| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
3 Induces Chemokines and Adhesion Molecules: Insight into the Role of LT in Inflammation and Lymphoid Organ Development1
*
Departments of Epidemiology and Public Health, Immunobiology, and
Internal Medicine (Cardiovascular Medicine), Yale University School of Medicine, New Haven, CT 06520; and
Department of Physiology, University of Michigan School of Medicine, Ann Arbor, MI 48109
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
preparation were evaluated. This cytokine preparation was
effective at inducing cytotoxicity of WEHI target cells with 50%
maximal killing observed with 1.2 ng/ml. mLT also induced the
expression of inflammatory mediators in the murine endothelial cell
line bEnd.3. rmLT induced expression of the adhesion molecules VCAM,
ICAM, E-selectin, and the mucosal addressin cellular adhesion molecule,
MAdCAM-1. When mLT, human (h) LT, and mTNF- were compared,
mLT was the most potent inducer of MAdCAM-1. None of these cytokines
induced the peripheral node addressin, PNAd. mLT also induced
expression of the chemokines RANTES, IFN-inducible protein 10 (IP-10),
and monocyte chemotactic protein 1 (MCP-1). mRNA levels peaked 4 h
following treatment with mLT and declined through the 24-h treatment
period. LT also induced chemokine protein within 8 h of
treatment, which increased through the 24-h treatment period. These
data demonstrate that the proinflammatory effects of LT3 may be
mediated in part through the induction of adhesion molecule and
chemokine expression. Further, LT3 may promote development of
lymphoid tissue through induction of chemokines and the mucosal
addressin MAdCAM-1. These data confirm previous observations in
transgenic and knockout mice that LT3 in the absence of LTß
carries out unique biologic activities. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
3 homotrimer or a
cell-associated LT1ß2 heterotrimer. The homotrimer interacts with
the (p55) TNFRI and (p75) TNFRII receptors, accounting for those
activities which are similar to those of TNF-; LT1ß2 binds to
the LTß receptor (LTßR) and thus carries out unique activities
(reviewed in 3 . It is possible that these ligands also bind to
one of the several new receptors that are being identified. In fact,
information derived from studies of mice deficient in LT or LTß
suggests that LT might also interact with an additional, as yet
undescribed, LTR to promote lymphoid organ development (reviewed in
4 . The LT/TNF family induces lymphoid organs through
combinatorial associations of the ligands and their receptors. Analysis
of mice deficient in the various ligands and receptors indicates that
the LT1ß2 complex contributes to the development of peripheral
lymph nodes (LN) and the organization of splenic architecture and
germinal centers because mice deficient in LTß or treated with LTßR
fusion protein lack these structures (5, 6). Other aspects of lymphoid
development, such as Peyer’s patch (PP) formation, are also mediated
as an LT3 complex through the TNFRI, because TNFRI-/-
mice either lack (7) or have abnormal (8) PP, whereas
TNF--/- mice have normal PP (8, 9). Mesenteric and
cervical LN development may be mediated by an LT-specific receptor
or alternatively as complementary signaling between LT3 and
LT1ß2 through TNFR1 and LTßR respectively. The mechanisms of
LT-induced lymphoid organ development have not been elucidated partly
because there are no developmentally appropriate in vitro systems to
study these effects, and partly because recombinant murine LT has
not been available for such systems.
LT has been implicated in inflammation from the time of its initial
description as an in vitro correlate of delayed type hypersensitivity
(10) and by its expression by Th1, but not Th2 T cells (11, 12). LT
induces inflammation in vivo when expressed under the control of the
rat insulin promoter (RIPLT mice) at the sites of transgene expression
in the pancreas and kidney (13). This occurs even in islets and kidneys
of RIPLT.LTß-/- mice (1) indicating that LT3 alone,
in the absence of LTß, induces this inflammation. Additional data
suggesting a proinflammatory role for LT derives from studies of
experimental allergic encephalomyelitis (EAE) in its correlation with
encephalitogenicity of myelin basic protein-specific T cell clones (14)
and the fact that LT-/- mice are resistant to
inflammation and clinical signs of EAE (15). The observation that
LTß-/- mice can develop EAE supports the concept that
LT3, in the absence of LTß, plays an important proinflammatory
role (15).
The studies presented here were designed to identify the mechanisms by
which LT promotes inflammation and lymphoid organ development. One
possible pathway is through the induction of adhesion molecules in
endothelial cells. Human (h) and murine (m) TNF- can induce adhesion
molecule expression in vitro (reviewed in 16 . Sikorski et al.
have demonstrated that mTNF- induces expression of VCAM-1, ICAM, and
mucosal addressin cellular adhesion molecule (MAdCAM-1) on the murine
endothelial cell line bEnd.3 (17). rhLT induces expression of ICAM
and E-selectin in human endothelial cells in vitro (18). Though no
studies have been conducted with rmLT, RIPLT mice exhibit
up-regulated expression of ICAM-1, VCAM-1, MAdCAM-1, and peripheral
node addressin (PNAd) in the vasculature of the inflamed pancreas and
kidney (19). RIPLT.RAG2-/- mice that lack the mature T
and B cell infiltrate continue to express VCAM, ICAM, and MAdCAM-1
(19), suggesting that LT in the absence of additional cytokines from
mature lymphocytes induces these molecules directly. RIPLT mice crossed
to mice deficient in LTß express ICAM, VCAM, and MAdCAM-1, but fail
to express PNAd.5 Together
these data suggest that LT3 induces VCAM, ICAM, and MAdCAM-1, while
LT1ß2 may be necessary for PNAd.
LT may also contribute to inflammation and lymphoid organ development
through induction of chemokines. Several chemokines are involved in
inflammation (20), and Burkitt’s lymphoma receptor 1 (BLR-1), a
chemokine receptor expressed on B cells, appears necessary for B cell
compartmentalization and LN development (21). TNF- induces
chemokines in vitro (22, 23, 24), and an effect in vivo is suggested by a
study in which Abs to TNF abrogate chemokine induction in a lung model
of inflammation (25). LT1ß2 has been reported to induce RANTES and
IL-8 from a melanoma-derived cell line (26). There has been one report
of RANTES induction by hLT in human mesangial cells (27), but no
such analyses have been reported for mLT.
Recently, Mackay and colleagues prepared rmLT that was cytotoxic to
the WEHI 164.13 cell line (28). mLT was less potent than mTNF- in
this study, leading the authors to question whether LT in the
absence of LTß has any activity and to suggest that all the
proinflammatory activity of Th1 cells could be ascribed to TNF- and
the developmental roles of lymphotoxin were mediated by the LTß
complex (28). The present studies address whether LT exhibits
independent biological activity in an in vitro target, the murine
endothelial cell line bEnd.3. This model system made it possible to
address several issues: 1) can in vitro studies with murine LT
provide insight into the mechanism by which LT promotes inflammation
and lymphoid organ development; and 2) does LT induce these effects
in the absence of LTß? We show here for the first time that mLT
induces expression of adhesion molecules VCAM-1, ICAM, and E-selectin
in murine endothelial cells. These activities are consistent with a
proinflammatory role of LT3. LT also induced expression of MAdCAM-1,
an adhesion molecule expressed on the vasculature of all developing LN
(29), mature mesenteric nodes, and PP. Neither LT nor TNF-
induced PNAd. LT also induced expression of the chemokines RANTES,
IFN-inducible protein 10 (IP-10), and monocyte chemotactic protein 1
(MCP-1). These studies, which provide potential mechanisms for the
roles of LT in development and inflammation, reaffirm the concept
that LT, in addition to its activities in the LT1ß2 complex,
plays unique roles in its LT3 form in the crucial biologic processes
of inflammation and lymphoid organogenesis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
rmLT was obtained from Dr. Jeffrey Browning (Biogen,
Cambridge, MA) (28). The soluble cytokine was produced in insect cells
from a cDNA provided by our laboratory (30), purified, and collected as
an eluate from a TNF-R55-Fc affinity column (31). rmTNF-, which was
produced in Escherichia coli, was obtained from Dr. Regina
Turetskaya (Engelhardt Institute, Moscow, Russia). rhLT was obtained
from Genentech (South San Francisco, CA).
WEHI 164 cell killing assay
Units of biologic activity were measured by using the sensitive nonadherent WEHI 164 fibrosarcoma cell line in the 3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide (MTT) dye reduction assay as previously described (32). This cell line and assay were included in this laboratory’s involvement in a study defining the International Standards for LT and TNF (33). WEHI 164 cells (5 x 103/well) were added to serial dilutions of test samples in 96-well microtiter plates in a 100-µl volume and incubated at 37°C/5% CO2 for a total of 48 h. After 44 h, 25 µl of 5 mg/ml MTT was added and 4 h later the cells were lysed by the addition of 150 µl 0.04 M HCL in isopropanol. The amount of MTT taken up and reduced was determined 24 h later by OD reading on a Vmax plate reader (Molecular Devices, Menlo Park, CA) at 570 nm with a 650-nm reference standard. Units were calculated as the highest dilution causing 50% cytotoxicity. Percent cytotoxicity was calculated as: 100 x (1.00 - OD of sample wells)/OD of control wells.
FACS analysis
The murine brain endothelial cell line, bEnd.3 (34) was obtained from W. Risau (Max Planck Institute, Bad Neuheim, Germany). Cells were grown to confluence in RPMI 1640 (Life Technologies, Grand Island, NY) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM L-glutamine, and 10% FCS (Life Technologies, Gaithersburg, MD). Cells were incubated with or without cytokines for various times, as indicated. For flow cytometry, cells were harvested with 0.25% trypsin and 1 mM EDTA, suspended in FACS buffer (PBS containing 1% BSA, 0.1% sodium azide, and 1% heat-inactivated rabbit serum), and 1 x 106 cells were delivered per tube for immunofluorescence staining. Cells were incubated on ice for 15–30 min with primary Abs. Abs used were: murine anti-ICAM YN 1/1.7 (35) (hybridoma was purchased from American Type Culture Collection, Manassas, VA, and the supernatant was used at a dilution of 1:10), anti-murine VCAM (PharMingen, San Diego, CA; 0.5 µg), anti-murine MAdCAM-1 (MECA 367 diluted 1:5–1:10; generously provided by E. Butcher) (36); anti-murine PNAd (MECA 79, diluted 1:5; provided by E. Butcher) (37), and anti-murine E-selectin (PharMingen; 1 µg). In all experiments, rat IgG was used as a negative control (Jackson Immunoresearch, West Grove, PA; 1 µg). After incubation with primary Ab, cells were washed and incubated with goat anti-rat IgG conjugated with phycoerythrin (PharMingen, 0.5–1 µg) as secondary Ab. Samples were analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Data were analyzed using CellQuest software (Becton Dickinson). The specific mean fluorescence for each sample was calculated by subtracting background (IgG).
Chemokine analysis
bEnd.3 cells were treated with 100 U/ml of rmLT for 4, 8, 16,
or 24 h. Total RNA was isolated using Trizol (Life Technologies)
according to manufacturer’s recommendations. Chemokine mRNA levels
were determined using multiprobe RNase protection assay system
(PharMingen). This system detects mRNA for lymphotactin, RANTES,
eotaxin, macrophage inflammatory protein (MIP) -1ß, MIP-1, MIP-2,
IP-10, MCP-1, and TCA-3 as well as the housekeeping genes L32 and
GAPDH. 32P-labeled riboprobes were synthesized from the
plasmid template set using T7 polymerase after which the DNA template
was digested with DNase. Five micrograms of total endothelial cell RNA
were hybridized with the riboprobes in a 10-µl volume overnight at
56°C. RNA samples were digested and electrophoresed on a 5%
polyacrylamide gel according to manufacturer’s recommendations. The
level of mRNA expression was quantified by densitometric analysis using
Imagequant software (Molecular Dynamics, Sunnyvale, CA).
Supernatants from LT-treated bEnd.3 cells were analyzed by ELISA for chemokine proteins. RANTES was detected with a Quantikine ELISA kit purchased from R & D Systems (Minneapolis, MN). MCP-1 was determined by sandwich ELISA. Nunc maxisorp ELISA plates (Fisher, Pittsburgh, PA) were coated with 4 µg/ml of hamster polyclonal anti-mouse MCP-1 Ab in 0.1 M NaHCO3 buffer, pH 8.2, overnight at 4°C. Plates were washed three times with PBS and blocked with 2% BSA/PBS/0.05% Tween for 2 h at room temperature. Then, 100 µl of rmMCP-1 standard (PharMingen) or supernatant (either undiluted or diluted 1:2 in 2% BSA/PBS) were added to the plate and incubated overnight at 4°C. Plates were washed four times with PBS/Tween and biotinylated anti-murine MCP-1-detecting Ab (PharMingen) was added at 1 µg/ml and incubated for 45 min at room temperature. Plates were washed six times with PBS/Tween. Alkaline phosphatase-conjugated avidin (Zymed, South San Francisco, CA) was added at 1:1000 and incubated for 30 min at room temperature. Plates were washed eight times with PBS/Tween and developed with p-nitrophenyl phosphate (Pierce, Rockford, IL; 10 µg/ml) in diethanolamine buffer. Absorbance was quantified on a Vmax plate reader at 405 nm. Samples were analyzed in duplicate. IP-10 was not analyzed, as matching Ab pairs were not available.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
induces killing of WEHI 164 cells
A fundamental biologic effect of LT is cytotoxicity, so the
activity of the rmLT was analyzed first. The WEHI 164 assay was used
to compare the cytotoxic activity of three preparations of
recombinant-derived cytokines, mLT, hLT, and mTNF-. The
results (Fig. 1
) indicate that all three
cytokines induced complete killing of WEHI 164 cells. The preparations
differed in potency in that 1.9 ng of mLT was needed for 50%
cytotoxicity, whereas comparable killing required only 1.2 pg hLT or
0.19 pg mTNF-. Treatment with mLT revealed a comparable
cytotoxicity in four separate experiments. Nevertheless, LT induced
cytotoxicity with starting material diluted as much as 1:20,000,
indicating that this preparation of mLT was very potent. This
demonstration of mLT3 cytotoxic activity validates the use of this
material for evaluating new biologic activities of LT.
|
induces expression of adhesion molecules on murine
endothelial cells
The ability of LT to modify adhesion molecule expression in
endothelial cells was investigated to obtain insight into direct
effects of LT in promoting inflammation and lymphoid organogenesis.
To establish sensitivity of murine endothelial cells to LT, the
ability of hLT to induce expression of adhesion molecules was first
examined using the murine endothelial cell line bEnd.3 (Fig. 2). hLT (100 U/ml) stimulated
expression of VCAM, ICAM, and MAdCAM-1. VCAM expression increased
6.7-fold above baseline levels within 4 h and persisted for
24 h. Similarly, ICAM expression increased 3.4-fold within 4
h and decreased slightly over 24 h. The most dramatic change in
adhesion molecule expression was evident with MAdCAM-1. MAdCAM-1
expression increased 3.1-fold within 4 h and continued to increase
over the 24-h assay period to a final increase of 15.5 times baseline
levels. PNAd was not induced by hLT at any time point. These
observations establish that LT can induce adhesion molecule
expression in murine endothelial cells. The kinetics of induction are
consistent with previous observations of dermal microvascular
endothelial cells that demonstrate early induction of VCAM and
persistent expression of E-selectin (38). The 18-h time point was
chosen for subsequent experiments.
|
3 to modify adhesion molecule expression in
endothelial cells was of particular interest to test whether this was
one mechanism whereby mLT promotes inflammation and lymphoid
organogenesis. mLT was evaluated for its ability to induce ICAM-1,
VCAM-1, E-selectin, MAdCAM-1, and PNAd. mLT (100 U/ml) induced VCAM,
ICAM, and MAdCAM-1 expression; however, 500 U/ml was necessary for
induction of E-selectin. A representative experiment with 500 U/ml
mLT is shown in Fig. 3. Although
bEnd.3 cells constitutively express relatively high levels of ICAM-1,
this level was increased 2.3-fold following mLT treatment. Baseline
expression of VCAM-1 was lower and was increased 25-fold following
treatment with mLT. E-selectin was induced 3-fold above baseline by
mLT. MAdCAM-1 expression was dramatically induced by
treatment with mLT. At 100 U/ml, MAdCAM-1 was induced 14-fold over
baseline. The induction was even more striking when cells were treated
with 500 U/ml. This treatment induced MAdCAM-1 expression 26-fold above
baseline. mLT did not induce expression of PNAd at any of the doses
tested (up to 1000 U/ml).
|
, hLT, and TNF- to induce adhesion
molecules was then tested. All three cytokines induced VCAM, ICAM,
E-selectin, and MAdCAM-1 but not PNAd (Fig. 4). The individual cytokines did differ
in their effectiveness at inducing different adhesion molecules. The
murine cytokines, mLT and mTNF-, were much more effective at
inducing VCAM expression than the hLT3. In contrast, all three
cytokines were comparable in their ability to induce ICAM expression
(2- to 2.5-fold induction). mTNF- was slightly more effective at
inducing E-selectin than either mLT or hLT. Importantly, this
comparative study demonstrated that mLT was the most potent inducer
of MAdCAM-1 expression. In this experiment, mLT induced
MAdCAM-1 expression to a level three times higher than that
induced by hLT or mTNF-. This experiment was conducted three
times with treatments of 100 or 500 U, and mLT was always more
effective at inducing MAdCAM-1 than the other two cytokines. None of
the three cytokines induced PNAd.
|
induces chemokine expression
To gain further insight into the mechanisms by which LT promotes
inflammation and lymphoid organogenesis, we analyzed the ability of
rmLT3 to induce chemokine mRNA and protein expression. bEnd.3 cells
were treated with 100 U/ml rmLT, and the chemokine expression
pattern was analyzed. RNase protection analysis of bEnd.3 cells
revealed a low constitutive accumulation of RANTES, IP-10,
and MCP-1 mRNA (Fig. 5A).
These increased dramatically within 4 h of treatment with mLT.
These mRNA levels decreased through the observation period, but still
remained elevated above untreated levels at 24 h. A similar time
course and chemokine profile was seen in two separate experiments.
Densitometric analysis was performed on a shorter exposure of the blot
depicted in Fig. 5A by normalizing the level of chemokine
mRNA expression to the level of GAPDH housekeeping gene. This analysis
confirmed LT-induced RANTES, IP-10, and MCP-1 mRNA expression peaked at
4 h, then decreased at 16h and 24 h (Fig. 5C).
Although RANTES, IP-10, and MCP-1 are the predominant chemokines
induced by LT, upon overexposure of the autoradiogram, MIP-1ß and
MIP-2 mRNAs were also detected (Fig. 5B, lane 7).
This expression profile is distinct from that observed in LN. RNase
protection analysis of that tissue revealed expression of a different
chemokine array, namely lymphotactin, RANTES, eotaxin, MIP-1, IP-10,
and TCA-3 (Fig. 5B, lane 8).
|
(Fig. 6) and
continued to increase through the 24-h evaluation period.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
3 has
significant biologic effects in vitro. mLT kills WEHI 164 cells and
induces expression of adhesion molecules associated with inflammation
(VCAM, ICAM, and E-selectin) and lymphoid organs (MAdCAM-1)
and chemokine mRNA and protein in a murine endothelial cell line,
bEnd.3. This is the first report that rmLT has any effects other
than killing. The studies have profound implications with regard to
determining the mechanisms by which LT induces inflammation and
lymphoid organ development and are supportive of recent work with LT
transgenic and knockout mice.
Though LT was one of the first cytokines to be discovered (39, 40, 41), and certainly the first of the still expanding TNF family,
analysis of its biologic activity has been hampered by the absence of
adequate quantities of recombinant murine material that might allow
absolutely unambiguous distinction between LT and TNF-. hLT
has been available, and, in fact, the first crystal structure analysis
of a TNF family member was based on the interaction of hLT (TNFß)
with TNFRI (42). Induction of cytotoxicity and adhesion molecule
expression by rhLT (18) and cytotoxicity with supernatants from
murine Ag-activated CD4TH1 clones that accumulate LT and TNF-
mRNA (3, 11, 32) are consistent with the conclusion that mLT and
mTNF- carry out similar activities through TNFR1. This is also
supported by the observations that the inflammation induced by LT is
qualitatively similar to that induced by TNF- in transgenic mice
(43) and that, at least in the case of LT, this occurs through TNFRI
(1). In the current studies, we document the biologic activities of
rmLT, namely the induction of adhesion molecules and
chemokines.
The data presented here clearly demonstrate that LT3 alone is
biologically active and induces inflammatory mediators independent of
LTß. This is consistent with previous publications from this
laboratory indicating that inflammation in RIPLT.LTß-/-
mice is quantitatively similar to that observed in RIPLT.wt mice (1).
In addition, RIPLT.LTß-/- mice expressed the same
chemokines expressed in the RIPLT.wt mice, namely RANTES, IP-10, and
MCP-1, at the sites of transgene expression. These data indicate the
the LTß complex is not required for LT-induced expression of these
chemokines.5 The current
study indicates that LT is less potent than mTNF- in signaling
through TNFR1 as more LT is necessary to induce 50% cytotoxicity. A
quantitative, but not qualitative, difference between these cytokines
is also observed in vivo by comparing the infiltrates of the islet in
RIPLT and RIPTNF transgenic mice (43). The cellular composition of the
inflammation is identical, but the RIPTNF islets contain more
infiltrating cells. One important difference between these cytokines is
that while TNF can either suppress or exacerbate the progression of
nonobese diabetic insulin-dependent diabetes mellitus (44, 45, 46),
LT in our hands does not have this effect (A. Kratz et
al., manuscript in preparation). This may be because LT has a lower sp.
act., and in most situations LT reaches biologic levels that are
proinflammatory and prolymphoid organogenic, but not
immunosuppressive.
The observations reported here confirm and extend in vivo studies on
the role of LT in inflammation (3, 19).5 They indicate
that LT alone, in the absence of an LTß complex, effectively
induces expression of adhesion molecules VCAM, ICAM-1, E-selectin, and
MAdCAM-1 and the chemokines RANTES, IP-10, and MCP-1. We have
previously demonstrated that VCAM, ICAM, MAdCAM-1, and PNAd are induced
in vivo in the RIPLT mouse model (19). The induction of adhesion
molecules is consistent with the marked inflammation induced in the
RIPLT mouse and other in vivo studies that strongly implicate LT in
the inflammation observed in EAE (14, 15). We have also recently
demonstrated that the RIPLT transgene induces the same array of
chemokines in vivo as the rmLT3 induced in these
studies.5 The array of chemokines induced by LT is
consistent with the type of cells that accumulate in RIPLT-induced
lesions, namely mononuclear cells. All three of these chemokines
attract T cells (47, 48, 49). In addition, MCP-1, and to a lesser extent
RANTES and IP-10, also attract monocytes (47, 50, 51, 52).
The in vitro data reported here provide insight into the mechanism by
which LT promotes lymphoid organ development through induction of
adhesion molecules expressed on high endothelial venules. One of the
most dramatic effects of LT in this study was its induction of
MAdCAM-1, a marker of all developing LN (53), mature mesenteric LN, and
PP (36), and also some instances of chronic inflammation (19, 54). The
fact that mLT does this more effectively than equal units of TNF-
is consistent with its crucial role in the development of LN and PP.
The data are consistent with our in vivo observation that LT induces
MAdCAM-1 through TNFRI in the absence of LTß,5 but may
also act through another as yet undescribed receptor. This also
suggests that LT may use more than one receptor to induce MAdCAM-1
expression. It is already apparent through studies of knockout mice
that LT induces PP both through an interaction with TNFRI and the
LTßR (5, 7, 8). Thus induction of MAdCAM-1 by LT could contribute
to these effects. TNF- does not appear to contribute in any way to
the development of LN or PP (8, 9) although in vitro it can induce
MAdCAM-1 (Ref. 17, and data provided here). Perhaps it does not
participate in LN development because it is not expressed at the
appropriate time and place in embryogenesis. None of the preparations
used here induced expression of PNAd from the bEnd.3 cell line. This is
consistent with in vivo data indicating that RIPLT mice need LTß to
induce expression of that adhesion molecule and consistent with the
fact that LTß-/- mice lack all peripheral LN (5),
though they have mesenteric and cervical LN. A test of the hypothesis
that LTß complex is necessary for peripheral LN in part through
induction of PNAd would be to determine whether LT1ß2 induces PNAd
in vitro.
LT may also contribute to lymphoid organ development through its induction of chemokines. Thus far only one chemokine, through its receptor, BLR-1, has been shown to play a role in lymphoid organ development, but it is likely that others will be described. Mice deficient in the gene that codes for blr1 have profound defects in B cell trafficking and lack inguinal LN and have missing or defective PP (21). It will be interesting to determine whether LT can induce BCA-1/BLC, the ligand for this receptor (55, 56), or other chemokines involved in lymphoid trafficking in development. The in vitro studies initiated here will continue to provide insight into the role of LT in inflammation and development by allowing the analysis of the molecular basis of its effects in these crucial and mechanistically related processes.
|
Acknowledgments |
|---|
3 and Regina Turetskaya for
rmTNF-. We also thank Matthew Hanson for helpful advice with flow
cytometry, Margot Iverson for technical assistance, Eugene Butcher for
his generous gift of Abs, and Walter Risau for the gift of the bEnd.3
cell line. |
Footnotes |
|---|
2 C.A.C. and J.S. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Nancy H. Ruddle, Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, P.O. Box 208034, New Haven, CT 06520-8034. E-mail address:
4 Abbreviations used in this paper: LT, lymphotoxin; LN, lymph node; PP, Peyer’s patch; RIP, rat insulin promoter; EAE, experimental allergic encephalomyelitis; MAdCAM-1, mucosal addressin cellular adhesion molecule; PNAd, peripheral node addressin; BLR-1, Burkitt’s lymphoma receptor; IP-10, IFN-inducible protein 10; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein.
5 Cuff, C. A., R. Sacca, and N. H. Ruddle. Differential regulation of leukocyte trafficking by LT3 and LTß elucidates potential mechanisms of mesenteric and peripheral lymph node development. Submitted for publication.
Received for publication May 18, 1998. Accepted for publication August 11, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
3 and membrane LT1ß2 in lymphotoxin-induced inflammation: critical role of TNFR1 signaling. J. Immunol. 160:485. in murine T cell clones activated through the TCR. J. Immunol. 152:4336.[Abstract]
and ß revealed in lymphotoxin ß-deficient mice. Immunity 6:491.[Medline]
/ß complex is required for the development of peripheral lymphoid organs. J. Exp. Med. 184:1.-deficient mice: a critical requirement for TNF in the formation of networks and germinal centers, and in the maturation of humoral immune response. J. Exp. Med. 184:1397. production by myelin basic protein-specific T cell clones correlates with encephalitogenicity. Int. Immunol. 2:539. and IL-1. J. Immunol. 151:5239.[Abstract]
. Am. J. Pathol. 145:913.[Abstract]
and transforming growth factor ß upregulate astrocyte expression of monocyte chemoattractant protein-1. J. Neuroimmunol. 57:193.[Medline]
induces expression of the chemoattractant cytokine RANTES in cultured mouse mesangial cells. Kidney Int. 44:795.[Medline]
and lymphotoxin-ß complexes. J. Immunol. 159:3299.[Abstract]
and ß. J. Bio. Chem. 271:8618. (hTNF-) and ß (hTNF-ß) and for murine tumour necrosis factor (mTNF-). J. Immunol. Meth. 170:1.[Medline]
production in islets leads to insulitis, not diabetes: distinct patterns of inflammation in TNF- and TNF-ß transgenic mice. J. Immunol. 149:4136.
and interleukin 1. Proc. Natl. Acad. Sci. USA 87:968. in islets prevents autoimmune diabetes in nonobese diabetic (NOD) mice by preventing the development of auto-reactive islet-specific T cells. J. Exp. Med. 184:1963. on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process. J. Exp. Med. 180:995.This article has been cited by other articles:
|
|
 |
|
  N. H. Ruddle and E. M. Akirav Secondary Lymphoid Organs: Responding to Genetic and Environmental Cues in Ontogeny and the Immune Response J. Immunol., August 15, 2009; 183(4): 2205 - 2212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. R. Roach, H. Briscoe, B. M. Saunders, and W. J. Britton Independent Protective Effects for Tumor Necrosis Factor and Lymphotoxin Alpha in the Host Response to Listeria monocytogenes Infection Infect. Immun., August 1, 2005; 73(8): 4787 - 4792. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Silva-Santos, D. J. Pennington, and A. C. Hayday Lymphotoxin-Mediated Regulation of {gamma}{delta} Cell Differentiation by {alpha}{beta} T Cell Progenitors Science, February 11, 2005; 307(5711): 925 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Drayton, G. Bonizzi, X. Ying, S. Liao, M. Karin, and N. H. Ruddle I{kappa}B Kinase Complex {alpha} Kinase Activity Controls Chemokine and High Endothelial Venule Gene Expression in Lymph Nodes and Nasal-Associated Lymphoid Tissue J. Immunol., November 15, 2004; 173(10): 6161 - 6168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Soderberg, M. M. Linehan, N. H. Ruddle, and A. Iwasaki MAdCAM-1 Expressing Sacral Lymph Node in the Lymphotoxin {beta}-Deficient Mouse Provides a Site for Immune Generation Following Vaginal Herpes Simplex Virus-2 Infection J. Immunol., August 1, 2004; 173(3): 1908 - 1913. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Corcione, S. Casazza, E. Ferretti, D. Giunti, E. Zappia, A. Pistorio, C. Gambini, G. L. Mancardi, A. Uccelli, and V. Pistoia Recapitulation of B cell differentiation in the central nervous system of patients with multiple sclerosis PNAS, July 27, 2004; 101(30): 11064 - 11069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Palmeri, F.-R. Zuo, S. D. Rosen, and S. Hemmerich Differential gene expression profile of human tonsil high endothelial cells: implications for lymphocyte trafficking J. Leukoc. Biol., May 1, 2004; 75(5): 910 - 927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Meissner, O. Zilles, R. Varona, K. Jozefowski, U. Ritter, G. Marquez, R. Hallmann, and H. Korner CC chemokine ligand 20 partially controls adhesion of naive B cells to activated endothelial cells under shear stress Blood, October 15, 2003; 102(8): 2724 - 2727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Drayton, X. Ying, J. Lee, W. Lesslauer, and N. H. Ruddle Ectopic LT{alpha}{beta} Directs Lymphoid Organ Neogenesis with Concomitant Expression of Peripheral Node Addressin and a HEV-restricted Sulfotransferase J. Exp. Med., May 5, 2003; 197(9): 1153 - 1163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Grant, S. Goddard, J. Ahmed-Choudhury, G. Reynolds, D. G. Jackson, M. Briskin, L. Wu, S. G. Hubscher, and D. H. Adams Hepatic Expression of Secondary Lymphoid Chemokine (CCL21) Promotes the Development of Portal-Associated Lymphoid Tissue in Chronic Inflammatory Liver Disease Am. J. Pathol., April 1, 2002; 160(4): 1445 - 1455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Schreyer, C. M. Vick, and R. C. LeBoeuf Loss of Lymphotoxin-alpha but Not Tumor Necrosis Factor-alpha Reduces Atherosclerosis in Mice J. Biol. Chem., March 29, 2002; 277(14): 12364 - 12368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Weyand, P. J. Kurtin, and J. J. Goronzy Ectopic Lymphoid Organogenesis : A Fast Track for Autoimmunity Am. J. Pathol., September 1, 2001; 159(3): 787 - 793. [Full Text] [PDF]
|
|
|
|
|
|
S. Takemura, A. Braun, C. Crowson, P. J. Kurtin, R. H. Cofield, W. M. O'Fallon, J. J. Goronzy, and C. M. Weyand Lymphoid Neogenesis in Rheumatoid Synovitis J. Immunol., July 15, 2001; 167(2): 1072 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hashi, H. Yoshida, K. Honda, S. Fraser, H. Kubo, M. Awane, A. Takabayashi, H. Nakano, Y. Yamaoka, and S.-I. Nishikawa Compartmentalization of Peyer's Patch Anlagen Before Lymphocyte Entry J. Immunol., March 15, 2001; 166(6): 3702 - 3709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Reddy, P. Chastagner, L. Fiette, X. Liu, and J. Theze IL-2-induced tumor necrosis factor (TNF)-{beta} expression: further analysis in the IL-2 knockout model, and comparison with TNF-{{alpha}}, lymphotoxin-{beta}, TNFR1 and TNFR2 modulation Int. Immunol., February 1, 2001; 13(2): 135 - 147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Roach, H. Briscoe, B. Saunders, M. P. France, S. Riminton, and W. J. Britton Secreted Lymphotoxin-{alpha} Is Essential for the Control of an Intracellular Bacterial Infection J. Exp. Med., January 15, 2001; 193(2): 239 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Hjelmstrom, J. Fjell, T. Nakagawa, R. Sacca, C. A. Cuff, and N. H. Ruddle Lymphoid Tissue Homing Chemokines Are Expressed in Chronic Inflammation Am. J. Pathol., April 1, 2000; 156(4): 1133 - 1138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Iizuka, T. Tanaka, M. Suematsu, S. Miura, T. Watanabe, R. Koike, Y. Ishimura, H. Ishii, N. Miyasaka, and M. Miyasaka Stage-Specific Expression of Mucosal Addressin Cell Adhesion Molecule-1 During Embryogenesis in Rats J. Immunol., March 1, 2000; 164(5): 2463 - 2471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.A. Warnock, J.J. Campbell, M.E. Dorf, A. Matsuzawa, L.M. McEvoy, and E.C. Butcher The Role of Chemokines in the Microenvironmental Control of T versus B Cell Arrest in Peyer's Patch High Endothelial Venules J. Exp. Med., January 3, 2000; 191(1): 77 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Juedes, P. Hjelmstrom, C. M. Bergman, A. L. Neild, and N. H. Ruddle Kinetics and Cellular Origin of Cytokines in the Central Nervous System: Insight into Mechanisms of Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis J. Immunol., January 1, 2000; 164(1): 419 - 426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. V. Kuprash23, M. B. Alimzhanov2, A. V. Tumanov2, A. O. Anderson, K. Pfeffer, and S. A. Nedospasov TNF and Lymphotoxin {beta} Cooperate in the Maintenance of Secondary Lymphoid Tissue Microarchitecture But Not in the Development of Lymph Nodes J. Immunol., December 15, 1999; 163(12): 6575 - 6580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Wu, Y. Wang, J. Wang, E. O. Hedgeman, J. L. Browning, and Y.-X. Fu The Requirement of Membrane Lymphotoxin for the Presence of Dendritic Cells in Lymphoid Tissues J. Exp. Med., September 6, 1999; 190(5): 629 - 638. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), IP-10 (
), and MCP-1 (
)
mRNA expression of the autoradiogram shown in A. Data
are normalized to the level of GAPDH expressed in each sample.
Representative data from one of two experiments.