < 0.01) and nephropathy without associated crescent formation or normal T cells (P < 0.01) were correlated with raised serum creatinine levels, and the numbers of both IL-2R+ MNC (P < 0.05) and macrophages controls [II. Moreover, the numbers of IL-2R+ MNC correlated with raised serum creatinine and decreased creatinine (P< 0.01) correlated with decreased creatinine clearance. Patients with inactive crescentic glomerulonephritis also showed increased levels of clearance, suggesting that local cellular immune activation is tuft MNC compared to normal kidneys, but significantly less intraglo- involved in the immunopathogenesis of IgA disease, particumerular MNC than biopsies from patients with active crescentic dis- larly in cases with crescent formation. ease. Thus, in inactive crescentic disease there was a four-fold increase The current study examined whether IL-2R+ MNC, T cells in glomerular MNC infiltration (total leucocytes 10.0 1.4, P < 0.01), due to macrophages (6.6 1.0, P < 0.01 compared to normals or active and macrophages were present within glomeruli and/or associcrescentic glomerulonephritis) and T cells (2.9 0.3, P < 0.01), and ated crescents in a larger series of biopsies from patients with including IL-2R+ MNC (2.2 0.3, P < 0.01 compared to normals or more common forms of crescentic glomerulonephritis, and active crescentic disease). The cellular composition of actual crescents was also determined. Leucocytes contributed more than 60% of the more importantly, whether the presence of such activated MNC total cells within active crescents, due primarily to the presence of was associated with deteriorating renal function. 0.7 cells/glomerular cross-section; mean
SEM). By contrast, in
macrophages (43.1% 5.1%) and T cells (7.1% 2.1%); 8% of these leucocytes within crescents were IL-2R+. Glomeruli with active cres-
Methods
cents contained extensive ruptures of Bowman's capsules. Inactive crescents, which generally displayed intact Bowman's capsules, presumeably as a result of fresh synthesis of basement membrane components, contained fewer leucocytes (19.1% 2.2%), including reduced 1.9%) and T cells (0 to 3, ranges). numbers of macrophages (7.2% Given the close regulation of IL-2R expression on activated MNC, and
Tissues
Renal biopsies from 19 patients with crescentic glomerulone-
Received for publication August 21, 1990 and in revised form December II, 1990 Accepted for publication December 12, 1990
phritis (> 60% crescents), were compared with biopsies from nine patients with IgA nephropathy with crescents, and six patients with other forms of proliferative glomerulonephritis and focal crescent formation (10 to 44%) (Table 1). Biopsies with predominantly fleshy, cellular crescents and associated fibrin deposition were further classified as active crescents (N = 15), whereas those with largely organized, fibrous or fibrocel-
© 1991 by the International Society of Nephrology
lular crescents and largely lacking fibrin deposits were classified
793
794
Li et a!: IL-2R+ MNC in human crescentic ON Table 1. Histologic classification of renal biopsies
No. of
Active Inactive biopsies Crescents crescentsa crescentsa
Diagnosis
Immune complex GN IgA nephropathy Focal proliferative ON Idiopathic GN Anti-GBM disease Pyelonephritis Membranoproliferative GN Totals
14 9 4 3 2
%
70—100%
8—71 % 35—44 % 70—100% 60—100%
1
30%
1
10%
34
6 8 2
8
1 1
2 2 2 0 0
19
15
1
0
a As defined in Methods
Renal function Serum creatinine, endogenous 24-hour creatinine clearance, and glomerular filtration rate were determined in each patient at the time of biopsy.
Statistical analysis Data were analyzed on a personal computer using programs from the CSS system (Statsoft, Tulsa, Oklahoma, USA). The numbers of labelled cells in different groups were compared using the two-tailed Student's t-test. The percentages of la-
belled cells within glomeruli or crescents in biopsies from different groups were compared using a two-tailed Wilcoxon Rank sum test, and correlations determined using Spearman's
test. Results
as inactive crescents (N 19) [2, 3]. Normal control tissues Intraglomerular MNC consisted of portions of cadaver nephrectomies not used for or kidneys removed because of localtransplantation (N = 7) Few leucocytes were present in normal renal tissue (2.4 0.7 ized tumors (N = 3). All biopsies were routinely assessed by cells/glomerular cross section); these consisted of monocytes light microscopy, immunofluorescence and electron micros- (1.4 0.4) and lesser number of T cells (0.3, range 0.1 to 0.4) copy, as well as by immunoperoxidase labelling using anti- (Table 2). No IL-2R+ cells were detected. In contrast, biopsies leucocyte and other monoclonal antibodies (mAbs). Specimens from patients with active crescentic glomerulonephritis showed for immunoperoxidase studies were fixed in PLP at 4°C, washed increased intraglomerular leucocytes (14.0 1.7, P < 0,01), in PBS containing 7% sucrose, snap frozen and stored at —80°C including macrophages (10.4 1.1, P < 0.01), T cells (3.7 until cryostat sectioning, as described previously [41. 0.6, P < 0.01) of both CD4+ and CD8+ phenotypes (P < 0.01),
Monoclonal antibodies
Infiltrating leucocytes were identified using the following mAbs: all leucocytes—PHMI (CD45); all T cells—Leu 4 and OKT3 (CD3); helper/inducer T cell subset—Leu 3A and OKT4 (CD4); cytotoxic/suppressor T cell subset—OKT8 (CD8); macrophages-FMC32 (CD14); IL-2R+ MNC—Tac-l (CD25); B cells—B 1 (CD2O), and NK cells—Leu 7 (CD57). Details of
and IL-2R+ MNC (4.0 0.7, P < 0.01; Table 2). Moreover, though the nature of IL-2R+ MNC was not directly established
in this study, there were strong correlations between the numbers of intraglomerular IL-2R+ MNC and both T cells (r = 0.76, P < 0.001, Fig. 1) and macrophages (r = 0.75, P < 0.001,
Fig. 2). Furthermore, the numbers of infiltrating macrophages and T cells were also highly correlated (r = 0.77, P < 0.001, Fig. 3). B cells and NK cells were rarely identified within glomeruli these mAbs and their use were presented previously [5]. of any group (< 0.1 cells/glomerular cross section). Biopsies from patients with inactive crescentic glomeruloneBiopsies were also labelled using a mAb to glomerular parietal phritis also showed increased numbers of intraglomerular leuand visceral epithelial cells (PHM5) 12, 61. Immunoperoxidase labelling
cocytes, though to a lesser extent than in active crescentic disease (Table 2). Thus, glomeruli in patients with inactive crescentic disease showed increased total leucocytes (10.0
Cryostat sections (4 m) were labelled using a four-layer 1.4, P < 0.01 vs. normal), due to increases in macrophages (6.6 immunoperoxidase (PAP) technique (4).
Evaluation of immunostained sections
Renal biopsies were examined for the presence of labelled cells within glomerular tufts and any associated glomerular crescents; biopsies contained 3 to 20 glomeruli. Labelled cells within each glomerulus were counted and results were expressed as the number of cells per glomerular cross section. Labelled cells within glomerular crescents were expressed as a percentage of total crescent cells. Cell counts were performed three times to reduce counting errors, and quantitative analysis of infiltrating MNC was performed independently and without knowledge of clinical or histopathologic data. Data were also expressed in terms of whether most crescentic glomeruli (> 75%) in a given biopsy retained intact Bowman's capsules, or whether such capsules were predominantly intact [7]; this was assessed by examination of silver-stained paraffin sections, prepared at three different levels from each biopsy, and by immunoperoxidase labelling with PHM5 mAb [6].
1.0, P < 0.01 compared to both normal and active crescentic disease) and T cells (2.9 0.3, P <0.01 vs. normal) of both CD4+ and CD8+ phenotypes, as well as IL-2R+ MNC (2.2
0.3, P < 0.01 compared to normal or active disease). The numbers of intraglomerular total leucocytes, macrophages, T cells and IL-2R+ MNC present in biopsies classified primarily as crescentic glomcrulonephritis, or proliferative glomerulonephritis with associated crescent formation, were significantly
increased compared with our previously studied groups of patients with IgA nephropathy with crescents (Fig. 4)111. Cells in glomerular crescents Leucocytes contributed some 60% of cells within crescents of patients with active crescentic disease (N 15) (Table 3).
Macrophages were the main cell type identified, contributing 5.1% of cells within active crescents, and in some 43.1% glomeruli up to 90% of crescent cells were macrophages. T cells were also present (7.1% 2.1%), including both CD4+ (4.9%
0.7%) and CD8+ (2.5% 0.5%) phenotypes, and IL-2R+ MNC
795
Li et a!: IL-2R+ MNC in human crescentic GN Table 2. Intraglomerular tuft MNC in biopsies from patients with crescentic glomerulonephritis (N =
Normal kidneys
10
Inactive crescentse Active crescentse
'
Total WBC
No.
Group
2.4a
10.0" 14.0"
19 15
T cells
Monocytes 1.4 0.4
0.7 1.4 1.7
a Expressed as cells/glomerular cross section (mean
6.6C
1.0
104c.d
1.1
34)
versus normal kidneys (N= 10)
CD8+
CD4+ cells
cells
IL-2R+ cells 0.0
03b
0.1
0.1
(0.1—0.4)
(0.1—0.2)
(0.1—0.2) l.2C 0.2
2.2"
0.3
0.3
4.0""
0.7
2.9" 3.7"
0.3 0.6
1.6" 27c.d
0.5 0.4
1.8"
SEM)
Ranges are
shown when numbers were very small P < 0.01, between normal and crescentic glomerulonephritis d P < 0.05, between inactive and active crescentic glomerulonephritis as defined in Methods
25 C
0
r = 0.755
C)
U,
0
0
9
20 -
P<0.0001
r0.77
P<0.0001
0
0
CC
15 C)
E
0
.
6
0
D)
.
C)
0
.
...
3
0 0
.
z 0
t10_
.. . .
8 E
5-
S
.. .
E
S
•..
..-• •
S
.
.• •
S
2
4
6
8
10
No. of L-2P + MNC/glomerular cross section Fig. 1. Correlation between the number of intraglomerular T cells and
IL-2R+ MNC in active crescentic disease, determined by Spearman's
test.
10 4 6 8 2 0 No. of L-2R + MNC/glomerular cross section Fig. 2. Correlation between the number of intro glomerular macrophages and JL-2R+ MNC in active crescent disease, determined by Spearman's test.
1.4%). No crescent cells expressed B cell or NK cell (P < 0.05), macrophages (P < 0.01) and IL-2R+ MNC (P < markers. 0.05) were correlated with decreased creatinine clearance. Nineteen patients with older, scierosed crescents were stud- However, none of these cells were correlated with urinary ied (Table 3). In contrast to biopsies with active, cellular protein or RBC excretion. crescents, the proportion of leucocytes in scierosed crescents (5.0%
was reduced to 19.1%
(P <
consisting of macrophages (7.2% 1.9%, P <0.01) and only very few T cells 2.2%
0.01),
Discussion
(range of 0 to 3%, P < 0.01) or IL-2R+ MNC (range of 0 to 2%,
This study investigated the contribution of leucocyte infiltration to the glomerular hypercellularity, accumulation of cells in
P < 0.01). Morphologically, most crescent cells at this stage
Bowman's space, and associated decreased renal function
appeared to be fibroblastoid or epithelioid in nature. The
characteristic of the various forms of human crescentic glomerulonephritis [31. We found that essentially all biopsies from patients with crescentic glomerulonephritis, regardless of etiology, but particularly those with active disease, showed signifi-
influence of the integrity of Bowman's capsule on the composition of crescent cells was also considered (Table 4). For each leucocyte population, the numbers of leucocytes within glomeruli with ruptured capsules were significantly greater (P < 0.01) than in those with intact capsules. Correlation between intro glomerular leucocytes and decreased renal function
cantly increased numbers of intraglomerular tuft leucocytes, consisting predominantly of macrophages and lesser numbers of T cells. Moreover, the findings that in active disease over 25% of these tuft leucocytes showed evidence of immune activation (Table 2), as indicated by expression of IL-2R, and that the presence of these cells correlated with decreased renal function (Table 5), were consistent with the developing concept
Intraglomerular MNC infiltration was strongly associated with decreased renal function (Table 5). Thus, the numbers of that MNC activation is important to the immunopathogenesis of total leucocytes (P < 0.01), monocytes (P < 0.01), T cells (P < human crescentic glomerulonephritis. 0.01) and IL-2R+ MNC (P < 0.05) were each correlated with Since our initial report [8], using glomerular culture, of increased serum creatinine, and the numbers of total leucocytes macrophage infiltration of glomeruli from patients with crescen-
Li et a!: IL-2R+ MNC in human crescentic GN
796
12 C
0
r= 0.784
a,
CO
U,
C
0
P< 0.0001
C,
C, U,
9
U,
0
CO
0
C-)
C, ci, ci,
E
6
.
0 DC
a)
F-
0 0
3
.
z 0
E
0
Di S
. .
.
C-)
U)
.
0 U)
0
5
10
15
20
No. of macrophages/glomerular cross section
3. Correlation between the numbers of intraglomerular macrophages and T cells, determined by Spearman's test. Fig.
Fig. 4. Numbers of intraglomerular total leucocytes and cell subsets present in biopsies classified as IgA nephropathy with crescents (IgA), other proliferative GN with crescents (GN), or crescentic GN (>60%
crescents, CGN). CGN and GN biopsies had significantly greater numbers of intraglomerular total leucocytes (U), macrophages ( cells (NJ) and IL-2R+ MNC (U) than IgA biopsies (P < 0.05).
), T
tic glomerulonephritis, a number of groups have confirmed that expression is controlled by cytokine(s) produced locally by leucocytes contribute to the glomerular hypercellularity usually activated helper T cells [19], the current results suggest a found in this group of diseases [reviewed in 31. Other groups possible source of cytokine(s) for such macrophage activation. Following activation, both IL-2R+ T cells and macrophages [9—111 have also reported that, based on the use of mAbs, small numbers of T cells accompany the more prominent influx of shed IL-2R into surrounding fluids, and measurement of soluble macrophages into the glomerulus during crescentic glomerulo- IL-2R levels is currently used for monitoring of a number of nephritis. However, the new and more important finding of the diseases with an immunologic basis [121. Yorioka et al [201 have current study was the demonstration that many of these MNC recently reported that serum IL-2R levels were significantly expressed IL-2R, a receptor whose induction and transient elevated in 55 patients with noncrescentic glomerulonephritis, expression play a key role in T cell activation [121. including IgA nephropathy, membranous glomerulonephritis In fact, a variety of leucocytes are now known to be capable and mesangial proliferative glomerulonephritis, compared to
healthy controls. Soluble IL-2R levels correlated with serum creatininc and decreased creatinine clearance [201. The current results, combined with our previous report of IL-2R+ MNC within glomeruli of patients with crescentic IgA disease [1], indicate that the presence of increased serum IL-2R levels may with induction of IL-2R, and production of a panopoly of reflect, at least in part, local immune activation and IL-2R biologically active cytokines, but notably IL-2, required for induction on MNC within damaged glomeruli. The basis for the correlations between increased numbers of clonal expansion of activated T cells [121, and interferonintraglomerular macrophages, T cells and IL-2R+ MNC and gamma, a potent activator of macrophages [13]. Macrophages can themselves be activated by IL-2 or interferon-gamma to increased serum creatinine and decreased creatinine clearance transiently express IL-2R [14, 151, and indeed, IL-2R+ macro- in these patients is unknown. However, it is of interest that phages are found in a variety of human diseases involving T glomerular cell cultures from patients with crescentic glomerucell-dependent immune responses, including allograft rejection lonephritis contain significant levels of IL-i [211, and that the [16] and sarcoidosis [17]. Hence, in the absence of double binding of IL-2 to IL-2R+ human macrophages is a potent labelling studies, it was unclear whether intraglomerular IL- inducer of IL-i [22] and TNF [23] production. It is possible that 2R+ MNC detected in the current study were actually activated intraglomerular production of IL-i and TNF (or additional T cells, macrophages or a mixture of both cell types; statisti- cytokines) may be responsible, at least in part, for the decally, the presence of both cell types was strongly correlated creased renal function, consistent with the effects o. these with IL-2R+ MNC, as well as with each other, and the cytokines on glomerular mesangial [241 and endothelial cells of expressing IL-2R. However, with relevance to the present study, IL-2R are expressed upon activation of both T cells and macrophages, but are absent from resting cells. T cells recognize antigens located on the surfaces of Class Il-bearing macrophages, and in the presence of IL-i, undergo rapid activation
morphologic features of IL-2R+ MNC in cryostat sections were indistinct. In addition, we have previously reported that intraglomerular macrophages in biopsies from patients with crescen-
tic glomeruionephritis expressed the procoagulant molecule,
[25—271 in vitro and in vivo.
Conversely, the decreased renal function may simply result from disruption of normal glomerular filtration due to accumulation and proliferation of cells in Bowman's space. We found
tissue factor, and that such procoagulant expression was that some 60% of crescent cells during active disease were closely associated with the fibrin deposition generally found in leucocytes, consistent with our previous findings [21, but that in these patients [181. Since induction of macrophage tissue factor addition to a large proportion of macrophages, these cells
797
Li et al: IL-2R+ MNC in human crescentic GN Table 3. Leucocytes within crescents of biopsies from patients with active or inactive crescentic glomerulonephritisa Group Active
(N=
Total WBC 60.5
50"
19.1
2.2
43.1
CD4+ cells
T cells
Monocytes 5.1"
7.1
2.1"
4.9
CD8+ cells
05b
2.5
0.7"
IL-2R+ cells 14b
5.0
15)
Inactive
7.2
1.9
0—2
0—3"
0—1
0—2
(N= 19) a Expressed as % of total crescent cells (mean SaM), and determined by immunoperoxidase labelling with mAbs. b p < 0.01, between active and inactive crescents
Ranges are shown when numbers very small
Table 4. Leucocytes within crescents of glomeruli with ruptured versus intact Bowman's capsules (BC) Group
No.
Total WBC
Monocytes
T cells
Ruptured BC Intact BC
20
635b 5.8
451b 55
75b 2.2
14
15.9
2.9
6.9
1.7
0—3"
a Leucocytes determined by mAb labelling and expressed as % total crescent cells (mean silver-stained paraffin sections and/or by immunoperoxidase labelling with PHM5 mAb. "P < 0.01, between groups with predominantly intact vs. ruptured BC "Ranges are shown when numbers were very small
CD4+ cells
48b
0.9
0—1.5
CD8+ cells
27b 0.5 0-0.8
IL-2R+ cells 55b
1.3
0—2
5EM); integrity of BC determined by evaluation of
Table 5. Correlations between in traglomerular MNC and renal function i n crescentic glomeronephritis (N = 34) Renal function Total CD4+ CD8+ WBC T cells cells cells parameter Monocytes r = 0.69 r = 0.68 r = 0.69 r = 0.62 r = 0.60 Serum creatinine P < 0.01 P < 0.01 P < 0.01 P < 0.05 P < 0.05 r = —0.68 r = —0.80 Creatinine clearance NS NS NS P < 0.05 P < 0.01 Urine protein excretion NS NS NS NS NS Urine RBC excretion NS NS NS NS NS
IL-2R+ cells
r = 0.61 P < 0.05
r=
—0.63
P < 0.05 NS NS
included significantly increased numbers of T cells and IL-2R+ rophages, as observed here, but increased proportions of epiMNC, compared to inactive disease. Moreover, in agreement thelial cells [3]. with the results of Boucher et al [71, such increases were usually In summary, these studies indicate that activated MNC are accompanied by tears in Bowman's capsule (Table 4). Hence, present within glomeruli and Bowman's space during active
whether these leucocytes originate from within damaged tuft human crescentic disease, and correlate with decreased renal capillaries, or migrate into Bowman's space through breaks in function, suggesting the importance of cellular immune activathe capsule, is unknown and warrants careful and serial analysis tion to the evolution of crescentic glomerulonephritis. Further studies are necessary to identify the precise nature of these using experimental models. Current concepts of crescent formation suggest that egress of IL-2R+ MNC, and to determine, initially using experimental MNC into Bowman's space may follow intense inflammation models, whether targeting of this critical subpopulation of within the tuft with release of proteolytic enzymes, superoxide activated MNC is relevant to the therapy of crescentic glomeranions, and cytokines which damage the glomerular capillary ulonephritis, analogous to the successful abrogation of transwalls [31. Hence, continuance of an active immune response plant rejection achieved through the administration of antiwithin Bowman's space, as reflected by the content of IL-2R+ IL-2R mAbs [16]. MNC in active disease, may contribute to this damage, for Acknowledgments example, by promoting macrophage production of IL-l and Parts of this work were presented at the 26th Annual Meeting of the TNF, and procoagulant activity. The latter suggestion is consistent with knowledge that crescent macrophages express Australasian Society of Nephrology, Sydney, February 1990, and at the tissue factor [18, 28], and the almost universal finding of fibrin XIth International Congress of Nephrology, Toyko, July 1990. deposition within Bowman's space during active disease [3]. Reprint requests to Professor Robert C. Atkins, M.D., Department of Moreover, -thrombin was recently reported to induce glomeru- Nephrology, Prince Henry's Hospital, St. Kilda Road, Melbourne, lar epithelial cell proliferation in vitro [29], suggesting a possible link between the initial active phase of crescentic disease, when T cell-induced macrophage activation and procoagulant expres-
sion may occur, and a later phase when crescents become organized and contain significantly decreased numbers of mac-
Victoria 3004, Australia.
References I. Li HL, HANCOCK WW, HOOKE DH, DOWLINO JP: Mononuclear
cell activation and decreased renal function in IgA nephropathy with crescents. Kidney mt 37: 1552—1556, 1990
Li et a!: IL-2R+ MNC in human crescentic GN
798
2. HANCOCK WW, ATKINS RC: Cellular composition of crescents in human rapidly progressive glomerulonephritis defined using monoclonal antibodies. Am J Nephrol 4:177—181, 1984 3. ATKINS RC, THOMSON NM: Rapidly progressive glomerulonephri-
tis, in Diseases of the Kidney, edited by SCHRIER RW, GOTTSCHALK CW, Boston, Little, Brown and Company, 1988, pp. 1903—1927
4. HANCOCK WW, ATKINS RC: Immunohistological studies with monoclonal antibodies, in Immunochemical Techniques: Hybridoma Technology and Monoclonal Antibodies. Methods in Enzymology (vol 121), edited by LANGONE JJ, VAN VUNAKIS H, Orlando,
C, HOGGN, TILNEY NL: Identification of IL-2R+ T cells and macrophages within rejecting rat cardiac allografts, and comparison of the effects of treatment with anti-IL-2R monoclonal antibody or cyclosporin. J Immunol 138:164—170, 1987 17. HANCOCK WW, K0BzIK L, COLBY AJ, O'HARA CJ, COOPER AG, GODLESKI JJ: Detection of lymphokines and lymphokine receptors in pulmonary sarcoidosis: Immunohistologic evidence that inflammatory macrophages express IL-2 receptors. AmfPathol 123:1—8, FR, DIJKSTRA
1986 18. HANCOCK WW, ATKINS RC: Activation of coagulation pathways
and fibrin deposition in human glomerulonephritis. Semin Nephro/
Academic Press, 1986, pp. 828—848
5. HANCOCK WW, GEE D, DE MOERLOOSE P, RICKLES FR, EWAN VA, ATKINS RC: Immunohistological analysis of serial biopsies
taken during ongoing human renal allograft rejection: Changing profile of infiltrating cells and involvement of the coagulation system. Transplantation 39:430—438, 1985 6. HANCOCK WW, ATKINS RC: Monoclonal antibodies to human glomerular cells: A marker for glomerular epithelial cells. Nephron 33:83—90, 1983 7. BOUCHER A, DROZ D, ADAFER E, NOEL LH: Relationship between
the integrity of Bowman's capsule and the composition of cellular crescents in human crescentic glomerulonephritis. Lab Invest 56:
5:67—77,
19. HELIN
cell-instructed monocyte procoagulant response is rapid and is elicited by HLA-DR. J Exp Med 158:962—967, 1983 20. YORIOKA N, HIRABAYASHI A, KANAHARA K, TAKEMASA A,
ODA
H, JOARDER ZH, YAMAKIDO M: Serum soluble interleukin-2 receptor in patients with glomerulonephritis. Am J Nephro! 10:181—185, 1990 21. MATSUMOTO K, DOWLING J, ATKINS RC: Production of interleukin
in glomerular cell cultures from patients with rapidly progressive crescentic glomerulonephritis. Am J Nephro! 8:463—470, 1988 1
Studies of the human and rat macrophage receptor for interleukin-2 (IL-2R): Induction and demonstra-
22. PLEAU ME, HANCOCK WW:
526—533, 1987 8.
1985
H, EDGINGTON TS: Allogeneic induction of the human T
ATKINS RC, HOLDSWORTH SR. GLASGOW EF, MATTHEWS FE:
tion that IL-2 boosts interleukin-l production by IL-2R+ macro-
The macrophage in human rapidly progressive glomerulonephritis. Lancet ii:830—83l, 1976
phages. Transplant Proc 21:140—141, 1989 23. HERRMANN F, GEBAUER G, LINDEMANN A, BRACH M, MERTELSMANN R: Interleukin-2 and interferon-gamma recruit different sub-
I, SI L, MADAN E, WHITESIDE T: Mononuclear cell in human renal disease. Enumeration in tissue sections with monoclonal antibodies, Clin Immunol Immunopathol 3:362—373,
9. STACHURA
sets of human peripheral blood and monocytes to secrete interleukin-I beta and tumour necrosis factor-alpha. Clin Exp Immunol
subsets
1984
F, CAMERON JS, HARTLEY B, COELHO GH, REUBEN R: Intraglomerular T cells and monocytes in nephritis: Study with
77:97—100, 1989
LOVETT DH, RYAN JL, STERZL B: Stimulation of
rat mesangial cell proliferation by macrophage interleukin 1. J Immunol 131:2830—
10. NOLASCO
24.
monoclonal antibodies. Kidney mt 31:1160—1166, 1987 11. BOLTON WK, INNES DJ, STURGILL BC, KAISER DL: T cells and
2835, 1983 25. BERTANI T, ABBATE M, ZOJA C, CORNA D, PERICO N, GHEZZI P, REMUZZI G: Tumor necrosis factor induces glomerular damage in the rabbit. Am J Pathol 134:419—430, 1989 26. HANCOCK WW, COTRAN RS: Induction of activation antigens on
macrophages in rapidly progressive glomerulonephritis: Clinicopathological correlations. Kidney mt 32:869—876, 1987 12. SMITH KA: Interleukin 2: Inception, impact and implications. Science 240:1169—1 176, 1988
13. ADAMS DO, HAMILTON TA: The cell biology of macrophage activation. Annu Rev Immunol 2:283—3 18, 1984 14. HANCOCK WW, MULLER WA, COTRAN RS: Interleukin-2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis, and are inducible by lymphokine treatment of normal human
human glomerular endothelium by interleukin 1 (IL-i), interferongamma (IFN-y), and endotoxin (LPS). (abstract) Kidney mt 3 1:322, 1987
27. HANCOCK WW: IL-l and TNF depress glomerular endothelial thrombomodulin expression in vitro and in vivo. (abstract) Kidney mt 38:557, 1990
lung macrophages, blood monocytes and monocyte cell lines. J 28. HANCOCK WW, RICKLES FR, EWANVA, ATKINS RC: lmmunohisImmunol 138:185—191, 1987 tological studies with AI-3, a monoclonal antibody to activated human monocytes and macrophages. J Immuno! 136:2416—2420, 15. HANCOCK WW, PLEAU ME, KOBZIK L: Recombinant granulocytemacrophage colony-stimulating factor down-regulates expression of IL-2 receptors on human mononuclear phagocytes by induction of prostaglandin E. J Immunol 140:3021—3025, 1988 16. HANCOCK WW, LORD RH, COLBY AJ, DIAMANTSTEIN T, RICKLES
1986 29.
HE CJ, RONDEAU E, LACAVE R, DELARNE F, SRAER JD: Thrombin
stimulates proliferation decreases fibrinolytic activity of human glomerular epithelial cells. (abstract) Kidney liii 37:195, 1990