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and -1ß in Macrophages1
*
Laboratory of Molecular Biology, G. Gaslini Institute, Genova, Italy;
DTP-Tumor Hypoxia Program and
Laboratory of Experimental Immunology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß (MIPs) mRNA
expression in mouse macrophages in a dose- and time-dependent fashion
and through a de novo protein synthesis-dependent process. The
induction by PA occurred within 3 h of treatment and reached a
peak in 12 h. The stimulatory effects of PA were selective for
MIPs because other chemokines, including monocyte chemoattractant
protein-1, RANTES, IFN-
-inducible protein-10, MIP-2, and
macrophage-derived chemokine, were not induced under the same
experimental conditions and were not an epiphenomenon of macrophage
activation because IFN- did not affect MIPs expression. Induction of
both MIP-1 and MIP-1ß by PA was associated with transcriptional
activation and mRNA stabilization, suggesting a dual molecular
mechanism of control. Iron chelation could be involved in MIPs
induction by PA because iron sulfate inhibited the process and the
iron-chelating agent, desferrioxamine, induced MIPs expression. We
propose the existence of a new pathway leading to inflammation
initiated by tryptophan catabolism that can communicate with the immune
system through the production of PA, followed by secretion of
chemokines by macrophages. These results establish the importance of PA
as an activator of macrophage proinflammatory functions, providing the
first evidence that this molecule can be biologically active without
the need for a costimulatory agent. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, CC or ß, C or , and CX3C or 
) and are
encoded by different sets of genes clustered on separate chromosomes
(2, 3, 4). Chemokines are produced by both immune and
nonimmune cells in response to inflammatory stimuli or tissue damage
(5, 6, 7, 8, 9, 10, 11) and are involved in an impressive array of
immunoregulatory and inflammatory functions, including leukocyte
migration and activation (12, 13, 14), myelopoiesis, and
neoangiogenesis (3, 4). A primary source for chemokines are activated mononuclear phagocytes, which are important mediators of cellular immunity against infections and tumors (15, 16), acting directly through the release of effector molecules (15) or indirectly by recruiting T lymphocytes and NK cells to target tissues through the release of chemokines (10, 17). The extent and magnitude of a local macrophage response are regulated by an interplay of stimulatory and inhibitor signals of various nature that include cytokines and metabolites produced by surrounding cells (16), microbial products present in an inflammatory environment (10, 18), or tissue-selective factors, such as changes in O2 tension and pH (19, 20). However, the contribution of stimuli not directly derived from the immune system to the induction of chemokines has not been fully elucidated.
Recent studies have suggested a role for amino acid catabolites as
important signals for mononuclear phagocyte physiology. The metabolism
of two essential amino acids, L-tryptophan
(L-TRP)3
and L-arginine, for instance, has been associated with the
tumoricidal and microbicidal activities of murine macrophages
(21). Enhanced breakdown of L-TRP has been
demonstrated in inflammatory reactions and implicated in antimicrobial
activity, T cell tolerance, and in some of the biological effects of
IFN- (22, 23, 24, 25), and increased expression of indoleamine
2,3-dioxygenase (IDO), the inducible enzyme controlling
L-TRP catabolic pathway in extrahepatic tissues
(26), was detected in inflammatory lesions and placenta
(21, 23, 24, 27). L-TRP catabolism, initiated
either by IDO or epatic enzymes, leads to the production of
metabolites, some of which are biologically active molecules
(21). Among them, PA, an end-product of L-TRP
degradation (28) detected in human milk, pancreatic juice,
and intestine (29, 30), is endowed with important
immunomodulatory properties involving activation of mononuclear
phagocyte effector functions (31). This molecule is a
potent costimulus for induction of macrophage-mediated cytotoxicity,
able to inhibit tumor growth in tumor-bearing mice through the
stimulation of macrophage tumoricidal activity (32, 33).
PA may also contribute to the microbicidal activity of macrophages in
vivo. Intraperitoneal and intracerebral administration of this
metabolite protects mice against a lethal intracerebral challenge with
the opportunistic pathogen Candida albicans
(34). Moreover, PA, in combination with IFN-, inhibits
retrovirus expression in macrophages, both in vitro (35)
and in vivo (31), and triggers the transcriptional
activation of the inducible isoform of the NO synthase gene,
stimulating production of NO (36), a major effector
molecule implicated in the expression of macrophage tumoricidal and
microbicidal activities (37).
Despite the growing body of evidence demonstrating the importance of PA
for the activation of macrophage lytic activities, there is currently
little information on its effects on mononuclear phagocyte
proinflammatory functions. Because increased catabolism of tryptophan
appears to be correlated to the inflammatory response, the present
study was designed to determine whether PA could trigger macrophage
expression of chemokines and thus contribute to the onset of
inflammation. To address this issue, we analyzed the expression pattern
of several - and ß-chemokine mRNA in both mouse peritoneal
macrophages and in the murine macrophage cell line ANA-1 in response to
PA stimulation. The data presented in this study demonstrate for the
first time that PA alone can potently and selectively induce the
coordinated expression of the CC-chemokines macrophage inflammatory
protein-1 (MIP-1) and MIP-1ß (MIPs) mRNA in murine
macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse macrophage cell line ANA-1 was established by infecting bone marrow-derived cells from C57BL/6 mice with the J2 recombinant retrovirus, carrying the v-raf/v-myc oncogenes (38), and was shown to display the phenotypic and functional features and the morphology of well-differentiated macrophages (36, 38). ANA-1 macrophages were cultured in DMEM (ICN Biomedicals, Aurora, OH) supplemented with 10% heat-inactivated FCS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Celbio, Milan, Italy). Peritoneal macrophages were obtained from C57BL/6 mice injected i.p. with 1 ml of 3% thioglycolate broth (Sigma, St. Louis, MO). After 4 days, the peritoneal exudate cells were collected by lavage of the peritoneal cavity with 10 ml of sterile PBS (ICN Biomedicals). Cells were washed, resuspended, and plated in RPMI 1640 (ICN Biomedicals) supplemented as described above. Macrophages were isolated by adherence to tissue culture dishes, and their purity was about 94%, as assessed by morphology on Giemsa-stained cytocentrifuge slide preparations. Viability, determined by trypan blue dye exclusion test, was greater than 99%. Cells were maintained at 37°C in a humidified incubator containing 20% O2, 5% CO2, and 75% N2. For experimental purpose, macrophages were cultured in 15-cm Costar plates (Costar, Cambridge, MA) at 1 x 106 cells/ml, and stimulated for different time points with the indicated factors. Special care was taken to ensure LPS-free conditions in all the experiments.
Reagents
Mouse IFN- (sp. act. 
107 U/mg) was
purchased from Life Technologies (Gaithersburg, MD). LPS (from
Escherichia coli serotype 011:B4) was purchased from Sigma.
PA, desferrioxamine, and ferrous sulfate were from Sigma. During the
course of experiments, several batches of PA were used, and all of them
gave consistent and reproducible results. PA was dissolved in PBS, and
the pH was adjusted to 7.4. The stock solution was then passed through
a 0.2-µm filter, aliquoted, and stored at -20°C. Actinomycin D
(ActD; Calbiochem-Novabiochem, La Jolla, CA) was dissolved in ethanol
at 1 mg/ml and used at a final concentration of 5 µg/ml for the times
specified in the text. Cycloheximide (Sigma) was used at 7.5 µg/ml
final concentration. The content of endotoxin, as determined by assay
with a chromogenic Limulus amebocyte lysate test (QCL-1000;
BioWhittaker, Walkersville, MD), was below the detection limit of 6
pg/ml in all of the reagents used.
RNase protection assay (RPA)
Total RNA was extracted from ANA-1 macrophages using the Trizol
isolation reagent (Life Technologies) and subjected to RPA analysis
using the RiboQuant MultiProbe RPA System from PharMingen (San Diego,
CA), according to the manufacturer’s instruction. Briefly, a
32P-labeled antisense RNA probe set specific for
different mouse /ß-chemokines (Mouse Chemokine Template Set,
mCK-5) was hybridized in excess to 10 µg of total RNA from each
sample in solution, after which free probe and other ssRNA were
digested with RNases. The remaining RNase-protected probes, annealed to
homologous sequences in the sample RNA, were purified by ethanol
precipitation and resolved on denaturing PAGE. Following separation by
PAGE, protected 32P-labeled probe fragments were
visualized by film autoradiography (Kodak XAR-5 films; Eastman Kodak,
Rochester, NY): the presence of the target mRNA in the sample was
revealed by the appearance of an appropriately sized fragment of the
probe. For quantification, autoradiographs were scanned using a light
densitometer (PhosphorImager; Molecular Dynamics, Sunnyvale,
CA).
Northern blot analysis
Total cellular RNA was purified from ANA-1 macrophages and from
thioglycolate-elicited peritoneal macrophages using the Trizol RNA
reagent (Life Technologies), according to the manufacturer’s
instructions. A total of 20 µg of RNA from each sample was
electrophoresed under denaturing conditions on a 1.2% agarose gel
containing 2.2 M formaldehyde, transferred onto Nytran membranes
(Schleicher & Schuell, Keene, NH), and cross-linked by UV irradiation.
Filters were hybridized with 32P-labeled probes
and autoradiographed, as previously described (36).
Different times of exposure were used to obtain comparable levels of
band intensities with different probes. For MIP-1 and MIP-1ß
detection, the mouse MIP-1 and MIP-1ß full-length cDNAs from the
pBR322 vector, kindly provided by Dr. Antonio Sica (Istituto Mario
Negri, Milan, Italy), were used. For MCP-1 detection, the full-length
JE cDNA from the pUC19 vector, obtained from Dr. Antonio Sica, was
used. The pBluescript II SK(-) vector containing
the mouse MDC cDNA was gently provided by Dr. Silvano Sozzani (Istituto
di Ricerche Farmacologiche "Mario Negri," Milan, Italy). The
pEMBL-8 vector containing the ß-actin cDNA was kindly provided by Dr.
Cecilia Garré (Istituto di Biologia e Genetica, Facoltá di
Medicina e Chirurgia, Universitá di Genova, Italy).
Nuclear run-on experiments
Nuclear run-on experiments were performed, as previously
described (36). Briefly, nuclei were isolated from 10
x 107 cells/sample by cell lysis and collected
by centrifugation. In vitro RNA elongation was performed by adding 2x
transcription buffer and 100 µCi of 800 Ci/mmol
[-32P]uridine triphosphate (NEN, Boston, MA)
to the nuclei suspension, incubating the mixture at 29°C for 30 min,
and for additional 10 min at 30°C after addition of
CaCl2 and RNase-free DNase I (Promega,
Gaithersburg, MD). Nuclei were lysed with 1 ml Trizol, and total RNA
was isolated according to the manufacturer’s procedure. Equal amounts
of labeled elongated transcripts were added in Hybrisol solution to
Nytran membranes, on which denatured pBR322 plasmid containing the
full-length MIP-1 cDNA or the full-length MIP-1ß cDNA, denatured
pEMBL plasmid containing the full-length ß-actin, and
EcoRI-linearized pBR322 were immobilized using a dot-blot
apparatus (Bio-Rad Laboratories, Hercules, CA). Hybridization was
performed at 42°C for 48 h, and filters were then washed as
described for Northern analysis. When needed, the autoradiographs were
scanned using a light densitometer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß mRNA
expression in mouse macrophages
Initial experiments were designed to study the effects of PA in
regulating the expression pattern of several - and ß-chemokine
mRNA in the murine macrophage cell line ANA-1. Cells were cultured for
18 h in medium alone, supplemented with PA, or LPS as a positive
control (2, 10). MultiProbe RPA was then performed on
total RNA preparations to analyze the expression of distinct chemokine
transcripts (Fig. 1
). We found little or
no constitutive expression of the mRNA for the ß-chemokines monocyte
chemoattractant protein (MCP-1/JE), MIP-1, MIP-1ß, and
RANTES, and the -chemokines IFN--inducible protein-10 and
macrophage inflammatory protein-2 (MIP-2). PA caused a significant
up-regulation of MIP-1 and MIP-1ß (MIPs) mRNA without affecting
the levels of the other chemokine mRNAs, providing the first suggestion
that PA alone can induce gene expression in macrophages and
differentially modulate the expression of different chemokine mRNA. As
previously reported (2, 10), LPS strongly induced the
expression of the message for all of the chemokines tested.
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A, low constitutive levels of
MIPs mRNA were detectable, although MIP-1ß seemed less expressed than
MIP-1 because longer blot exposure was required to obtain bands of
comparable intensity. PA treatment caused a major enhancement of
MIP-1 and MIP-1ß mRNAs. MIPs mRNA induction, ranging from 8- to
15-fold, was consistently observed in five independent experiments
performed, although slight fluctuations in the baseline levels were
detected. Under the same conditions, PA did not affect the constitutive
expression of MCP-1 mRNA (Fig. 2A). To determine whether
this response was a general consequence of macrophage activation, the
effects of other macrophage activators, IFN- (100 IU/ml) or LPS (10
ng/ml), on MIPs mRNA levels were also investigated. IFN- failed to
increase MIPs expression, while potently inducing MCP-1 mRNA
accumulation, whereas LPS greatly up-regulated the expression of all
three chemokines tested (Fig. 2A).
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B).
Thioglycolate-elicited peritoneal exudate macrophages from C57BL/6 mice
were analyzed to extend the results obtained with ANA-1 cells to
primary macrophage cultures. Control macrophages expressed low
constitutive levels of MIPs mRNA (Fig. 2C). Stimulation with
PA increased MIP-1 and MIP-1ß mRNA accumulation, although to a
lesser extent than in ANA-1 cells. A similar pattern of results was
consistently observed in three independent experiments, with the
induction ranging from 4- to 8-fold over control.
The kinetics of MIP-1 and MIP-1ß mRNA induction by PA is shown in
Fig. 3A. MIP-1 and MIP-1ß
transcripts were detectable as early as 3 and 6 h after treatment,
respectively, reached plateau levels at 12–24 h, and declined
thereafter.
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and MIP-1ß
mRNA expression was assessed after stimulation for 12 h with
increasing amounts of PA (Fig. 3B). Plateau levels of
expression were obtained with 4 mM PA, a dose previously shown to be
optimal for mouse macrophage activation (36). At the
concentrations used, PA did not affect cell viability, as determined by
the trypan blue dye exclusion test (data not shown).
These results provide the first evidence that PA is a potent and
selective inducer of MIP-1 and MIP-1ß mRNA expression in murine
macrophages.
PA augments the transcriptional activity of MIP-1 and MIP-1ß
genes and enhances the mRNA stability of both chemokines
Run-on experiments were conducted on nuclei isolated from ANA-1
cells stimulated for 4 h (Fig. 4A) or 6 h (Fig. 4B) with medium alone, PA, or LPS, to study the mechanism
responsible for MIP-1 and MIP-1ß mRNA induction. MIP-1 gene was
transcriptionally active in medium-treated cells, and susceptible to
augmentation in response to LPS. A 2.6-fold increase in the rate of
MIP-1 gene transcription was observed in cells stimulated with PA
for 4 h (A) or 6 h (B). In contrast,
constitutive transcription of MIP-1ß gene was not detected, and
induction of transcription was not observed after 4 h
(A), but only after 6 h (B) of treatment
with PA. In conclusion, the up-regulation of MIPs mRNA expression by PA
was associated with the transcriptional activation of the genes,
although the kinetics of MIP-1ß induction was delayed compared with
that of MIP-1.
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/ß mRNA induced by PA, ANA-1
cells were incubated in the presence or absence of PA for 12 h,
and mRNA expression was tested immediately or after addition of 5
µg/ml of ActD for the indicated lengths of time to block further RNA
transcription. As indicated in Fig. 5,
MIPs mRNAs decayed with different kinetics in untreated and PA-treated
cells. The levels of MIP-1 and MIP-1ß transcripts in unstimulated
macrophages decreased by 50%
(t1/2) after 2.5 and 1.2
h, respectively. PA-treated cells displayed a greater MIPs mRNA
stability. The t1/2 of MIP-1 mRNA
was increased to more than 8 h, whereas that of MIP-1ß was
increased to 2 h by PA. In addition, 30% of MIP-1ß mRNA was
still detectable after 8 h of exposure to ActD in PA-treated
cells, whereas in control cells it became undetectable after 4 h
of exposure to the drug. We concluded that PA stabilized MIP-1 and
to a lesser extent MIP-1ß mRNAs.
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and MIP-1ß
mRNA expression (Fig. 6). Similar results
were observed in three independent experiments.
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and MIP-1ß mRNA in ANA-1 macrophages is controlled
through a dual molecular mechanism involving both the transcriptional
activation of the genes and the posttranscriptional stabilization of
the messages, and that it requires active protein synthesis. Iron chelation is involved in PA-dependent induction of MIPs gene expression
It has been previously reported that the biological effects of PA
involved chelation of iron (41, 42). To determine the
requirement for iron chelation in PA-dependent induction of MIPs
expression, ANA-1 cells were stimulated for 12 h with PA, alone or
in the presence of increasing concentrations of
FeSO4, and the expression of MIP-1 and
MIP-1ß mRNA was analyzed. One representative experiment of three
performed is depicted in Fig. 7A. The addition of
FeSO4 caused a major and dose-dependent
suppression of MIPs mRNAs in PA-stimulated macrophages, detectable at
100 µM and maximal at 300 µM. FeSO4 alone did
not have any effect on the expression of MIPs mRNA at any of the
concentrations tested (Fig. 7A). We conclude that chelation
of iron was part of the mechanism of action of PA.
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and MIP-1ß expression. ANA-1 macrophages were
treated with DFX, and the expression of MIPs mRNA was then tested (Fig. 7B). DFX selectively increased MIP-1 and MIP-1ß, but
not MCP-1, mRNA expression (10- to 15-fold above the baseline), and
addition of FeSO4 caused a reduction in
DFX-mediated MIPs up-regulation greater than 90%.
These results show that DFX is a stimulus for the induction of MIP-1
and MIP-1ß in macrophages and that FeSO4
inhibits the activating properties of PA and DFX, indicating that iron
chelation is involved in the regulation of MIPs expression in
macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß mRNA in murine macrophages.
The murine macrophage cell line ANA-1 expressed low constitutive levels
of MIP-1 and MIP-1ß mRNA, and PA caused a major up-regulation of
their expression. MIP-1 increased earlier than MIP-1ß, but both
chemokines reached plateau levels 12 h after PA stimulation.
Slight fluctuations in the baseline levels of MIPs mRNA were observed,
probably due to cell adherence to plastic (16). However,
the constitutive levels of MIP-1ß were lower than those of MIP-1,
even if PA induced a comparable up-regulation of the two transcripts.
Similar results were observed using fresh murine peritoneal exudate
macrophages, excluding an idiosyncratic behavior of the ANA-1 cell
line. Interestingly, PA stimulatory activity was selective for MIP-1
and MIP-1ß because it was not exerted on several other ß- and
-chemokines, including MCP-1, RANTES, IFN--inducible protein-10,
MIP-2, and MDC. Furthermore, the augmentation of MIPs mRNA expression
by PA was not a general response to macrophage stimulation. In fact,
IFN-, a potent activator of mononuclear phagocyte functions
(16, 36), did not increase MIP-1 or MIP-1ß mRNA,
despite its ability to induce MCP-1 expression. We can rule out the
possibility that endotoxin contamination contributed to the activation
of MIPs expression, because PA preparations did not contain any
detectable levels of LPS, and addition of polymyxin B sulfate, which
binds to and neutralizes LPS (44), did not reduce PA
stimulatory activity (data not shown). Moreover, other chemokines
besides MIPs were strongly induced by LPS, but not by PA. The
demonstration of MIPs mRNA augmentation is the first evidence of the
ability of PA alone to trigger gene expression in macrophages without
the need for a costimulus.
Transcriptional activation is the primary mechanism of regulation of
chemokine gene expression in different cell types (18, 45, 46, 47). Transcriptional induction of MIP-1 and MIP-1ß gene
expression in macrophages has been previously reported (3, 19, 48). We found that MIP-1 gene was transcriptionally active,
and that stimulation with PA induced a 2.6-fold increase in its
transcription already after 4 h. In contrast, activation of
MIP-1ß gene transcription by PA was delayed and became evident only
6 h after stimulation. These results provide the first evidence
that PA by itself can induce gene transcriptional activation, and raise
the issue of the promoter element(s) involved.
MIPs induction by PA is also due to the stabilization of the mRNA that
decayed at a slower rate in PA-treated macrophages than in control
cultures. These results are consistent with the findings that MIP-1
and MIP-1ß contain variants of the AUUUA consensus sequence in their
3' untranslated region (3, 49), present in the 3' UTR of
many cytokine genes, and controlling mRNA stability and translation
(50, 51, 52). Posttranscriptional regulation of MIP-1 and
MIP-1ß mRNA expression in macrophages has been previously shown
following stimulation by bacterial endotoxin (49), in
response to oxidative stress (19) or IL-10
(47).
PA-dependent increase of MIPs mRNA was abolished by addition of protein-synthesis inhibitors, demonstrating a requirement for active protein synthesis and suggesting that MIPs expression may be controlled by a de novo synthesized transcriptional enhancer(s) and/or by a factor(s) with a short t1/2 involved in the regulation of mRNA stability.
The mechanism of action of PA is not known, although it has been
reported that this agent interferes with several biochemical pathways
(31). PA binds iron and interferes with iron uptake
(31, 41), and iron chelation may contribute to PA
activities (41, 42). In this study, we show that iron
sulfate suppressed PA-induced MIP-1 and MIP-1ß mRNA up-regulation,
suggesting a role for iron chelation in the induction of MIPs. This
conclusion is supported by the demonstration that DFX, a synthetic iron
chelator (53, 54), is similar to PA in selectively
inducing MIP-1 and MIP-1ß mRNA, and that this effect is abrogated
by iron sulfate. Induction of MIP-1 and MIP-1ß by DFX may have
practical clinical implications because this drug is used for the
treatment of several pathological conditions, including iron overload
(54), cancer (55), and Alzheimer’s disease
(53), and, interestingly, several lines of evidence
indicate that chemokines might contribute to the pathogenesis of these
diseases (3).
In conclusion, the results presented in this study identify a novel
connection between L-TRP degradation, its end-product PA,
and the expression of MIP-1 and MIP-1ß chemokines in macrophages,
extending previous findings on the important role of
L-TRP catabolism in regulating host immune mechanisms
and inflammatory processes (21, 22, 25, 27, 56, 57).
Expression and activation of IDO, a key enzyme in the degradation of
L-TRP along the kynurenine pathway (26), in
placenta and human macrophages have been associated with inhibition of
T cell immune responses (24, 58). To which extent IDO
activation leads to PA production and whether such pathway is relevant
in mouse macrophages are currently being investigated. A recent report
demonstrating that PA significantly decreases IDO expression and
activity in mouse macrophages (59) suggests a role for
this catabolite as an inhibitor of IDO-mediated T cell
immunosuppression in local tissue microenvironment, and is consistent
with our results showing the importance of PA in the activation of
macrophage proinflammatory functions.
The levels of PA detected in vivo in biological fluids varies from 3 (30) to 300 µM (29), and we have measured micromolar concentrations of PA (from 10 to 80 µM) in the serum of patients with chronic liver diseases (Dazzi, et al., submitted manuscript4), although the concentrations of PA in tissues are still unknown. The stimulatory effects of PA in vitro require concentrations of about 2–4 mM (36 and this study), and our unpublished observations indicate that millimolar concentrations of PA are necessary in vitro to achieve detectable intracellular quantities of PA in ANA-1 macrophages (detection limit 100 pM). Levels of PA higher than those detected in human serum may be achieved in vivo in the intercellular space or could be locally produced during inflammatory responses, as suggested by elevation of L-TRP catabolites in localized compartments under pathologic conditions (60). Moreover, it is possible that the in vivo environment may alter the accessibility of PA to the cell, decreasing the biologically active concentration. In fact, the association of PA with serum proteins and/or with divalent cations (41) may modify the permeability of the compound. Finally, the possibility exists that PA may act endogenously in the producing cells and that its biologic activity may not correlate with the circulating levels.
MIP-1 and MIP-1ß are important mediators of the inflammatory
reactions (3, 12, 13) and potent T cell chemoattractants,
able to regulate T cell trafficking during an immune response in vivo
(3, 4, 14, 61). The modulation of MIPs expression is
a crucial set point for the control of the kinetics and composition of
the cellular infiltrate in target tissues (3, 17). The
demonstration that PA can induce MIP-1 and MIP-1ß expression
indicates that this molecule can regulate the inflammatory response at
multiple levels. In a simplistic view, PA alone elicits MIPs chemokine
expression in macrophages and, consequently, T cell recruitment, and,
in conjunction with T cell products such as IFN-, triggers the fully
activated phenotype.
It was recently demonstrated that MIP-1 and MIP-1ß can
inhibit HIV-1 infection by binding to their receptor CCR5, which
functions as a cell membrane fusion cofactor with CD4 for the virus
gp120 envelope protein, and, thus, by blocking HIV-1 entry into the
cells (62, 63). Schmidtmayerova et al. (64)
showed elevated levels of MIP-1/ß expression in HIV-1-infected
monocytes and in microglia and astrocytes from the brain tissue of
patients with HIV encephalitis. On the other hand, decreased levels of
circulating L-TRP and accumulation of L-TRP
catabolites, such as kynurenine and quinolinic acid, have been detected
in the cerebrospinal fluid of patients infected with HIV-1,
particularly in those with neurologic deficits and AIDS dementia
complex (25, 65). These results raise questions on the
role of L-TRP catabolites in AIDS, particularly of PA that,
by up-regulating MIPs expression, could exert protective effects on the
host. Evaluation of PA levels in the fluids of AIDS patients is
currently underway.
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Acknowledgments |
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and MIP-1ß full-length cDNAs and the
pUC19 vector containing the full-length JE cDNA; Dr. Silvano Sozzani
(Istituto di Ricerche Farmacologiche "Mario Negri") and Dr. Cecilia
Garré (Institute of Biology and Genetic (IBiG), Facoltá di
Medicina e Chirurgia, Universitá di Genova, Genova, Italy) for
kindly providing the pBluescript II SK(-) vector
containing the mouse MDC cDNA and the pEMBL-8 vector containing the
ß-actin cDNA, respectively. We also thank Fulvia Lerone for editing
the manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Maria Carla Bosco, Laboratorio di Biologia Molecolare, Istituto Giannina Gaslini, L.go Gerolamo Gaslini 5, 16147 Genova Quarto, Italy. E-mail address:
3 Abbreviations used in this paper: L-TRP, L-tryptophan; ActD, actinomycin D; CHX, cycloheximide; DFX, desferrioxamine; IDO, indoleamine 2,3-dioxygenase; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; PA, picolinic acid; RPA, RNase protection assay.
4 C. Dazzi, G. Candiano, A. Ponzetto, and L. Varesio. A new HPLC method for the detection of picolinic acid, a catabolite of L-tryptophan, in biological fluids. Submitted for publication.
Received for publication August 27, 1999. Accepted for publication January 3, 2000.
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