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Short Communications |
From the Department of Medicine,*
Division of Nephrology
and Hypertension, Henry Ford Hospital, Detroit; and the Department of
Pathology,
the University of Michigan Medical
School, Ann Arbor, Michigan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
whereas an additive effect occurs when both are applied in combination.
Our results indicate that stretch-induced ICAM-1 may provide a direct
link between hypertension and inflammation in the progression of injury
and glomerulosclerosis in diabetes, renal ablation, and
other forms of glomerulonephritis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to the glomerular hemodynamic alteration, leukocyte
infiltration is a factor that plays an important role in the initial
kidney damage and consequent sclerosis that is associated with various
forms of glomerular injury. Phagocytic leukocytes (neutrophils,
monocytes, and macrophages) infiltrate glomeruli early in acute
glomerulonephritis and play a key role in its
pathogenesis.9
It is now clear that an influx of
phagocytic leukocytes into the glomerulus is also a response to renal
ablation10-12
and to experimental
diabetes.13
The process of glomerular inflammation
requires migration in response to a chemokine gradient, diapedesis
through an endothelial barrier, and interaction with resident renal
cells via adhesion molecules.14
Intercellular adhesion
molecule-1 (ICAM-1), a transmembrane glycoprotein and member of the
immunoglobulin superfamily, seems to be the most important adhesion
molecule in this process.15
It is expressed on many
hematopoietic and nonhematopoietic cells and its ligands, or
counterreceptors, on leukocytes including the ß integrins, CD11a/CD18
and CD11b/CD18.16
Although the glomerular expression of
ICAM is normally low or absent, it is up-regulated in the mesangium in
many forms of glomerulonephritis including focal segmental
glomerulosclerosis, early rapidly progressive glomerulonephritis,
mesangioproliferative glomerulonephritis, IgA nephropathy, and lupus
nephritis, as shown in both human17,18
and animal
models.15,19,20
Recent studies in rat glomeruli have also
shown an up-regulation of ICAM-1 after streptozotocin-induced diabetes
and 5/6 nephrectomy.21,22
The mechanisms for induction of
ICAM in the glomerulus are not clear. However, the low constitutive
ICAM-1 expressed on MCs in culture is increased after exposure to tumor
necrosis factor (TNF) or interferon (IFN)-
, or interleukin (IL)-1,
suggesting that certain inflammatory cytokines released from
infiltrating leukocytes are key regulatory factors in the
glomerulus.14,23
A role for hypertensive force in the expression of ICAM has not been reported. However, it has been shown that after renal ablation in rats, treatments with the immunosuppressive agent mycophenolate mofetil, that blocks ICAM-1 up-regulation, correspondingly prevents proteinuria without an effect on arterial pressure.24 Also, sheer stress, another form of mechanical force, has been reported to increase ICAM expression on endothelial25 and epithelial26 cells. We hypothesized that cyclic strain resulting from glomerular hypertension would up-regulate the production and localization of MC ICAM-1, thus allowing increased interaction with leukocytes. This physical interaction would then result in MC activation and/or injury with subsequent glomerular damage occurring via inflammatory processes and acceleration of ECM accumulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MCs, previously characterized by us, were a cloned line
derived from outgrowths of Fischer rat glomeruli.7
On
serial passage, they continue to express key markers of
MCs,7
including Thy-1. They demonstrate the same high
sensitivity to phorbol-stimulated neutrophil adhesion and lysis, as do
early passage MCs indicating continued and like expression of essential
cell surface receptors.16
The medium used was RPMI 1640
with penicillin and streptomycin and 8 mmol/L glucose and (unless
otherwise noted) 20% Nu-Serum from Collaborative Research, (Bedford,
MA). Human peripheral blood leukocytes were obtained from healthy adult
volunteers and isolated as previously described.27
The
cells obtained were 
98% polymorphonuclear granulocytes and were
suspended in RPMI 1640 culture medium, supplemented with 0.5% bovine
serum albumin.
Application of Cyclic Mechanical Strain
Experiments testing the effects of mechanical strain were
performed as previously described.7
In brief, MCs were
seeded into six-well, Flex I plates (5 x
104
cells/25-mm diameter well) with flexible
collagen-coated bottoms from Flexcell International (McKeesport, PA)
and cultured in a 1 ml/well of growth medium. After 24 hours,
experimental cultures were subjected to cyclic stretching whereas the
control cultures were maintained in a static environment under
identical conditions. To mimic conditions of MC stretch during possible
low-frequency oscillations in intraglomerular pressure, all experiments
were performed using alternating cycles of 10 seconds of stretch and 10
seconds of relaxation (50 mHz).28
Vacuum intensity was set
to provide a 19% maximum elongation at the periphery of the well
(9% average elongation).
Adhesion Assay
MCs cultured on Flex I plates were washed after stretch or cytokine treatment and 8 x 105 neutrophils were added per well. After 1 hour at 37°C cultures were washed gently but extensively to remove the nonadherent cells. The remaining adherent cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, dried, then placed at -30°C until the time of staining. Cells were stained using Meyer’s hematoxylin and the number of neutrophils per field was determined. At least nine fields per well were examined.
Analysis of ICAM-1 Expression
For analysis of mRNA, total RNA was isolated as previously described29 and Northern blots hybridized and probed using 32P-labeled cDNA of ICAM-130 or ß-tubulin. Autoradiograms were digitized by scanning densitometry and quantified with image analysis.7 The analysis of ICAM-1 protein expression was determined by immunostaining, using an alkaline-phosphatase avidin-biotin assay.29 While attached to the supporting circular membrane, cells were fixed and pie-shaped wedges of this membrane were cut out. The primary antibody (1A29) used was ICAM-1-specific.31 The slides were counterstained in Mayer’s hematoxylin. Photographs were obtained at a x100 magnification under identical illumination. To obtain the level of stain intensity, digitized 8-bit RGB color images were divided into quadrants, separately analyzed under identical density calibration, and the quantity of red color present determined using image analysis.
Statistical Analysis
Unless otherwise noted, differences between two groups were analyzed by the Student’s paired t-test because of the cloned nature of the MCs studied. In the case in which results were normalized to corresponding control values, the data were analyzed by a one-sample t-test with a hypothesized mean of 100% to compare the test group with the control. A two-sample, paired t-test was used to examine differences between multiple test groups. In both cases, a post hoc Holm’s test was applied to adjust for multiple comparisons and the level of significance was set at 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the level of constitutive MC ICAM-1 message and
whether its expression is altered by cyclic strain, cells were exposed
to various periods of stretching or allowed to remain under static
conditions before harvesting cell layers for mRNA analysis. Control,
unstretched MCs expressed detectable amounts of ICAM-1 message (Figure 1)
. Expression was markedly up-regulated with
cyclic stretching. The induction was detected after 4 hours and
progressively increased with longer periods of stretch, reaching 66 and
116% greater than the levels in static cultures at 8 and 12 hours,
respectively (Figure 1)
.
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Next determined was the level of ICAM-1 protein present in the MCs
and whether the observed changes in ICAM-1 mRNA levels were associated
with corresponding changes in protein expression. To accomplish this,
MCs were fixed after cyclic strain or static incubation and the
flexible substrates removed from the wells. Culture membranes were then
immunostained for ICAM-1 protein. Because the force of elongation is
not uniform across a stretched well, but increases from the center to
the periphery, this attribute was used to determine the relationship
between the amplitude of strain and the level of ICAM-1 present. Three
assigned zones of the stretched cultures, experiencing 4%, 12%, and
18% elongation, respectively, were examined. Results were then
compared to those from the outermost zone of static cultures. Static
cultures showed weak staining in only a few MCs (Figure 2)
. In contrast, stretched MCs demonstrated
localization of cell-associated ICAM-1 in amounts that were directly
related to the increased elongation amplitude. To quantify this
dose-related induction of ICAM-1 protein and to determine whether it
was the result of a larger number of positive-staining cells, or a
greater deposition of the molecule per cell, multiple fields were
examined by image analysis. Figure 3A
shows
that although the number of positive-staining cells was near zero in
static cultures, this number markedly increased in stretched cultures
even in zones of minimal force. In areas of increasing amplitude of
strain, there were corresponding increases in the number of
positive-staining cells, reaching 90% in the zone of maximal stretch.
Interestingly, the greatest change (1 to 50%) occurred in cells with
lesser levels of strain (Figure 3A)
. When the intensity of staining per
cell was analyzed, it increased significantly, even under minimal
elongation and a dose response to increasing force also was evident
(Figure 3B)
. However, in contrast to the proportion of positive
staining cells, the cellular content of ICAM-1 demonstrated the
greatest change at the higher levels of strain. Similar results were
obtained when ICAM-1 expression was adjusted for the area of cytoplasm
(Figure 3C)
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The observed up-regulation of both ICAM-1 mRNA and protein
demonstrated a stretch-induced increase in production and
cell-associated deposition. However, not determined was whether cyclic
strain increased functional ICAM-1 on the cell surface, thereby
increasing the interaction with leukocytes. To test this, we subjected
MCs to either cyclic strain, or to TNF-, a known stimulator of ICAM
expression and ICAM-mediated leukocyte adherence. Freshly isolated
human neutrophils were then added and the number of attached cells was
determined. As expected, treatment with TNF- markedly stimulated
neutrophil adherence (225% of control) (Figure 4)
. Cyclic stretch also potently induced
leukocyte adherence, producing levels (282%) equal to or greater than
that stimulated by TNF-. MC cyclic stretch in the presence of
TNF- resulted in an additive effect on adherence (329%) that was
significantly greater than that of TNF- or stretch alone
(P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50% were positive at a low 4% average elongation
and increased to >90% positive at 18% elongation. Measurement of the
amount of cell-associated ICAM-1 per cell showed that although
relatively little force is required to induce the molecule, larger
increases in expression require higher levels of elongation (18%).
Importantly, our finding that the response was similar whether
expressed per cell or per area of cytoplasm indicated that the observed
effects were not the result of stretch-induced hypertrophy. The
observed dose-response induction of ICAM-1 is similar to what we have
previously reported for stretch-induced transforming growth factor-ß,
collagen I, fibronectin, and laminin,7,29
whereas, the
time course for induction of ICAM-1 mRNA and protein is similar to that
reported by others in response to IFN-, TNF, and
IL-1.23
The finding in nonstretched MCs of measurable
constitutive ICAM-1 mRNA but little, or no, detectable ICAM-1 protein
is not surprising. A strict correlation between the level of mRNA and
the level of a corresponding protein, is not always the
case.32
Alternatively, this may simply reflect the
differences in sensitivity of the two different assays.
The relevance of stretch-induced MC ICAM-1 expression to functional
activity was demonstrated by a corresponding increase in
ICAM-associated adherence. The same regimen of stretch used to induce
expression of ICAM-1 produced a threefold increase in the adhesion of
neutrophils. This stimulated adhesion was at least as great as that
induced by exposure to TNF-, a known inducer of MC
ICAM.23
It was interesting that an additive effect of
stretch in combination with TNF- was produced. This supports the
idea that MC exposure to cytokines in combination with mechanical
strain may form the basis for the accelerated development of
glomerulosclerosis that occurs when intraglomerular hypertension and
inflammation are simultaneously present. Although the leukocyte ligand
for ICAM-1 was not determined in the present study, it has been
previously shown that TNF- or IL-1-induced adhesion of MCs to
phagocytes is abrogated by monoclonal antibodies directed against
either MC ICAM-1, or leukocyte CD11/CD18.14
In addition,
we have previously reported that leukocyte-mediated, oxidant-induced
injury of both human and rat (the same line used in the present study)
MCs are also blocked by monoclonal antibodies to
CD11/CD18.16
Our findings in the present study with
neutrophils are likely to be extended to monocyte/macrophage-MC
interactions as well, because it has been previously shown that
monocytes are also induced by TNF- to up-regulate adherence to MCs
via ICAM-1.14
The mechanism(s) for stretch-induced ICAM-1 expression is (are)
unknown. However, in endothelial cells, the induction of ICAM-1 along
with VCAM-1 and E-selectin, has been reported to be regulated at the
level of gene transcription and to require binding of nuclear
transcription factor 
B to the respective promoters of these
genes.33
Further, TNF--induced ICAM-1 in mouse Sertoli
cells seems to be mediated through the activity of a mitogen-activated
protein kinase, Jun N-terminal kinase/stress-activated protein kinase
(JNK/SAPK), but not p42/p44 or p38.33
In MCs, the
signaling pathways for mechanotransduction, including those leading to
cytokine or ECM production, have not been clarified at this time.
However, protein kinase C and protein tyrosine kinases are rapidly
activated in MCs with stretch, followed by the induction of
c-fos mRNA, and changes in this transcription factor have
been linked to increased ECM production.34,35
Mitogen-activated protein kinase is also activated by mechanical
strain. One group has reported that both extracellular signal-related
kinase and JNK were activated in a time- and intensity-dependent (10 to
30% elongation) manner.36
However, another group reported
that high levels of MC stretch (29%) activate p/44/42 and p38/HOG but
not JNK, whereas the p38 activation is lost at reduced levels of
stretch (20%).37
Therefore, it remains to be determined
whether the signaling pathways for stretch-induced ICAM-1 are like
those for stretch-stimulated cytokines and/or ECM production, or for
cytokine-induced ICAM-1.
Glomerular injury is a likely consequence of sustained up-regulation of
ICAM-1. The possible mechanisms for ICAM-1-mediated damage are
indicated by several recent studies. We have shown that phorbol
myristate acetate-stimulated neutrophils adhere to cultured MCs through
an ICAM-1 mediated interaction.16
This stimulated
adherence results in MC injury mediated by leukocyte oxidants,
primarily H2O2. The
requirement for physical contact was shown by the substantial blockade
of stimulated cytolysis that occurred when adhesion was prevented by
treatment with CD11/CD18 antibody.16
In addition to these
potentially lethal products, phagocytic leukocytes also produce
inflammatory and prosclerotic cytokines including IL-1, IFN-, and
transforming growth factor-ß. These may act to stimulate MC growth,
hypertrophy, and/or ECM metabolism subsequent to cell-cell interaction.
Alternatively, monocytes are activated by soluble CD40 to induce MCs to
produce high levels of MCP-1, IL-6, and to up-regulate
ICAM-1.38
The up-regulation of IL-6 and ICAM-1 required
physical interaction of the two cell types. This indicates that an
induction of ICAM-1 on MCs and their interaction with phagocytic
leukocytes can lead to MC production of cytokines and chemokines
capable of exacerbating glomerular inflammation. Nitric oxide also
seems to be a product of phagocytic leukocyte/MC interaction, and may
have both beneficial and damaging effects in the kidney. In large
quantities it may have a direct toxic effect on MCs or act indirectly
on the afferent arteriole and MCs to alter glomerular
microcirculation.39
Last, the up-regulation of ICAM-1 may
directly alter MC-mediated effector function. MCs can function as
antigen presenting cells for T-cell stimulation.23
This
requires stimulated expression of MHC class II molecules on the MCs as
well as ICAM-1.23
Holland and Owens40
have
recently shown that the cross-linking of ICAM-1 on a B lymphoma line
induces a rapid increase in tyrosine phosphorylation of cellular
proteins, the activation of the mitogen-activated protein kinase, and
the up-regulation of class II major histocompatibility complex. This
indicates that ICAM-1 functions not just as an adhesive molecule, but
is also capable of transducing biochemical signals and suggests a
mechanism for stretch-induced, ICAM-1-mediated antigen presentation by
MCs. Our overall results in vitro are in agreement with the
findings from recent studies of glomerular disease in both 5/6
nephrectomy24
and in STZ-induced diabetic rat
models.21
They showed that the specific inhibition of
ICAM-1 expression resulted in reduced leukocyte
infiltration/proliferation and was associated with prevented
deterioration of renal function.
In summary, these results demonstrate that cyclic stretch of cultured MCs produces a time- and dose-related up-regulation of ICAM-1 expression and a subsequent increase in neutrophil adherence. A sustained ICAM-1 up-regulation in vivo would therefore be expected, and explained, under conditions that produce increased glomerular size and/or pressure such as renal ablation, diabetes, or certain forms of glomerulonephritis. ICAM-1 could act downstream to mediate MC injury, enhance inflammation, and increase ECM accumulation. It may be that other cell types respond in a similar manner, thus facilitating their interaction as targets for leukocyte interaction. This could have important implications in many other diseases in which inflammation is the basis, including atherosclerosis.
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Acknowledgements |
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Footnotes |
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Supported by Juvenile Diabetes Foundation International grants 1921461 and 1-1998-243 (to B. L. R.) and the John Kontich Renal Research Fund, and was presented in part at the annual meeting of the American Society of Nephrology on November 1996 in New Orleans, LA.
Accepted for publication September 15, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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