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: modification of the current `maturation' paradigm and implications for in vivo immunoregulation
* Immunotherapy Laboratory and
Clinical Support Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Division of Clinical Sciences, SAIC-Frederick, Frederick, Maryland 21702, USA; and
Medical Poliklinik, Ludwig-Maximilian-University, Munich, Germany
1Correspondence: Immunotherapy Laboratory, NCI-FCRDC, SAIC-Frederick, Bldg. 1050, Boyles St., Frederick, MD 21702, USA. E-mail: enelson{at}mail.ncifcrf.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The current paradigm holds that
this DC maturation event is required for full functional capacity and
represents terminal differentiation of this cell type, culminating in
apoptotic cell death. This provides a possible mechanism for avoiding
dysregulated immunostimulatory activity, but imposes constraints on the
capacity of DCs to influence subsequent immune responses and to
participate in immunological memory. We report that the cell surface
and functional effects induced by TNF- are reversible and
reinducible. These effects are accompanied by a concordant modulation
of cytokine mRNA expression that includes the induction of
proinflammatory factors (IL-15, IL-12, LT-, LT-ß, TNF-, RANTES)
which is coincident with the down-regulation of counter-regulatory
cytokines (IL-10, TGF-ß1, TGF-ß2, IL-1 RA, MCP-1). The resultant
net effect is a dendritic cell activation state characterized by a
transient proinflammatory posture. These results demonstrate that
1) human DCs do not undergo terminal `maturation' in
response to TNF-, 2) DC phenotypes are more
pleiotropic than previously thought, and 3) DCs are
potential immunoregulatory effector cells with implications for control
of immune responses in both in vivo and in
vitro systems.—Nelson, E. L., Strobl, S., Subleski, J.,
Prieto, D., Kopp, W. C., Nelson, P. J. Cycling of human
dendritic cell effector phenotypes in response to TNF-: modification
of the current `maturation' paradigm and implications for in
vivo immunoregulation. Cycling of human dendritic cell effector
phenotypes in response to TNF-: modification of the current
`maturation' paradigm and implications for in vivo
immunoregulation.
Key Words: antigen presenting cells • cytokines • chemokines • and dendritic cell activation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. DCs have also been reported to
participate in the maintenance of immunological memory and the
induction of immunological tolerance (4
5
6
7)
. The precise
lineage and functional characteristics of these cells, along with the
related Langerhans cell, have been debated since their initial
description (8
, 9)
. Recently, various subsets of DCs have
been characterized. It is thought that different DC subsets may play
distinct roles in different immunological responses (1
, 2)
. As there is currently no single, widely accepted surface
marker common to all human DCs, their identification depends on the
demonstration of a characteristic morphology, the expression of a
constellation of cell surface markers, and enhanced functional capacity
(1
2
3)
. This lack of a lineage-specific marker has
complicated the study of human DCs because the number of cells required
to fully evaluate the various cellular phenotypes recovered for
functional experiments is often prohibitive.
Several methods for the isolation and/or generation of DCs have been
described including conditions for generating the recently
characterized `immature' and `mature' DC phenotypic subsets
(1
2
3
, 10
11
12
13
14
15)
. Tumor necrosis factor (TNF-) is one of
several agents that have been reported to induce the phenotypic changes
characteristic of mature DCs. The existing paradigm of dendritic cell
action holds that the `job' of immature resident dendritic cells is
to continuously sample their surrounding tissue environment for
antigens. In response to specific signals produced during inflammation,
such as TNF-, the immature dendritic cells become activated and
undergo an `irreversible maturation' to a new phenotype. It has been
proposed that this maturation event is required for DCs to acquire
their full functional capacity and represents the terminal
differentiation of this cell type (1
2
3
, 12
, 15
16
17
18
19)
. This
maturation process leads to a cessation of environmental sampling, the
expression of new surface antigens, and migration of the mature
dendritic cell to the lymph nodes where it can present its complement
of `sampled' antigens to T cells. After antigen presentation in the
lymph node, it has been postulated that apoptotic death of the mature
dendritic cell occurs and thus allows for down-regulation of the
specific immune response (11
, 15
, 17
, 19)
. This model,
however, does not easily accommodate a role for dendritic cells in
immunological memory; it suggests limited potential for
immunoregulation and appears to be biologically inefficient especially
for rare cells with limited proliferative capacity. Therefore, we
hypothesized that the process identified as `maturation' might be a
reversible phenomenon and would constitute an activation state rather
than a state of terminal differentiation. Such a reversible capacity
would be biologically more economic in that induction of immune
responses by DCs would not be an obligatory one-time event. A
transiently activated DC phenotype could also play a more global role
in the regulation of subsequent immune processes, e.g., as in the
recently described `temporal bridge' between helper lymphocytes and
cytotoxic effectors (20)
. We generated large numbers of
human dendritic cells from peripheral blood monocytes and extensively
evaluated the functional and phenotypic effects of TNF- in these
preparations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100 U/ml) were added at day 0. TNF- (20 ng/ml, 200 U/ml)
was added to aliquots of DC cultures at 10–12 days (or as indicated)
for 48 h to obtain activated DCs. All cytokines were obtained
through the Biological Resources Branch of the National Cancer
Institute-Frederick Cancer Research and Development Center (Frederick,
Md.). Cultures were fed every 6–7 days by removing one-half of the
culture volume and adding an equal volume of fresh media containing
sufficient GM-CSF and IL-4 for the entire culture volume.
Cell surface markers
After blocking in 5% human serum, cells were incubated for 15
min at 4°C in phosphate-buffered saline (PBS), 2% bovine serum
albumin, and 0.1% sodium azide with FITC- or PE-conjugated monoclonal
antibodies to CD3, CD4, CD8, CD11b, CD14, CD19, CD56, HLA DR, CD80,
CD95 ligand (Becton Dickinson, San Jose, Calif.), CD32, CD40, CD86,
CD95, pan MHC class I (PharMingen, San Diego, Calif.), CD16 (20 µl of
1:100 3G8, Medarex, Annandale, N.J.), CD11a, CD11c, CD33, CD34, CD54,
CD83, CD154 (Coulter Immunotech Inc., Westbrook, Maine), and CD1a
(clones: SFC119Thy1A8 Coulter Immunotech; OKT6, Ortho Diagnostic
Systems Inc. Raritan, N.J.; M-T102 PharMingen; or NA1/34-HLK, Serotec,
Washington, D.C.). CD1b, CD1c, and CD1a (clones: B17.20.9, and BL6)
(Coulter Immunotech) were unconjugated and detected with
FITC-conjugated Fab'2 goat anti-mouse IgG incubated with 25 µl of a
1:20 dilution (Boehringer Mannheim Biochemicals, Indianapolis, Ind.).
After washing, cells were resuspended in l% paraformaldehyde,
evaluated on a FACScan (Becton Dickinson), and analyzed using FlowJo
software (Tree Star, San Carlos, Calif.).
Allogeneic mixed lymphocyte reactions
Allogeneic responding T cells from normal donors were
cultured at 1.0 x 105 cells/well in 96-well
flat-bottom microplates (Costar Corp., Cambridge, Mass.) with graduated
numbers of irradiated (3000 rad from a 137Cs
source) stimulator cells, either baseline DCs, TNF--activated DCs,
or autologous freshly thawed, cryopreserved monocytes. Cells were
pulsed for 18 h with 0.5 µCi/well [methyl
3H] thymidine (specific activity 5 Ci/mmol,
Amersham Life Science, Arlington Heights, Ill.) on day 5 of culture.
Cells were harvested using the Mach IIIm Harvester 96 (Tomtec Inc.,
Orange, Conn.) and specific activity was measured by liquid
scintillation on the MicroBeta Trilux liquid scintillation counter
(Wallac, Inc., Gaithersburg, Md.). The use of flat-bottom microplates
results in somewhat lower proliferative responses, but better
accommodates the large and more adherent TNF--activated DCs.
Naive antigen immune responses
All cultures for the evaluation of naive antigen immune
responses were performed in AIM V, serum-free media supplemented with
sodium pyruvate (1 mM), nonessential amino acids (1x) (Gibco BRL Life
Technologies), and 2-mercaptoethanol (50 µM; Sigma). APCs, either day
12 dendritic cells or autologous freshly thawed, cryopreserved
monocytes, were incubated for 18–24 h with `endotoxin free KLH'
(Calbiochem, La Jolla, Calif.) at a concentration of 10 µg/ml; the
resulting endotoxin level of this lot was less than 0.04 EU (endotoxin
unit) per milliliter. Activated DCs were exposed to TNF- for 48 h prior to their addition to responding lymphocytes. All APCs were
washed three times with PBS before addition to responding cells.
Responding autologous lymphocytes were mixed with graduated numbers of
keyhole limpet hemocyanin (KLH) pulsed, washed, stimulator dendritic
cells or monocytes. Cells were cultured together for 10 days. These
`primed' T cells (1x105 cells/well) were then
restimulated with a second set of identically KLH pulsed, washed,
irradiated, autologous APCs (either dendritic cells or cryopreserved
monocytes from the same initial preparation) and cultured together.
After 5 days of culture, cells were pulsed for 18 h with
3H thymidine, harvested, and measured as
described above.
Ribonuclease protection assays
Total RNA from DC preparations, cryopreserved monocytes,
and cryopreserved lymphocytes was isolated using Trizol reagent (Life
Technologies) according to the manufacturer's instructions. The
Riboquant RPA kit and Multi-probe sets (PharMingen) were used according
to the manufacturer's instructions with the exception that
32P (Amersham Life Sciences) labeled probes were
purified using TE Micro Select-D, G-25 microcentrifuge spin columns (5
Prime–3 Prime, Inc., Boulder, Colo.) to analyze 2–5 µg of total
RNA. Protected fragments were resolved on 6% polyacrylamide sequencing
gels and quantitated on a BAS 1000 PhosphorImager (Fuji Medical
Systems, Fairfield, N.J.). Signal strength was normalized to the GAPDH
housekeeping gene message signal (essentially identical results were
obtained if the L32 ribosomal RNA signal was used for normalization).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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as `TNF--activated DCs' (TNF- DCs). The
generation of BL DCs from monocytes was complete after 8 to 10 days,
resulting in a morphologically and phenotypically uniform population of
DCs with consistent yields of 10 to 25% relative to the initial cell
number placed into culture. This yield was reproduced in more than 30
separate normal donor preparations. Monocyte preparations that
demonstrated pronounced aggregation during elutriation resulted in very
low yields of DC conversion; they were considered to be partially
activated and were not used in subsequent studies. The DCs were
routinely maintained in culture in excess of 28 days without
significant cell loss while maintaining both their cell surface
phenotype and functional capacity. Significant proliferation was not
demonstrated as exemplified by background levels of
3H thymidine incorporation over this period (data
not shown). These BL DCs had the typical morphology (Fig. 1
A), with large irregular shapes and extensive cytoplasmic
filopods and the pattern of cell surface marker expression reported for
immature DCs (Fig. 2
). Notably, these cellular preparations did not express monocyte (CD14),
lymphocyte (CD3 and CD19), natural killer cell (CD56), myeloid
precursor (CD34), or mature DC (CD83) markers (21)
. BL DCs
differed further from autologous monocytes in having elevated cell
surface molecule expression of MHC class I and MHC class II antigens,
CD80, and low but detectable CD1a. The interpretation of possible
attenuated expression of CD11a, CD11b, and CD33 was complicated by the
significantly higher intrinsic autofluorescence of cultured DCs vs.
autologous freshly thawed, cryopreserved monocytes. The addition of
TNF- to the culture media resulted within 48 h in an altered
morphology (Fig. 1B
), with filopods being more coarse, more
adherent, elongated, and with more extensive arborization. These
changes were coincident with a shift in cell surface phenotype to that
which has been reported for mature DCs (1
2
3
, 10
11
12
13
14
15
, 17)
,
including enhanced expression of CD40, CD54, and CD80 and the induction
of CD83 expression (Fig. 2)
. Donor-dependent changes after exposure to
TNF- included variably attenuated expression of CD1a, CD11a, CD11b,
CD32, and CD33, along with variable induction of CD86, whereas the
remainder of the panel of cell surface molecules (CD1b, CD1c, CD11c,
CD16, and CD95) were unchanged. The effects of TNF- on cell surface
phenotype were dose dependent within the concentration range of 0.05
ng/ml to 20 ng/ml (data not shown).
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Characterization of allogeneic and naive antigen T cell responses
elicited by BL DCs and TNF- DCs.
Baseline and TNF--activated DCs were evaluated for their
immunostimulatory capacity in allogeneic mixed lymphocyte reactions
(allo-MLRs) and for their ability to induce (prime) naive antigen
responses. In allo-MLRs, both BL DCs and TNF- DCs showed markedly
enhanced stimulatory capacity relative to autologous freshly thawed,
cryopreserved monocytes (Fig. 3
A). TNF- DCs were two- to threefold more potent as
stimulators than the BL DCs. We also evaluated our DC preparations for
their capacity to prime an autologous, naive antigen immune response
using endotoxin free KLH as the naive antigen (Fig. 3B
).
Both baseline and TNF--activated DCs primed naive antigen immune
responses, albeit BL DCs less efficiently than TNF- DCs, whereas
autologous freshly thawed, cryopreserved monocytes showed no such
ability.
|
The TNF--induced DC cell surface phenotype and increased
functional capacity are reversible and reinducible.
To test the hypothesis that the effects of TNF- may be
reversible, we evaluated the cell surface molecule expression
(Fig. 4
) and functional capacity in allo-MLRs (Fig. 5
) of DCs transiently exposed to TNF-, followed by continued culture
in the usual DC media without TNF-. Single donor BL DCs were divided
at day 10, with one part of the preparation exposed to TNF- for
48 h. We analyzed a portion of each of the resulting 12-day-old
preparations, and both showed the expected cell surface phenotype and
enhanced functional capacity (data not shown). The remainder of these
baseline and TNF--activated DC cultures were washed twice with PBS
and placed into standard DC culture media without TNF-. Seven to 10
days after removal of the TNF-, there was no discernible difference
in either the cell surface phenotype or the allo-MLR stimulatory
capacity between the DCs previously exposed to TNF- and autologous
DCs maintained in the absence of TNF-. Re-exposure of the formerly
activated DCs to TNF- for 48 h again elicited the same
activated cell surface phenotype (Fig. 4)
and the enhanced
immunostimulatory function (Fig. 5)
. These phenotypic shifts were
comparable to those seen in the control 19-day-old DCs, maintained in
GM-CSF and IL-4 over the entire `washout' period and exposed for the
first time to TNF- for 48 h. Thus, these effects of TNF- are
both reversible and reinducible.
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Characterization of cytokine and chemokine expression profiles in
the base line and TNF--induced dendritic cell phenotypes.
To further characterize this TNF--induced phenotype
(activation state), we investigated the expression of cytokine mRNAs in
BL DCs and TNF- DCs using ribonuclease protection assays (RPA)
(Fig. 6
). The mRNA levels for the chemokine RANTES (regulated on activation,
normal T cell expressed and secreted) and the cytokines IL-15, IL-12
p40, TNF-, LT-, and LT-ß were all induced by 2- to 30-fold
after 48 h of TNF- exposure whereas IL-10, TGF-ß1, TGF-ß2,
IL-1 RA, MIP-1ß, and MCP-1 mRNA levels were attenuated to levels 50%
to 20% of `baseline'. To evaluate the reversibility of the
TNF--induced modulation of cytokine expression patterns, we
performed identical washout experiments to those described above. The
results demonstrated a reversible and reinducible modulation of
cytokine/chemokine expression that was entirely concordant with the
functional and cell surface phenotypic changes (Fig. 6)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced cell surface phenotype
and enhanced immunostimulatory capacity are both reversible and
reinducible. Our baseline and TNF--activated dendritic cells have
the combination of morphological, phenotypic, and functional
characteristics currently used to define human dendritic cells. BL DCs
showed the typical morphology of immature DCs, did not express CD83,
and modulated their cell surface phenotype and functional capacity when
exposed to TNF- in a manner consistent with that reported by others
as being associated with maturation (1
2
3
, 10
11
12
13
14
15
, 17)
. As
previously described for immature DCs (13
, 15)
, these BL
DCs were significantly more active in macropinocytosis than TNF-
DCs, (data not shown). The capacity of our BL DCs to prime for naive
antigen responses does not in and of itself indicate a mature
phenotype, particularly in light of the other immature characteristics
seen in these preparations. The capacity of immature DCs to prime naive
antigen responses has been controversial, with several groups reporting
this capacity to be limited to mature DCs; recently, however, two
groups have demonstrated the priming of naive antigen responses by both
mature and immature DCs (10
, 22)
.
It is our interpretation that the transient effects of TNF-
constitute true reversion and not apoptotic cell death of mature DCs
with repopulation from a pool of immature DCs. This conclusion is based
on the maintenance of stable cell numbers over the course of these
experiments, without demonstrable cell death or proliferation, and the
demonstration that the preparations did not contain two discrete cell
populations, mature (CD83+) and immature (CD83-). Furthermore, it is
unlikely that CD34+ cells could account for generation of the mature
dendritic cells as all monocyte and DC preparations were negative for
CD34+ cells by flow cytometry and did not undergo proliferation.
Heterogeneous populations of cells containing DCs of the mature
phenotype have been generated by the culture of CD34+ progenitor cells
with various cytokines including the combination of GM-CSF and TNF-
both with and without IL-4 (23
24
25
26)
. The somewhat
prolonged period required for reversion of the activated phenotype is
not surprising given the paracrine induction of TNF- message, the
limited proliferative capacity of these cells, and their ability to be
maintained in culture for at least a month without attenuation of any
defining properties. Previous reports of irreversible TNF--induced
maturation in human DCs were based on detecting no change in phenotype
in several days (27
, 28)
, 3 days (29)
or at 2
to 3 days (22)
. One of these groups (22)
noted partial attenuation of the enhanced DC immunostimulatory capacity
after culturing the DCs in the absence of TNF- for 5 or 3 days, but
they did not re-expose their DC preparations to TNF-. Notably, we
observed only partial reversion of the cell surface phenotype 5 days
after the 48 h pulse of TNF- (data not shown). Thus, the
apparent conflicting observations and interpretation of the effects of
TNF- on human DCs appear to be based on differences in experimental
design.
In our DC preparations, the transient modulation of cytokine and
chemokine mRNA expression by TNF- may provide some insight into the
potential immunoregulatory capacity of human DC subsets and lend
further support to our suggestion that this mature phenotype represents
an activation state. The cytokine mRNA expression pattern of DCs
exposed to TNF- as described in this and previous reports is in
general agreement (30
31
32
33
34
35)
. However, previous reports did
not directly evaluate modulation of cytokine mRNA expression associated
with the maturation of DCs. The TNF--induced cytokine mRNAs reported
here are characteristic of proinflammatory immune mediators—IL-15,
IL-12, LT-, LT-ß, TNF-, RANTES—whereas the attenuated
mRNAs are generally representative of counter-regulatory or
immunosuppressive cytokines: TGF-ß1, TGF-ß2, IL-1 RA, IL-10
(36
, 37)
. Thus, exposure of human DCs to TNF- results
not only in the induction of proinflammatory mediators but also in the
disengagement of a `physiological brake' on the immune response by
down-regulation of counter-regulatory cytokines. This reversible
pattern of cytokine expression suggests that the accentuated
immunostimulatory capacity seen with TNF--activated DCs may be due
to the modulation of immunoregulatory cytokines as well as induction of
cell surface accessory/costimulatory molecules. The gradual in
vitro reversion to a `baseline' state, which includes
reinduction of counter-regulatory immune mediators, may be necessary
and critical to appropriate regulation of the in vivo immune
response, thereby providing an alternative mechanism to apoptosis of
mature DCs for avoiding persistent, dysregulated stimulation of the
immune response.
Our data suggest that the selection of dendritic cell preparations for
use in eliciting any particular in vivo or in
vitro immunomodulation may be critical. The expression of IL-10
and other counter-regulatory immune mediators by BL DCs would suggest
that preparations like these would be of benefit for purposes of
attenuating autoimmune phenomena (38)
or providing
a Th2/humoral immune response, but less likely to produce benefit where
a robust, Th1-biased immune response is desired such as in anti-tumor
immunotherapies (7
, 39)
. Furthermore, the ability of other
cytokine combinations to substitute for TNF- in the generation of
DCs from CD34+ progenitors and in the induction of the mature DC
phenotype suggests that other immune mediators will be able to directly
activate DCs, although not necessarily with identical modulation of
cytokine expression.
Based on the observations detailed here, the existing model of
dendritic cell functional development can be expanded to include the
capacity to cycle between DC effector phenotypes, Fig. 7
. Dendritic cells have been shown to undergo cell death or reversion to
macrophage morphology (28
, 29
, 40)
upon withdrawal of all
cytokine support. Recently it was demonstrated that persistent
stimulation with GM-CSF can sustain immature DCs upon removal of IL-4
(41)
. The current view that, in vivo,
mature (activated) DCs undergo apoptotic cell death after migration to
lymphoid tissues has not been demonstrated directly and is based in
part on the relative absence of DCs in efferent lymph of antigen primed
animals (42
43
44
45)
. Thus, the presence or absence of various
trophic factors (46)
and immune mediators may well
determine the ultimate fate of activated (mature) DCs within lymphoid
tissues. This could range from apoptosis, which might occur in the
presence of continuous or overwhelming activating stimuli
(circumventing a failure to reinduce counter-regulatory cytokines), to
quiescent or `memory' states, or even return to the periphery as
reverted BL DCs with resumption of antigenic surveillance. Our data
suggest that for human dendritic cells there is a dynamic balance
between antigen capture, processing, presentation, and
immunostimulatory capacity that can shift in response to the immune
mediators, such as TNF-, present in the microenvironment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. J. Exp. Med. 184,695-706 is required for accumulation of dendritic cells in draining lymph nodes and for optimal contact sensitization. Immunology 84,31-35[Medline]
in modulating the quantity of peripheral blood-derived, cytokine-driven human dendritic cells and its role in enhancing the quality of dendritic cell function in presenting soluble antigen to CD4+ T cells in vitro. Blood 91,4652-4661This article has been cited by other articles:
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 T. Kruger, D. Benke, F. Eitner, A. Lang, M. Wirtz, E. E. Hamilton-Williams, D. Engel, B. Giese, G. Muller-Newen, J. Floege, et al. Identification and Functional Characterization of Dendritic Cells in the Healthy Murine Kidney and in Experimental Glomerulonephritis J. Am. Soc. Nephrol., March 1, 2004; 15(3): 613 - 621. [Abstract] [Full Text] [PDF]
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 C. Paolucci, S. E. Burastero, P. Rovere-Querini, C. De Palma, S. Falcone, C. Perrotta, A. Capobianco, A. A. Manfredi, and E. Clementi Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway J. Leukoc. Biol., February 1, 2003; 73(2): 253 - 262. [Abstract] [Full Text] [PDF]
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 M. Lindstedt, B. Johansson-Lindbom, and C. A. K. Borrebaeck Global reprogramming of dendritic cells in response to a concerted action of inflammatory mediators Int. Immunol., October 1, 2002; 14(10): 1203 - 1213. [Abstract] [Full Text] [PDF]
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 G. Pietra, R. Mortarini, G. Parmiani, and A. Anichini Phases of Apoptosis of Melanoma Cells, but not of Normal Melanocytes, Differently Affect Maturation of Myeloid Dendritic Cells Cancer Res., November 1, 2001; 61(22): 8218 - 8226. [Abstract] [Full Text] [PDF]
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D. Yang, Q. Chen, S. Stoll, X. Chen, O. M. Z. Howard, and J. J. Oppenheim Differential Regulation of Responsiveness to fMLP and C5a Upon Dendritic Cell Maturation: Correlation with Receptor Expression J. Immunol., September 1, 2000; 165(5): 2694 - 2702. [Abstract] [Full Text] [PDF]
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 C. Paolucci, P. Rovere, C. De Nadai, A. A. Manfredi, and E. Clementi Nitric Oxide Inhibits the Tumor Necrosis Factor alpha -regulated Endocytosis of Human Dendritic Cells in a Cyclic GMP-dependent Way J. Biol. Chem., June 23, 2000; 275(26): 19638 - 19644. [Abstract] [Full Text] [PDF]
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