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Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-
and cocultured with MC. CD40L-activated monocytes induced higher levels
of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1
synthesis by MC. Separation of CD40L-activated monocytes from MC by a
porous membrane decreased the mesangial synthesis of IL-6 by 80% and
ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against
the ß2 integrins, LFA-1 and Mac-1, decreased IL-6
production by 40 and 50%, respectively. Ligation of mesangial surface
ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous
neutralization of soluble TNF- and IL-1ß decreased MCP-1
production by 55% in membrane-separated cocultures of
MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated
monocytes (to preserve membrane integrity but prevent secretory
activity), cocultured with MC at various ratios, induced IL-6, MCP-1,
and ICAM-1 synthesis by MC. Plasma membrane preparations from activated
monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition
of plasma membrane enhanced TNF--induced mesangial IL-6 production
by 
4-fold. Together, these data suggest that the CD40/CD40L is
essential for optimal effector function of monocytes, that
CD40L-activated monocytes stimulate MC through both soluble factors and
cell-to-cell contact mediated pathways, and that both pathways are
essential for maximum stimulation of MC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence implicating macrophages directly in renal pathology arise from animal studies whereby depletion of macrophages inhibits the induction or progression of renal injury (5). Studies in humans have suggested a correlation of the extent of glomerular macrophage infiltration with glomerular hypercellularity, histologic damage, proteinuria, and loss of renal function (6). Macrophages are also thought to play a vital role in crescent formation and tissue injury in crescentic GN (7).
Macrophages exhibit critical regulatory, as well as effector, functions
during inflammatory responses. They communicate with adjacent
inflammatory and mesenchymal cells through secretion of soluble factors
or by direct cell-to-cell contact (8, 9). In vitro studies
have shown that macrophage-derived IL-6, platelet-derived growth
factor, and TGF-ß promote mesangial expansion (10, 11).
Macrophage-derived proinflammatory cytokines such as TNF- and
IL-1ß induce mesangial cells (MC) to secrete chemokines, such as
monocyte chemoattractant protein-1 (MCP-1), and express adhesion
molecules, such as ICAM-1, which facilitate further infiltration and
accumulation of macrophages into glomeruli (12, 13, 14).
In contrast with soluble factors, the role of direct cell-to-cell contact in mediating tissue injury by monocytes has received little attention. MC are known to interact with extracellular matrix through a variety of integrins (15, 16, 17), but whether they also interact with local macrophages has not been adequately investigated. This is particularly important in view of data suggesting that direct cell-to-cell interactions mediate important biologic effects, which, in some cases, are distinct from those mediated by soluble factors, and that these interactions may have a pathogenic role in a variety of inflammatory diseases (18, 19, 20, 21).
In the present study, we sought to critically evaluate potential
mechanisms whereby macrophages might contribute to renal pathology
through their interactions with resident renal cells. We employed an in
vitro experimental system with several unique features. First, we used
cytokines of likely importance in the pathogenesis of GN (GM-CSF,
IFN-) to prime peripheral blood human monocytes
(22, 23, 24, 25). Second, to activate primed monocytes, we used
soluble CD40 ligand (CD40L). CD40L is a membrane glycoprotein that is
transiently expressed on the surface of activated
CD4+ T cells. Interaction of CD40L with its
counterreceptor CD40 (which is constitutively expressed by macrophages)
is essential for the development of the effector function of
macrophages and has been implicated in the pathogenesis of autoimmune
GN, such as lupus nephritis (26, 27, 28, 29). Third, we employed
coculture experiments that used a semipermeable membrane to physically
separate cells and thereby delineate the relative contribution of
cell-to-cell contact. These experiments were extended using
paraformaldehyde-fixed monocytes (which preserves membrane integrity
but prevents soluble factor secretion), together with purified plasma
membrane preparations, to further characterize the relative
contributions of direct cell-to-cell contact and soluble factors. Our
data suggest that CD40L-activated monocytes amplify the glomerular
inflammatory response through both soluble and cell contact-dependent
mechanisms and identify key molecules involved in these
interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs, recombinant cytokines, and proteins were used
in these experiments: monoclonal mouse anti-human TNF- (R&D
Systems, Minneapolis, MN); monoclonal mouse anti-human IL-1ß
(Genzyme, Cambridge, MA); monoclonal mouse anti-human CD11a/LFA-1
(PharMingen, San Diego, CA); monoclonal mouse anti-human
CD11b/Mac-1 (PharMingen); monoclonal mouse anti-human CD49d/very
late activation Ag-4 (VLA-4) (Immunotech, Miami, FL); monoclonal mouse
anti-human ICAM-1 (Dako, Carpinteria, CA); R-PE-conjugated
F(ab)2 fraction of goat anti-mouse Igs
(Dako); recombinant human IFN- (Genzyme); recombinant human GM-CSF
(R&D Systems); recombinant human TNF- (R&D Systems); and trimeric
human CD40L/leucine-zipper fusion protein (Immunex, Seattle,
WA).
Human MC
Human MC were established and characterized as reported previously (30), and cultured in Waymouth’s medium (Life Technologies, Grand Island, NY) supplemented with 17% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 2 mM sodium pyruvate, 1% (v/v) nonessential amino acids, and 26 µg/ml of bovine insulin (all Life Technologies). Four independent cell lines were employed in passages 5–10.
Monocyte purification and culture
Purified monocytes were isolated using counterflow centrifugal elutriation from normal human leukocyte concentrates obtained from the Department of Transfusion Medicine, National Institutes of Health by automated apheresis. Monocyte purity was assessed by FACS analysis using fluoresceinated mAb (Becton Dickinson, Mountain View, CA) with specificity for the following cell surface markers: CD14 (monocytes), CD3 (T cells), and CD19 (B cells). The monocyte fraction routinely consisted of >80% CD14-expressing cells, <0.5% CD19-expressing cells, and <3% CD3-expressing cells. Purified monocytes were cultured in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Monocyte-MC coculture
Monocytes were cultured at 2 215’5f
106/ml for 24 h in 10% FBS/RPMI 1640 medium
in the presence or absence of IFN- (500 U/ml) and GM-CSF (10 ng/ml)
in a 24-well or 6-well plate. Preliminary experiments established these
concentrations of cytokines as optimal. Subsequently cells were
cultured with or without 3 µg/ml of trimeric CD40L or 10 ng/ml of
TNF- for 24 h. Supernatants were collected and stored for
TNF-, IL-1ß, and IL-12 synthesis determination, while activated
monocytes were washed three times and resuspended to a final
concentration of 1, 5, and 20 215’5f 105/ml in
Waymouth’s medium containing 17% FBS. In some experiments, monocytes
were fixed for 2 h in 1% paraformaldehyde (Electron Microscopy
Sciences, Washington, PA) in PBS at 4°C with gentle agitation; this
preserves membrane integrity but prevents secretory activity. MC were
harvested at 80% confluence and cultured in 24-well plates at 6
215’5f 104 cell/0.6 ml/well in triplicate for
16 h. Then, 0.6 ml of prepared activated monocytes were added to
MC. In some experiments, identical parallel cultures were established
in which monocytes were separated from MC by a 0.4-µm pore
semipermeable membrane (Biocoat, Falcon, Becton Dickinson Labware,
Bedford, MA), while sharing the same medium. After 24 h,
supernatants were harvested and stored at –20°C for IL-6 and MCP-1
determination, and cells were harvested and subjected to ICAM-1
expression estimation by FACS analysis.
Inhibition of cytokine synthesis by Ab
Abs against CD11a/LFA-1, CD11b/Mac-1, CD49d/VLA-4, TNF-,
IL-1ß, or IgG1 of irrelevant specificity (Sigma, St. Louis, MO) were
incubated at 10 µg/ml with activated monocytes for 30 min at room
temperature. Cells were added to MC as described before. Cytokine
synthesis after 24 h was determined by ELISA.
Membrane preparation from monocytes
Crude plasma membrane was prepared as described before (19). Briefly, monocytes were disrupted by sonication (five 5-s bursts of 90 W each) in PBS containing 0.68 M sucrose, 200 mM PMSF, and 5 mM EDTA. The lysate was centrifuged for 15 min at 4000 215’5f g to discard nuclei and intact cells. The supernatant was centrifuged for 45 min at 100,000 215’5f g, and the pellet containing the membrane fraction was resuspended at the theoretical concentration of 20 215’5f 106 cell equivalent/ml in Waymouth’s medium.
ICAM-1 cross-linking
MC were harvested and cultured in 96-well plates at 1 215’5f
104 cell/0.1 ml/well in triplicate for 16 h.
Subsequently, 0.1 ml of prepared medium, including 10 µg/ml of
anti-ICAM-1 Ab and 6.7 µg/ml of F(ab')2 of
goat anti-mouse IgG, Fc specific, were added to cross-link
mesangial ICAM-1. An irrelevant IgG was used as control instead of
anti-ICAM-1 Ab. After 24 h incubation, supernatants were
harvested and stored at –20°C for IL-6 and MCP-1 determination.
Determination of IL-6 and MCP-1 production and mesangial ICAM-1 expression
IL-12 p70, IL-1ß, IL-6, and MCP-1 were measured by ELISA (Endogen, Woburn, MA). ICAM-1 expression was determined by FAC analysis. Briefly, MC were harvested with trypsin/EDTA, and 1 215’5f 105 of cells were labeled by indirect immunofluorescence using anti-human ICAM-1 and R-PE-conjugated F(ab')2 goat anti-mouse Igs. ICAM-1 expression was analyzed using a Becton Dickinson FACSort running Lysis II. MC were carefully gated using forward scatter and side scatter to minimize the contamination by monocytes. In preliminary experiments, the extent of contamination by monocytes labeled with FITC-conjugated anti-CD14 was consistently <5%. The significant difference in the profile of the cells by forward scatter and sice scatter between MC and monocytes made it possible to distinguish MC from monocytes in subsequent experiments without labeling monocytes.
Statistical analysis
Statistical significance was determined by Student’s t test. A value of p < 0.05 was considered to represent a statistically significant difference between group means.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-12
production by primed monocytes
We first optimized the activation of peripheral blood-derived
monocytes by measuring cytokine synthesis after various stimuli. Both
IFN- and GM-CSF are known to activate monocytes and independently
up-regulate CD40 expression (31). In preliminary
experiments, a combination of IFN- and GM-CSF was much more
effective in priming monocytes than either protein alone, as judged by
IL-12 synthesis (data not shown). Primed monocytes were further
incubated in the presence or absence of CD40L or TNF-. After 24
h, supernatants were harvested to determine IL-1, TNF-, and IL-12 by
ELISA. CD40L induced high levels of IL-1ß, TNF-, and IL-12 p70
(Fig. 1
, A–C) production by
primed monocytes. Primed monocytes without CD40L produced low levels of
IL-1ß but failed to produce detectable level of TNF- and IL-12.
Although TNF--activated monocytes produced comparable levels of
IL-1ß with CD40L-activated monocytes, they failed to secrete
significant amounts of IL-12 p70. Stimulation of monocytes with
IFN-, GM-CSF, TNF-, or CD40L alone failed to induce detectable
levels of IL-1ß, TNF-, and IL-12 production. Increasing TNF- to
50 ng/ml or CD40L to 15 mg/ml did not lead to further synthesis of
IL-1ß, TNF-, or IL-12 p70 (data not shown). Together, these data
indicate that CD40L activates monocytes to produce inflammatory
cytokines such as TNF- and IL-1ß. The effect of CD40L on monocyte
activation is distinct from that of TNF- because only the former
induces IL-12 production.
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We next examined whether CD40L-activated monocytes could, in turn,
stimulate MC. IFN-/GM-CSF-primed monocytes were stimulated with
CD40L or TNF- for 24 h, then harvested, washed, and cocultured
with MC, which had been precultured to facilitate adherence. After
24 h, IL-6 and MCP-1 production was determined by ELISA, and
ICAM-1 expression was determined by FACS. In preliminary studies, we
established the optimal macrophage:MC ratio and duration for coculture
(not shown). Monocytes activated with CD40L consistently induced higher
levels of IL-6, MCP-1, and ICAM-1 synthesis by MC than did monocytes
activated with other stimuli, including TNF- (Fig. 2, A–C and Fig. 3). Production of TGF-ß, an important
growth factor for matrix expansion that is constitutively secreted in
vitro by cultured MC (10), was not affected by adding
activated monocytes (data not shown). Similarly, platlet-derived growth
factor, a potent mitogen for MC (10), or RANTES, a
chemokine produced by MC (32), were not induced during
coculture (data not shown). Thus, CD40L-activated monocytes enhanced
mesangial synthesis of IL-6, MCP-1, and ICAM-1 but not of TGF-,
platlet-derived growth factor, and RANTES. Although in this experiment
it is difficult to precisely evaluate the relative contribution of the
two cell types to IL-6 and MCP-1 production, we assumed that the major
source of cytokines produced are MC because 1) in the absence of MC,
monocytes activated maximally by IFN-, GM-CSF, and rCD40L failed to
induce significant amounts of IL-6 and MCP-1 (Fig. 2, A and
B), and 2) as shown in Fig. 2C, ICAM-1 expression
on MC parallels the up-regulation of IL-6 and MCP-1 in Fig. 2, A and B.
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We next investigated the relative contribution of soluble factors
and cell-to-cell contact in MC activation by monocytes. Primed
monocytes were stimulated with CD40L and cocultured with MC as
previously described at a ratio of 1:1. In parallel cultures, activated
monocytes were separated from MC by a porous (0.4 µm) membrane in
otherwise identical conditions. Twenty-four hours later, supernatants
were collected for cytokine determination and cells were harvested for
ICAM-1 determination by FACS. Separation of monocytes from MC markedly
decreased the synthesis of IL-6 (Fig. 4A) and ICAM-1 (Fig. 4C), but had little effect on MCP-1 synthesis (Fig. 4B). The obligatory contribution of cell-to-cell contact was
calculated (Fig. 4D) and was found to be 80% for IL-6 and
45% for ICAM-1 enhancement. These data clearly indicate that direct
cell-to-cell contact is essential for enhancement of mesangial IL-6
production by CD40L-activated monocytes, whereas it is not necessary
for MCP-1 production.
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We next sought to identify the cell-surface molecules involved in
these interactions between activated monocytes and MC. Previous studies
have suggested that ß2 integrins such as LFA-1
and Mac-1 may play critical roles in interactions between 1) monocytes
and endothelial cells and 2) monocytes and MC (13, 33).
CD40L-activated monocytes were preincubated with neutralizing mAbs
specific for LFA-1 and Mac-1 or with irrelevant control Ig. After
thorough washing, monocytes were added to MC and cultured for 24
h. Blockade of LFA-1 and Mac-1 decreased IL-6 production by 50 and
60%, respectively (Fig. 5A).
No additive effect was observed when LFA-1 and Mac-1 were
simultaneously blocked. Inhibition of VLA-4/VCAM-1, another important
pathway for monocyte adhesion (34), by neutralizing Ab
against VLA-4 did not affect IL-6 synthesis by MC. These data directly
implicate the ß2 integrin/ICAM-1 pathway in
MC/monocyte cell-to-cell interactions.
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and IL-1ß
neutralization
TNF- and IL-1ß are critical proinflammatory monokines for the
induction of glomerular injury in experimental GN (35). To
identify the role of soluble factors released by monocytes, we examined
the effects of TNF- and IL-1ß neutralization in membrane-separated
cocultures of monocyte/MC on MCP-1 production. We used MCP-1 production
as a read-out because its production was more soluble factor dependent
than IL-6 and ICAM-1. Neutralizing cytokine-specific or irrelevant
control Abs were used during monocyte preincubation and active
coculture. Simultaneous blockade of TNF- and IL-1ß led to
significant inhibition of mesangial production of MCP-1 (up to 60%;
Fig. 5
B), whereas neutralization of either TNF- or
IL-1ß alone did not significantly affect its production. These data
indicate that activated monocytes stimulate MC, at least in part,
through soluble TNF- and IL-1ß. Moreover, functional redundancy
likely exists, as single cytokine targeting was insufficient.
Cell-to-cell contact alone is sufficient to induce IL-6, MCP-1, and ICAM-1 synthesis by MC
Although these data established the importance of cell-to-cell
contact between activated monocytes and MC, soluble factors were
present in all cultures. Whether cell-to-cell contact-mediated signals
alone might be sufficient to induce inflammatory responses remained
unclear. Therefore, activated monocytes were fixed with 1%
paraformaldehyde to preserve membrane integrity but prevent soluble
factor secretion. Fixed monocytes were cultured with MC either in
contact with or separated from MC at variable ratios. Fixed
CD40L-activated monocytes induced IL-6, MCP-1, and ICAM-1 synthesis by
MC in a dose-dependent manner, which was abrogated by membrane
separation (Fig. 6). Plasma membrane
preparation from CD40L-activated monocytes also induced IL-6 and MCP-1
production by MC (Fig. 7A). As
before, CD40L-activated monocytes induced higher responses than
TNF--activated monocytes (data not shown). Compared with previous
live cocultures, the monocyte:MC ratio was increased 20-fold to induce
comparable mesangial responses. In addition, membrane preparations from
CD40L-activated monocytes induced an 5-fold increase in
TNF--induced IL-6 production by MC (Fig. 7B), suggesting
that cell contact-mediated signals operate synergistically with soluble
factors. Intriguingly, MCP-1 synthesis was induced by cell-to-cell
contact and was not blocked by addition of TNF-- and
IL-1ß-specific mAbs (data not shown), indicating that membrane-bound
cytokine may not replace soluble cytokine in this system and that
proinflammatory monokine "leakage" from fixed cells is unlikely to
explain our observations. Together, these data clearly demonstrate that
monocyte cell-surface molecules are potent inducers of inflammatory
mediator production by MC in the absence of soluble factors. This
activity is significantly enhanced in CD40L-activated, compared with
TNF--activated, monocytes, suggesting that the former activation
pathway may be of prime importance in vivo.
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There is increasing evidence that ICAM-1 acts not only as an
adhesion molecule, but also has signaling functions (36, 37). The finding that the blockade of the
ß2 integrin/ICAM-1 pathway reduced MC IL-6
synthesis (Fig. 5A), and that cell-to-cell contact alone was
sufficient to induce it, suggested that ICAM-1 ligation might directly
activate MC. Therefore, we investigated whether stimulation of MC
through ICAM-1 induced proinflammatory responses. ICAM-1 on the surface
of MC were cross-linked by simultaneously adding mouse monoclonal
anti-ICAM-1 Ab and the F(ab')2 of goat
anti-mouse IgG Fc fragment. After 24 h, IL-6 and MCP-1
synthesis by MC were determined by ELISA. ICAM-1 ligation induced IL-6
production, at a level comparable to that induced by 10 ng/ml of
TNF- (Fig. 8). Of interest, ICAM-1
ligation failed to induce MCP-1 production by MC. These data clearly
demonstrate that the ß2 integrin/ICAM-1 pathway
plays a critical role, not only for monocyte/MC adhesion, but also for
direct mesangial activation. These data may also partly explain the
differential contribution of cell-to-cell contact to IL-6 and MCP-1
synthesis by MC (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß,
TNF-, IL-6, and IL-8, and promote monocyte rescue from apoptotic
death at inflammatory sites (26). Macrophages and MC are
vital to the initiation and progression of renal injury in a variety of
GN models (6, 33, 38, 39). However, less is known about
those pathways through which macrophages might regulate the
proinflammatory activity of MC. The present study sought to define such
interactions between "physiologically activated" macrophages and MC
in modifying the biologic response of the latter.
To study these interactions, we used an in vitro system to quantify the
relative contribution of soluble and cell-to-cell contact-dependent
pathways, achieved either through cell separation using semipermeable
membranes or by effector cell fixation and utilization of isolated
plasma membranes. The latter methods facilitate the assessment of cell
contact-dependent pathways, independent of secreted bioactivities.
Similar in vitro approaches have yielded useful information about the
pathogenesis of several other human autoimmune diseases
(18, 19, 20, 21, 22). We have shown that activated monocytes amplify
glomerular responses by promoting proinflammatory activity in MC
through discrete but nonexclusive pathways. That these included
elaboration of soluble factors, such as TNF and IL-1ß, was
expected. However, our data provide strong evidence that the regulatory
activities of macrophages in the glomerulus extend beyond monokine
secretion to include significant contributions by cell-to-cell contact.
Previous reports have demonstrated that ß2
integrins, such as LFA-1 and Mac-1, are involved in cell-to-cell
interactions, which contribute to the glomerular accumulation of
inflammatory cells and subsequent injury. LFA-1 forms an adhesion
bridge with ICAM-1 on endothelial cells and MC, and the ICAM-1/LFA-1
pathway is important for various homotypic as well as heterotypic
cell-to-cell interactions. Abs to ICAM-1 or LFA-1 prevent glomerular
injury in a variety of experimental models of GN (40, 41, 42).
Our findings suggest that such effects might be mediated through
blockade of critical macrophage/MC interactions.
In the present study, nonprimed peripheral blood monocytes failed to
induce any significant response in MC, allowing us to define
physiologically relevant priming requirements before coculture. Our
data illustrate the importance of CD40 ligation in assuring maximum
effector functions in monocytes. Thus, in contrast to IL-1ß,
stimulation via CD40 was essential for the production of IL-12, a
cytokine essential for functional Th1 responses (Fig. 1). It is of
interest that most T cell clones isolated from lupus GN, in which high
levels of CD40L are expressed, are predominantly of the Th1 phenotype
(43). CD40L-activated monocytes also resulted in higher
biologic responses from MC when compared with those stimulated by
TNF-. MC activation was assessed by measuring the synthesis of
molecules with a reported pathogenic role in GN. Thus, IL-6 is a
sensitive indicator of mesangial activation and has been implicated in
the proliferation of MC (11). MCP-1, a chemotactic
cytokine that is highly specific for lymphocytes and monocytes, is
excreted in excessive amounts in the urine of patients with GN
(44, 45, 46). Animal data suggest that MCP-1 is involved in
glomerular crescent formation and interstitial fibrosis
(47, 48, 49). ICAM-1 enhances monocyte adhesion and retention
within the mesangium and facilitates cellular interaction with
infiltrating mononuclear cells (13).
Previous investigation of macrophage/MC interactions, which used U-937 myelomonocytic leukemia cells, suggested that the latter may cause cytotoxic damage to and subsequent proliferation of MC (50). Although ICAM-1, LFA-1, and VLA-4 were implicated in this system, the relative contribution of secreted and membrane-bound factors was not defined, nor were differential effects on MC function determined, as was the case in the present study. Moreover, the use of leukemic cells limits the generalization of these observations to physiologic systems. In contrast, we endeavored to employ primary monocytes, priming protocols, and measures of MC activation with direct physiologic relevance to human GN.
The differential effects of cell-to-cell contact, as compared with
those of soluble factors, were striking. Whereas IL-6 production
appeared largely cell contact dependent, MCP-1 production was
unaffected by cell separation. Functional redundancy was evident in the
latter cultures because optimal neutralization of neither TNF nor
IL-1ß alone was sufficient to modify total MCP-1 output. Moreover,
when the ratio of monocytes to MC was increased from 1:1 to 5:1, MCP-1
production was clearly induced by a cell contact-dependent pathway.
This effect was not evident in vitro until fixed and live macrophage/MC
cocultures were compared. Similarly, ICAM-1 expression appeared
dependent on both pathways. Stimulation of MC by activated macrophages
enhanced ICAM-1 expression. This, in turn, can increase adhesion of
macrophages to MC, thus creating a positive feedback loop. Our
experimental system does not exactly mimic the kinetics of in vivo
responses, and it is probable that soluble and contact factors may
contribute differentially through the evolution of an inflammatory
lesion. Thus, soluble factors might up-regulate ICAM-1 expression,
which thereby facilitates enhanced cell contact-dependent effector
function. Certainly, when cells were fixed, we observed a significant
increase (>5-fold) in the macrophage cell number required to activate
MC, suggesting that optimal activation of MC likely required both
soluble and cell contact-dependent factors. Thus, cell contact may
increase the efficiency of cytokine-mediated bystander activation by
facilitating paracrine mechanisms, perhaps through modification of
cytokine receptor expression. However, Abs to TNF- did not modify
cell contact-dependent function in our culture system, which argues
against a role at least for membrane-bound TNF- (9, 51). The differential secretion of cytokines and chemokines that
we observed further suggests that cell-to-cell contact uses different
intracellular pathways than those activated by soluble factors in MC.
This has potential relevance in therapeutic targeting of signaling
pathways.
Up-regulation of CD40 in resident renal cells and CD40L expression in
infiltrating mononuclear cells have been observed in renal biopsies of
patients with proliferative, but not membranous, lupus nephritis
(26). In animal models of lupus nephritis, anti-CD40L
Abs ameliorate nephritis even when administered after disease onset
(52). CD40-mediated signals induce secretion of chemokines
by resident renal cells, such as IL-8, MCP-1, and RANTES, and promote
tissue damage and fibrosis (53). To this end, we envision
the following scenario in vivo (Fig. 9).
T cells activated by specific Ags up-regulate CD40L expression, which
in turn activate macrophages either in the peripheral blood, in lymph
nodes, or in situ in the kidney. Activated peripheral blood macrophages
enter the glomerulus either through the mesangial area (or though holes
in the Bowmanns’ capsule in the case of crescentic GN) where they
activate MC and epithelial cells. Under certain conditions, activated
macrophages may also express CD40L (54), raising the
possibility of local fraternal activation of macrophages in the
glomerulus. It is conceivable that in animal models, interruption of
the CD40/CD40L pathways, in addition to preventing autoantibody
production, inhibits macrophage activation and resultant renal injury.
Thus, CD40L-targeted approaches may exert salutary effects on
immune-mediated GN above and beyond its effects on autoantibody
production
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: GN, glomerulonephritides; MCP-1, monocyte chemoattractant protein-1; CD40L, CD40 ligand; VLA-4, very late activation Ag-4; MC, mesangial cells.
Received for publication March 3, 1999. Accepted for publication June 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v integrins in mesangial cell adhesion to vitronectin and von Willebrand factor. Kidney Int. 51:1900.[Medline]
production in rheumatoid arthritis. Nat. Med. 3:189.[Medline]
-interferon. J. Exp. Med. 166:798. is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J. Clin. Invest. 101:364.[Medline]
receptor signaling is essential for the initiation, acceleration, and distraction of autoimmune kidney disease in MRL-Fas (lpr) mice. J. Immunol. 161:494. stimulates monocyte adhesion to glomerular mesangial cells: the role of intercellular adhesion molecule-1 gene expression and protein kinases. J. Immunol. 156:2571.[Abstract]
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 A.-J. Ruth, A. R. Kitching, T. J. Semple, P. G. Tipping, and S. R. Holdsworth Intrinsic Renal Cell Expression of CD40 Directs Th1 Effectors Inducing Experimental Crescentic Glomerulonephritis J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2813 - 2822. [Abstract] [Full Text] [PDF]
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 C.D. Garlichs, S. Kozina, S. Fateh-Moghadam, B. Tomandl, C. Stumpf, S. Eskafi, D. Raaz, A. Schmeisser, A. Yilmaz, J. Ludwig, et al. Upregulation of CD40-CD40 Ligand (CD154) in Patients With Acute Cerebral Ischemia Stroke, June 1, 2003; 34(6): 1412 - 1418. [Abstract] [Full Text] [PDF]
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 C. S. Rinder, M. Fontes, J. P. Mathew, H. M. Rinder, and B. R. Smith Neutrophil CD11b upregulation during cardiopulmonary bypass is associated with postoperative renal injury Ann. Thorac. Surg., March 1, 2003; 75(3): 899 - 905. [Abstract] [Full Text] [PDF]
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T. Tanaka, T. Kuroiwa, H. Ikeuchi, F. Ota, Y. Kaneko, K. Ueki, Y. Tsukada, I. B. McInnes, D. T. Boumpas, and Y. Nojima Human Platelets Stimulate Mesangial Cells to Produce Monocyte Chemoattractant Protein-1 via the CD40/CD40 Ligand Pathway and May Amplify Glomerular Injury J. Am. Soc. Nephrol., October 1, 2002; 13(10): 2488 - 2496. [Abstract] [Full Text] [PDF]
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 C. D. Garlichs, S. John, A. Schmei{beta}er, S. Eskafi, C. Stumpf, M. Karl, M. Goppelt-Struebe, R. Schmieder, and W. G. Daniel Upregulation of CD40 and CD40 Ligand (CD154) in Patients With Moderate Hypercholesterolemia Circulation, November 13, 2001; 104(20): 2395 - 2400. [Abstract] [Full Text] [PDF]
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 B. L. Riser, J. Varani, P. Cortes, J. Yee, M. Dame, and A. K. Sharba Cyclic Stretching of Mesangial Cells Up-Regulates Intercellular Adhesion Molecule-1 and Leukocyte Adherence : A Possible New Mechanism for Glomerulosclerosis Am. J. Pathol., January 1, 2001; 158(1): 11 - 17. [Abstract] [Full Text] [PDF]
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T. Kuroiwa, R. Schlimgen, G. G. Illei, I. B. McInnes, and D. T. Boumpas Distinct T Cell/Renal Tubular Epithelial Cell Interactions Define Differential Chemokine Production: Implications for Tubulointerstitial Injury in Chronic Glomerulonephritides J. Immunol., March 15, 2000; 164(6): 3323 - 3329. [Abstract] [Full Text] [PDF]
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