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with a Th2-Associated Expression Pattern1
Klinik und Poliklinik für Dermatologie, Universitätsklinikum Benjamin Franklin, Freie Universitat Berlin, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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with a cDNA and protein sequence homology
of 55% and 59%, respectively. However, the expression pattern of
AMAC-1 is directly opposite to that of MIP-1. While MIP-1 is
induced by classical macrophage mediators such as LPS and is inhibited
by IL-4 and glucocorticoids, AMAC-1 is specifically induced in
macrophages by alternative macrophage mediators such as IL-4, IL-13,
and IL-10. Expression of AMAC-1 is inhibited by IFN-
while
glucocorticoids exert a slightly positive synergistic effect in
combination with IL-4. Peripheral blood monocytes do not express
AMAC-1; time course experiments show that monocyte-to-macrophage
differentiation is a prerequisite for AMAC-1 expression. Expression of
AMAC-1 by granulocyte--macrophage CSF/IL-4-induced, monocyte-derived
dendritic cells is complex; in mature adherent dendritic cells,
however, only minor AMAC-1 mRNA expression was found. In vivo, AMAC-1
is expressed by alveolar macrophages from healthy persons, smokers, and
asthmatic patients. In conclusion, AMAC-1 is a novel CC-chemokine whose
expression is induced in alternatively activated macrophages by
Th2-associated cytokines; thus, AMAC-1 may be involved in the
APC-dependent T cell development in inflammatory and immune
reactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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and LPS, activation of macrophages by
agents such as IL-4 or glucocorticoids
(GCs),3 was classified as alternative.
Besides IFN- and IL-4, macrophage activation and differentiation is
influenced by other cytokines. The effects of TNF- and IL-12
partially overlap with those of IFN-. IL-10 and IL-13 resemble IL-4,
while TGF-ß acts similarly to GC. Nevertheless, IFN- and IL-4 are
the agents best known to exert a wide range of antagonistic effects on
macrophages; for example, expression of the three species of FcRs
(2, 3) is induced by IFN-, but is inhibited by IL-4. On the other
hand, expression of the macrophage mannose receptor (1, 4) and of
15-lipoxygenase (5) is induced by IL-4, but is inhibited by IFN-.
Furthermore, expression and synthesis of proinflammatory cytokines such
as IL-1 (6, 7), IL-6 (8), and TNF- (6, 7) are inhibited by IL-4. In
contrast, IL-4 and IFN- sometimes exert synergistic effects such as
accumulation of cytoplasmic CD23 (9) or inhibition of CD14 expression
(10). Vice versa, GC effects on macrophages are sometimes antagonistic
to IL-4; in contrast to IL-4, GCs, for example, inhibit macrophage
expression of CD23 (9, 11). In general, IL-4-induced inflammatory
macrophages adopt an alternative phenotype characterized by a high
capacity for endocytic clearance and by reduced proinflammatory
cytokine secretion (1).
In previous reports, we have shown that MS-1 high m.w. protein
(MS-1-HMWP) (9, 12, 13, 14) and RM 3/1 Ag (15, 16) characterize alternative
macrophage phenotypes (17) in that their expression is induced by IL-4
and GC and inhibited by IFN- (9, 18). In vivo,
MS-1-HMWP+, RM 3/1+ alternatively activated
macrophages are found during the healing phase of acute inflammatory
reactions (19), in chronic inflammatory diseases such as rheumatoid
arthritis (20) and psoriasis (21), and in wound healing tissue (9). In
addition, alternatively activated macrophages are the cells of origin
in cutaneous macrophage-derived tumors (14, 18). In contrast to
IFN--induced classically-activated macrophages that occur during
early phases of inflammation (19) and in high turnover reactive
granulomas (14, 18, 22), alternatively activated macrophages are
associated with a high degree of vascularization in vivo (9, 14, 18)
and seem to be angiogenic in vitro (23). Furthermore, alternatively
activated macrophages do not costimulate, but actively inhibit
mitogen-induced proliferation of PBL and CD4+ T cells by an
unknown mechanism (24). Therefore, we reasoned that alternatively
activated macrophages may host a not yet fully unraveled molecular
repertoire important in modulating diverse inflammatory and immune
responses.
Here, we report the identification, cloning, and expression analysis of a novel CC-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1, induced by Th2-associated cytokines such as IL-4, IL-13, and IL-10 in alternatively activated macrophages in vivo and found in alveolar macrophages in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Skin and bowel specimens were taken at routine surgery and bronchoalveolar lavage cells were harvested at routine BAL after informed consent was obtained. Cell lines used were monocytic leukemia cell line THP-1 and melanoma cell line MEWO cultured in RPMI 1640 supplemented with 10% FCS (Biochrom, Berlin, Germany) and appropriate concentrations of penicillin/streptomycin, glutamine, and nonessential amino acids (all from Biochrom); human umbilical vein endothelial cells were isolated as described (25) and cultured in Clonetics endothelial basal medium (Clonetics, Walkersville, MD) supplemented with 10 µg/ml human epidermal growth factor, 1 µg/ml hydrocortisone, bovine brain extract 12 µg/ml, 2% FCS and appropriate concentrations of gentamicin and amphotericin B. Isolation and culture of human monocytes/macrophages and monocyte-derived dendritic cells as well as PBL was performed largely as described (9, 18, 26). The cells were purified using EDTA-anticoagulated blood from single donors or pooled buffy coats. A total of 35 ml of blood were layered on top of 15 ml Ficoll-Paque (Biochrom) in a 50-ml Leuco-sep centrifuge tube (Greiner, Nürtingen, Germany) and were centrifuged in a swing out rotor for 40 min at 650 x g at room temperature without using the brake. PBMC were collected at the serum/Ficoll interface and washed three times in sterile Ca2+ and Mg2+ free PBS (Biochrom). 3 ml of a suspension containing 5 to 8 x 108 PBMC in PBS were layered on top of 30 ml of a preformed Percoll gradient (Pharmacia, Freiburg, Germany) in a 50-ml tube; the Percoll gradient (13.5 ml Percoll, 1.5 ml 10x Earle’s MEM, 15 ml Spinner’s medium supplemented with appropriate concentrations of penicillin/streptomycin, glutamine, and nonessential amino acids (all from Biochrom)) had been preformed in an SS-34 rotor of an ultracentrifuge (Sorvall, Frankfurt, Germany) at 11950 x g for 12 min at 20°C without using the brake. Percoll gradients with the cells on top were centrifuged at 650 x g for 40 min at 20°C without using the brake. The upper cell layer contained approximately 80 to 90% monocytes while the lower cell layer contained approximately 90 to 95% PBL. Both layers were collected separately, and the cells were washed three times in PBS and further used or frozen in liquid nitrogen.
For culture, monocytes were resuspended in McCoy’s medium (Biochrom) supplemented with 10% FCS and appropriate concentrations of penicillin/streptomycin, glutamine, and nonessential amino acids, and were then transferred into Teflon-coated, UV-irradiated (1 min; transiluminator 2011 macrovue (LKB, Germany) plastic bags (Biofolie, Heraeus, Hanau, Germany), which were sealed using Polystar 410 HM (Rische und Herfurth, Hamburg, Germany). Cell numbers varied between 0.2 x 106 and 2 x 106 (usually 1.3 x 106) monocytes/ml according to the length of the culture period (3 and 6 days). Mediators were added directly to the medium in the combinations indicated at the beginning of the culture period or at the time points indicated. In some experiments, monocytes were also cultured in T75 tissue culture plastic flasks (Falcon). Incubation was at 7.5% CO2. Before harvest, the plastic bags were put on ice for at least 30 min and were lightly hit with a stick for a while to get the lightly adherent cells back into suspension. The bags were cut open, the supernatant was collected and the cells were washed in PBS and either used directly for coculture, frozen as pellets for RNA isolation, or used for flow cytometric analysis or to prepare cytospin preparations in a cytocentrifuge at 700 rpm for 3 min using 104 cells/cytospin. Monocytes cultured in plastic flasks were harvested using trypsin-EDTA and were otherwise treated similarly.
Mediators
Human IFN-, used at 1000 U/ml, was from Sigma (Deisenhofen,
Germany); human rTNF-, used at 20 ng/ml, and human recombinant
GM-CSF, used at 200 U/ml, were from Tebu Peprotech (Frankfurt/M.,
Germany); human rIL-2, used at 100 U/ml, was from Chiron (Ratingen,
Germany); human rIL-3, used at 10 ng/ml, human rIL-6, used at 200 U/ml,
human rIL-12, used at 100 U/ml, human rIL-13, used at 50 ng/ml, and
human rIL-1, used at 100 U/ml, were from R&D Systems (Wiesbaden,
Germany); human recombinant macrophage CSF (M-CSF), used at 100 U/ml,
was from Cellular Products (Buffalo, NY); human rIL-4, used at 15 ng/ml
for induction of alternative macrophage differentiation and at 45 ng/ml
for derivation of dendritic cells from monocytes, was from Serva
(Heidelberg, Germany); human rIL-10, used at 50 ng/ml, was from Biomol
(Hamburg, Germany). Dexamethasone, used at 5 x 10-7
M or as indicated, and LPS from Escherichia coli serotype
055:B5, used at 25 µg/ml, were from Sigma.
Isolation of cDNA clones
Total RNA from alternatively activated macrophages was isolated
using Stratagene isolation kits. The poly(A)+ fraction was
separated with Oligotex-dT (Qiagen, Hilden, Germany) and 2 µg were
used for synthesis of cDNA library using 
ZAP Express
EcoRI/XhoI vector cloning kit (Stratagene,
Heidelberg, Germany). The library consisted of 1.2 x
105 original clones, 86% of which contained inserts. This
first cDNA library was amplified up to a titer of 2.5 x
108 pfu/ml. The resultant alternatively activated
macrophage cDNA library and a commercially available human spleen cDNA
library (gt10, Clontech, Heidelberg, Germany) were differentially
screened as follows.
A total of 2 x 106 pfu of each cDNA library were
plated on NZY plates and three replicas on Nylon filters (Millipore,
Eschborn, Germany) or GeneScreen (DuPont/NEN/Life Science,
Cologne, Germany) were taken. For differential screening,
32P-labeled first cDNA was prepared using 2 to 5 µg
poly(A)+ from alternatively and classically activated
macrophages and from control macrophages. Poly(A)+ was
isolated with use of Oligotex-dT and was reverse transcribed overnight
at 42°C in 1 mM dATP, dGTP, dTTP and 0.05 mM dCTP 200 µg/ml
oligo(dT)18-mer (Pharmacia), 10 U AMV reverse transcriptase
(Invitrogen, Leek, The Netherlands), 60 U RNasin (Ambion, Austin, TX),
and 125 µCi [-32P]dCTP with a specific activity of
800 µCi/mmol (Amersham Buchler, Braunschweig, Germany).
Unincorporated radioactivity was separated using Sephadex-25-filled
NAP-5 columns (Pharmacia). Filters were prehybridized for 2 to 6 h
at 42°C in 10% dextransulfate, 5x Denhardt’s solution, 2x SSC,
1% SDS, 50% deionized formamide, and 100 µg/ml salmon sperm DNA.
For hybridization, denatured labeled first cDNA probes were added and
hybridized for 2 days at 42°C. After hybridization, the filters were
washed twice in 2x SSC, 1% SDS at room temperature, twice in 2x SSC,
1% SDS at 65°C for 30 min, and one final wash was in 0.1 x SSC at
room temperature. Filters were dried, put into saran wrap, and exposed
to X-AR Film (Kodak, New Haven, CT). After the exposure, differentially
expressed clones were picked. After amplification of these clones,
DNA was isolated using a commercially available kit (Qiagen) and was
PCR-amplified with T3/T7 primer for ZAP clones and gt10
forward/reverse primers for spleen cDNA clones using GeneAmp XL PCR kit
(Perkin Elmer, Branchburg, NJ). In order to identify the extreme 5' end
of the AMAC-1 mRNA, 5'RACE system version 2.0 (Life Technologies,
Eggenstein, Germany) was used with the following primers: REV (5'-TCA
CAG TGA GAA TGC TGG TTT ACC TTT TAT; 751–780); REV1 (5'-GAG TTG AAG
GGA AAG GGG AAA GGA TGA TAA; 584–613); REV3 (5'-CTC CAG GGT GGC AGG
GCC ATT GCC CT; 406–431) (see Fig. 1
).
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). The sequencing reaction was separated on 6%
polyacrylamide/urea sequencing gels. Northern hybridization
Multiple tissue Northern blots of normal organs including
lymphoid tissues were purchased from Clontech. For macrophages and some
other cell lines and tissues, total RNA was isolated using RNA
Midi-Isolation kit (Qiagen). Ten micrograms of total RNA were
electrophoresed in formaldehyde/agarose gels and blotted onto nylon
membrane Gen screen plus (DuPont). After 2 h at 80°C, filters
were hybridized with an AMAC-1 specific 200-bp PCR-generated DNA probe
(primers used were FORW and REV; see Fig. 1). The probe was labeled
using a ready-to-go random priming kit (Pharmacia) with 50 µCi
[-32P]dCTP with a specific activity of 6000
µCi/mmol (Amersham Buchler), and nonincorporated radioactivity was
separated using G50 Sephadex-filled Nick columns (Pharmacia). The
filters were prehybridized for at least 2 h in 1 M NaCl, 1% SDS,
and 10% dextran sulfate at 60°C. Denatured probe and 100 µg/ml
salmon sperm DNA were then added and hybridized at 60°C overnight.
After hybridization, the filters were washed twice in 2x SSC, 1% SDS
at room temperature, twice in 2x SSC, 1% SDS at 60°C for 30 min,
and the final wash was in 0.1 SSC at room temperature. Filters were
dried, put into SaranWrap, and exposed to X-AR film.
RT-PCR
Total RNA was isolated using Trizol (Life Technologies), and
first-strand cDNA was synthesized using commercially available kits
(Pharmacia; or Invitrogen, Leek) according to the manufacturers’
protocols. Several primer pairs were tested. Of these, putative
intron-spanning primer pair FORW5 and REV (see Fig. 1) turned out to
give the most specific and reliable results. Primers were tested for
exclusion of amplification of genomic sequences using 200 ng and 1 µg
genomic DNA from PBMC (not shown). PCR was performed in a 50-µl total
reaction volume containing 25 to 250 ng cDNA templates, 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 1.25 U of
Amplitaq DNA polymerase (Perkin-Elmer Cetus, Überlingen,
Germany), and oligonucleotide primers (0.2 µM each). The
amplification profile for the primer pair was: 95°C, 45 s;
60°C, 45 s; 72°C, 2 min for 40 cycles. Amplification was
terminated by 10 min of final extension at 72°C. Twenty-microliter
aliquots of the resulting PCR reaction mixture were separated on
ethidium bromide-stained 1.5% agarose gels (Life Technologies) by
electrophoresis. Gels were photographed under UV light (Polaroid 665
film, Sigma). Specific signals were adjusted according to expression of
housekeeping genes ß-actin or glyceraldehyde-3-phosphate
dehydrogenase.
Bioinformatics
The DNA sequence and the deduced protein sequence of the clone were compared with public databases using the internet programs BLASTN and BLASTP at http://www.ncbi.nlm.nih.gov to find similar sequences. The database set was a nonredundant combination of GenBank, EMBL, DDBJ, PDB, dBEST, and SWISSPROT sequences (27).
Sequences of known cytokines were obtained from the ENTREZ server at National Centre for Biotechnology Information at http://www.ncbi.nlm.nih.gov/Web/Search/index.html. The sequences were then aligned using the ClustalW program (28) with standard parameters, and the resulting data set was imported into the GeneDoc4 program. The phylogenetic trees were drawn with Treeview5 using the guide tree file of the ClustalW program. Modeling of the three-dimensional structure of AMAC-1 was possible using the Swissmodel server at ExPasy (http://www.expasy.ch/swissmod/SWISS-MODEL.html.) (29, 30) based on the known structures of the following sequence entries from the Brookhaven database: 11HUM.pdb (HS_MIP-1ß), 11DOK.pdb, 11MCA.pdb, 11DOM.pdb, 11DOL.pdb, 13IL8.pdb (HS_IL8), 11IKL.pdb, and 11HRJ.pdb. The obtained coordinates for AMAC-1 were visualized with the Rasmol program (Roger Sayle, Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, U.K.).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-induced,
classically activated macrophages and of IL-4- and GC-induced,
alternatively activated macrophages using differential hybridization,
several clones were found to be specifically expressed in alternatively
activated macrophages. The greater part of these clones showed positive
cross-hybridization reactions using Southern blotting, and a high
frequency of cross-hybridizing clones was detected in our cDNA library
made from alternatively activated macrophage RNA, but not in a spleen
cDNA library. Several of these cross-hybridizing clones were sequenced
from their 5' and 3' ends and from various internal primers and showed
partially overlapping sequences. In order to identify the extreme 5'
end of the respective mRNA, 5' RACE products were also sequenced.
Taking all this sequence information together, a contig for a sequence
of 803 bp was constructed. Finally, a full length clone (ZAP Express
clone 202) was identified and fully sequenced from both ends and using
several internal primers. The resulting sequence fully confirmed the
previously established contig sequence (Fig. 1; accession no. Y13710).
The isolated clone showed an open reading frame of 267 bp coding for 89
amino acid residues. An analysis for the presence of a signal peptide
sequence gave three possible cleavage points after residues 18, 20, or
21 with the highest probability for cleavage between residues 20 and 21
resulting in an N terminus for the mature protein with the sequence M k
g l a a a l l v l v c t m a l c s c || A Q V (Fig. 1). Further
sequence analysis pointed toward a possible CC-chemokine of 69 amino
acid residues and a predicted m.w. of 7855.
By scanning the sequence against a nonredundant database set on the
internet, the highest homology to our clone was found with the human
and mouse DNA sequences of the CC-chemokine MIP-1 (55% and 51%,
respectively). Several expressed sequence tags (ESTs) and other cDNA
clones (accession nos. R83915, T89961, AA031820, AA031821, N44551,
N33793, and G907797) with very high homology to our sequence were also
found; however, our sequence is the longest reported to date. When
using only the coding region of our cDNA sequence from nucleotide 71 to
337, a perfect match of the protein sequence to protein sequence 3 from
U.S. patent No. 5504003 (accession no. I19356) was found. This protein
sequence was tentatively named macrophage inflammatory protein (MIP)-4
(31). However, we prefer to use the term alternative macrophage
activation-associated CC-chemokine (AMAC)-1 for our clone since we feel
that it better reflects the expression pattern of our gene that is
opposite to other macrophage inflammatory proteins, especially,
MIP-1.
In order to identify the relationship of AMAC-1 to other known
chemokines, an alignment of related sequences obtained from a BLAST
search was made with ClustalW and processed in Treeview and Genedoc.
The resultant dendrogram of protein sequences of known CC-chemokines
(Fig. 2A) shows that the
strongest relationship of AMAC-1 is to human and mouse MIP-1 (59%
and 51% homology, respectively). The next closest similarity of AMAC-1
is to human and mouse MIP-1ß (39% and 45% homology, respectively).
Together with MIP-1, MIP-1ß, RANTES, and macrophage-derived
chemokine (32), AMAC-1 constitutes one group of CC-chemokines that is
clearly separated from two other groups in the dendrogram; one of these
two latter groups comprises human HCC-1 (33), MIP-3/MPIF-1 (34),
MIP-3, MIP-3ß (35), MIP-5/HCC-2 (accession no. R91733) and murine
C10 (36), and MIP-1 (37) while the other comprises human eotaxin and
MCP-1, -2, -3, and -4 (38). Between these groups, overall homology is
in the 25% range. An optimized alignment of these chemokines together
with the human CXC-chemokine IL-8 (Fig. 2B) reveals six
absolutely conserved positions in both CC- and CXC-chemokines
comprising the four cysteines, Val-58, and Leu-65 (numbered according
to mature AMAC-1 protein and marked in blue). Within the mature
proteins, there are 14 residues more or less well conserved among the
CC chemokines (green). These include regions of putative importance for
dimerization and structure determination (i.e., WV motive at position
57, 58) (39, 40).
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C). The
structure-determining residues as well as the four cysteines are well
conserved in their position in the three-dimensional structure between
AMAC-1 and MIP-1ß resulting in conservation of all decisive
structural features between these two chemokines. These features are
the 310-helical turn, the three antiparallel ß-sheets
ß1, ß2, and ß3 as well as the C-terminal -helix.
With respect to receptor selectivity of chemokines, a two-site paradigm
has been postulated (41). Site I serves initial complex formation
(address) while site II is important for agonist activity of the ligand
(message). The region of AMAC-1 known from MIP-1ß to be directly or
indirectly involved in interaction at site I is centered around Tyr27
(including also Asp26, Glu29, and Ser32) and is highly conserved in
most CC-chemokines including AMAC-1 (40). In CXC-chemokines such as
IL-8, Tyr27 is uniformly replaced by leucine. This receptor recognition
site is located within ß-sheet ß1 opposite to the dimerization
region preceding the 310-helical turn. Alteration of this
sequence motif by site-directed mutagenesis influences receptor
specificity in case of IL-8 (41). In contrast, the flexible N terminus
of AMAC-1 involved in agonist activity at site II is considerably
different from all other CC-chemokines and rather resembles the N
terminus of CXC-chemokines such as IL-8 including an incomplete ELR
motif and a leucine/isoleucine at position 12 vs a
tyrosine/phenylalanine in other CC-chemokines (AMAC-1:
TNKEL-C-CL; IL-8:
SAKELRCQCI; MIP-1:
DTPTA-C-CF).
Expression of AMAC-1 was studied by Northern analysis in a variety of
cultured cells and tissues and was found highly specific for
alternatively activated macrophages (Fig. 3A). In contrast, most
chemokines including MIP-1 expressed by macrophages are induced by
classical macrophage activators such as LPS (not shown) or IFN-,
and, interestingly, MIP-1 with its high homology to AMAC-1 is
frankly inhibited by alternative macrophage activators such as IL-4,
IL-13, and GC (42, 43, 44). Classically activated macrophages, lymphocytes,
endothelial cells, melanoma cells, and normal human organs do not
express AMAC-1 (Fig. 3A). However, in some specimens,
especially endothelial cells and melanoma cells, weak
cross-hybridization was seen with an as yet unidentified mRNA species
of 1350 bp. RT-PCR with carefully selected putative intron-spanning
AMAC-1-specific primers revealed only the expected single band in
alternatively activated macrophages (Fig. 3B). Due to the
high sensitivity of RT-PCR, weak AMAC-1 expression was also detected in
some unstimulated control macrophage specimens and very weakly in
spleen.
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A). In contrast to
macrophage migration inhibitory factor (45), GC do not induce AMAC-1
even if they are used at lower concentrations than usual
(10-8 to 10-16 M) (Fig. 3C). In
contrast to other chemokines such as MIP-1 (44) and to other
proinflammatory cytokines such as IL-1 or TNF-, AMAC-1 expression is
not inhibited by GC.
In time course experiments, AMAC-1 expression by alternatively
activated macrophages was shown to start only at day 2 and to be fully
developed at day 3 when IL-4 was present throughout the culture period
(Fig. 3D). When IL-4 was added only at day 2 of culture,
expression of AMAC-1 was nevertheless fully developed at day 3 (Fig. 3E) indicating that monocyte-to-macrophage differentiation
is a prerequisite of AMAC-1 expression. This is also reflected by the
finding that expression of AMAC-1 by the monocytic leukemia cell line
THP-1 requires preceding induction of macrophage differentiation by
phorbol esters (Fig. 3B). Similarly, even monocytes of
atopic patients with high serum IgE levels do not express AMAC-1 (not
shown). Besides IL-4, other mediators of alternative immunologic
macrophage activation such as IL-13 and IL-10 are able to induce
expression of AMAC-1 while all other cytokines tested such as IFN-,
IL-1, IL-2, IL-3, IL-6, IL-12, macrophage-CSF, GM-CSF, and TNF- are
not (Fig. 4A). Besides lacking
correspondence in the homology-based CC-chemokine dendrogram, these
findings confirm that C10 is not the murine homologue of human AMAC-1,
since C10 is inducible by IL-3 and GM-CSF (36) in addition to IL-4. On
the other hand, IFN- strongly inhibits expression of AMAC-1 (Fig. 4B), while TNF- does rather enhance AMAC-1 expression in
IL-4- and GC-stimulated macrophages (Fig. 4D). This latter
result is unexpected, since expression of alternatively activated
macrophage Ags MS-1-HMWP and RM 3/1 is inhibited by both IFN- and
TNF- (9, 18).
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A), while expression of AMAC-1 at days 6 and 8 was less
intense and was further down-regulated by addition of TNF- (Fig. 4C).
In addition to cultured human cells, a variety of inflamed human
tissues was examined for expression of AMAC-1 including acute
appendicitis, Crohn’s disease, ulcerative colitis, lesional skin from
psoriasis, and bronchoalveolar lavage cells from various donors. Only
alveolar macrophages from asthmatics, smokers and healthy persons, but
not inflammatory skin and bowel disease specimens strongly expressed
AMAC-1 in vivo (Fig. 4E).
During preparation of this manuscript, Adema et al. (47) reported on the identification and analysis of a novel CC-chemokine, DC-CK1, which was cloned from a dendritic cell-derived cDNA library. Based on a limited expression analysis, Adema et al. claim that DC-CK1 is a dendritic cell-selective chemokine in vitro and in vivo and they show that it functions in selectively attracting and activating naive T cells. Summarizing, Adema et al. hold that DC-CK1 is part of the potent immunostimulatory armament of dendritic cells during the development of naive T cells into Th1 effector cells.
Interestingly, DC-CK1 and AMAC-1 show sequence identity. Insofar, the
data presented in this paper help to broaden and correct the
oversimplified view of DC-CK1/AMAC-1 put forward by Adema et al. (47).
In contrast to Adema et al., we have shown here that AMAC-1 is
preferentially expressed by IL-4-induced alternatively activated
macrophages; admittedly, AMAC-1 is also expressed by
GM-CSF/IL-4-induced monocyte-derived dendritic cells, but the level of
expression is much lower and decreases with dendritic cell maturation.
The common denominator for expression of AMAC-1 in alternatively
activated macrophages and monocyte-derived dendritic cells obviously is
its inducibility by IL-4. This notion is substantiated by our finding
that AMAC-1 is also inducible by other Th2 cytokines such as IL-10 and
IL-13 and that its expression is inhibited by the Th1 cytokine IFN-.
Whether dendritic cells such as Langerhans cells that have not seen
IL-4 and do not express the IL-4-inducible macrophage mannose receptor
(48) will express AMAC-1 remains to be determined. In further contrast
to Adema et al. (47), we have found that AMAC-1 expression in vivo is
strong in peripheral organs such as lung (alveolar macrophages) and
placenta (not shown) and is therefore not restricted to some sparsely
distributed putative dendritic cells in lymphatic organs.
In summary, AMAC-1 is a novel CC-chemokine with several unconventional
features. In contrast to most other known CC-chemokines, AMAC-1
expression is specifically induced in alternatively activated
macrophages by Th2-associated cytokines in vitro and is naturally
present in alveolar macrophages in vivo. Interestingly, both
alternatively activated and alveolar macrophages are known to be
involved in suppression of Th1-associated immune reactions (24, 49, 50, 51, 52). To a lesser extent, AMAC-1 is also expressed in
monocyte-derived dendritic cells. As Adema et al. have shown (47),
AMAC-1 may be functional in preferentially attracting and activating
naive T cells. Unexpectedly, double-negative knockout mice lacking the
CC chemokine receptor 1, which is the major receptor for MIP-1,
experience a shift of the Th1/Th2 balance toward Th1 predominance (53)
indicating that MIP-1 supports Th2 reactions. Due to the fact,
however, that both Th2-associated, preferentially anti-inflammatory
alternatively activated macrophages and Th1-associated, potent
immunostimulatory dendritic cells express AMAC-1, AMAC-1 may not be
directly involved in determining Th1 or Th2 skewing during T cell
development. Full clarification of this open question must await
identification of the AMAC-1 receptor. Special structural features of
AMAC-1 at sites influencing receptor specificity suggest unusual
CC-chemokine receptor-ligand interactions which might comprise unknown
or orphan chemokine receptors (54, 55) or even chemokine receptor
antagonism.
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Footnotes |
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2 Address correspondence and reprint requests to Professor S. Goerdt, Klinik und Poliklinik für Dermatologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany.
3 Abbreviations used in this paper: GC, glucocorticoids; MS-1-HMWP, MS-1 high m.w. protein; AMAC-1, alternative macrophage activation-associated CC-chemokine; MIP, macrophage inflammatory protein; GM-CSF, granulocyte-macrophage CSFpfu, plaque-forming units.
4 Nicholas, K. B., and H. B. Nicholas, Jr. 1997. GeneDoc: a tool for editing and anotating multiple sequence alignments. Distributed by the author.
5 Page, R. D. M. 1996. TreeView: tree drawing software for Apple Macintosh and Microsoft Windows. Distributed by the author at: http://taxonomy.zoology.gla.ac.uk/rod/rod.htm.
Received for publication September 2, 1997. Accepted for publication October 20, 1997.
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