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Departments of
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Immunobiology and
Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many chemokines are expressed constitutively and/or during inflammation
in the lung as well as in other tissues. Examples include epithelial
cell-derived neutrophil activating protein-78 (ENA-78) (4, 5), eotaxin
(6), macrophage inflammatory protein-3
(MIP-3)3 (7), RANTES (8),
IL-8, (9) and many others.
The redundancy in chemokine production by resident and/or infiltrating cells in the lung may reflect the need for rapid cell recruitment in response to the large number of Ags that penetrate the lungs.
During the last few years, one of our goals has been to identify and
characterize new chemokines. We initially reported human MIP-3 (7, 10, 11), which was recognized as a chemokine that is abundantly
produced in several organs (7). While trying to isolate a cDNA clone
encoding mouse MIP-3 by screening lung cDNA libraries, we
unexpectedly found a new mouse CXC chemokine that we have designated
Lungkine.
Lungkine differs from all previously described chemokines because its expression is restricted to lung bronchoepithelial cells and is up-regulated during inflammation. The subfamily of the ELR-CXC chemokines is known to induce the migration of neutrophils and, in some cases, T cells, although the latter is controversial (12, 13). Acute inflammation is characterized by a prevalent neutrophilic infiltrate, which are the first leukocytes to enter a site of allergic inflammation. For this reason, we studied the regulation of Lungkine mRNA expression in normal or in various murine inflamed lung tissues, including lungs from OVA- and Aspergillus-challenged mice, which are recognized asthma models. Our data suggest that this new lung-specific chemokine may play a role in neutrophil trafficking during normal and inflammatory conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 150,000 clones from a RAG-1-/- lung
mouse cDNA plasmid library were screened using a human MIP-3 cDNA
probe (7). The hybridization was conducted in Churches solution at
65°C (14), and filters were washed at medium stringency (1x
SSC/0.1% SDS at 65°C). One positive clone (clone 20) was isolated
and sequenced following the second round of screening. The new cDNA
clone was used as a probe in the screening of a Nippostrongylus
brasiliensis-infected mouse lung cDNA library to obtain the
full-length Lungkine cDNA. Three positive clones were obtained after
screening 100,000 clones. Most of the work described here was done with
the full-length clone N4C. The nucleotide sequences of Lungkine cDNA
clones were confirmed by both-strands automated sequencing using an
Applied Biosystems 373 sequencer (Foster City, CA). All sequences
obtained were imported into and analyzed using Sequencher (Genecodes,
Ann Harbor, MI).
Murine Lungkine mRNA distribution
Total RNA was made from different tissue sources and cell lines using RNAzol B solution (Tel-Test, Friendswood, TX). A total of 15 µg of RNA was loaded in each lane, transferred to a Hybond-N membrane (Amersham, Arlington Heights, IL), hybridized, and washed at high stringency (0.2x SSC/0.1% SDS at 65°C). A mouse tissue Northern blot from Clontech (Palo Alto, CA) was hybridized to confirm mRNA distribution. Lungkine cDNA (1 kb) and a 0.4-kb cDNA fragment of hypoxanthine phosphoribosyltransferase (HPRT) were used as probes in all Northern blot analyses.
Southern blot analysis of cDNA libraries
A panel of mouse cDNA libraries was analyzed by Southern blot as described previously (15).
Chromosomal mapping
The 1-kb Lungkine cDNA probe was used by Genome Systems (St. Louis, MO) to determine the chromosomal localization by fluorescent in situ hybridization (ISH). The cDNA probe was labeled with digoxigenin dUTP by nick translation. Labeled probe was combined with sheared mouse DNA and hybridized to normal metaphase chromosomes derived from mouse embryo fibroblast cells in a solution containing 50% formamide, 10% dextran sulfate, and 2x SSC. Specific hybridization signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin Abs followed by counterstaining with 4',6-diamidino-2-phenylindole.
Immunohistochemistry
Lungs from normal mice and OVA-sensitized and -challenged mice were treated as described previously (16). Sections were incubated with a rabbit polyclonal affinity-purified Ab raised against the Lungkine peptide CLDPDAPWVKATVGPITNRFLPEDLKQKE-COOH (Genemed, South San Francisco, CA). The negative controls used in this experiment were sections incubated in the absence of primary Ab or incubated with a blocking mix of peptide and primary Ab. The peptide was used in a 10 molar excess in respect to the Ab to ensure blocking.
In situ hybridization
ISH was performed as described previously (17). Briefly, fetuses and tissues were fixed in 4% paraformaldehyde in PBS overnight, dehydrated, and infiltrated with paraffin. Serial sections at a thickness of 5–7 µ were remounted on gelatinized slides. Sections were deparaffinized in xylene, rehydrated, and postfixed. The sections were digested with proteinase K, postfixed, treated with triethanolamine/acetic anhydride, washed, and dehydrated. Complementary RNA (cRNA) was prepared from linearized cDNA templates to generate antisense and sense probes. The cRNA transcripts were synthesized according to the manufacturer’s instructions (Ambion, Austin, TX) and labeled with [35S]UTP (>1000 Ci/mmol; Amersham). cRNA transcripts of >200 nucleotides were subjected to alkali hydrolysis to give a mean size of 70 bases. Sections were hybridized overnight at 52°C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM NaPO4, dextran sulfate, 1x Denhardt’s solution, 50 µg/ml total yeast RNA, and 50,000-75,000 cpm/µl 35S-labeled cRNA probe. The tissue was subjected to stringent washing at 65°C in 50% formamide, 2x SSC, and 10 mM DTT and washed in PBS before treatment with 20 µg/ml RNase A at 37°C for 30 min. Following washes in 2x SSC and 0.1x SSC for 10 min at 37°C, the slides were dehydrated, dipped in Kodak NTB-2 nuclear track emulsion, and exposed for 2–3 wk in light-tight boxes with desiccant at 4°C. Photographic development was conducted in Kodak D-19. Slides were counterstained lightly with toluidine blue and analyzed using both the light and dark field optics of a Zeiss Axiophot microscope. Sense control cRNA probes (identical with the mRNAs) indicate the background levels of the hybridization signal.
Recombinant protein expression and purification
Escherichia coli (strain SG220014) was transformed with pMBD101012 plasmid carrying Lungkine cDNA. Cells were grown in freshly prepared 2x Luria-Bertani medium, induced at time 0, and grown at 37°C until the A560 was 2.5. Next, cells were harvested by centrifugation, and the inclusion bodies were isolated by fluidizing the cells in 50 mM Tris buffer (pH 8.5), 5 mM EDTA, and 1 mM Pefa Bloc. The inclusion bodies were washed in 50 mM Tris buffer (pH 8.5) and 5 mM EDTA containing 1% Triton X-100 and subsequently in Tris buffer containing 1 M guanidine HCl. The washed inclusion bodies were solubilized in 50 mM Tris buffer (pH 8.5) containing 8 M guanidine HCL, 5 mM EDTA, 10 mM DTT, and 1 mM Pefa Bloc. The solubilized inclusion bodies were renatured by dilution (100x) in 50 mM Tris (pH 8.5) containing 1.5 mM glutathione, 0.5 mM glutathione disulfide, 0.4 M guanidine, and 5 mM EDTA. The protein was allowed to renature for 16 h and subsequently concentrated and diafiltered into 50 mM Tris (pH 8.5) and 5 mM EDTA. The refolded protein was purified by chromatography on a Baker C4 reverse phase column. The fractions containing Lungkine were recognized by gel electrophoresis and confirmed by Western blot analysis using an anti-peptide Ab. The Lungkine-containing samples were pooled and run over the same Baker C4 column again to remove endotoxin (final concentration of 2 endotoxin units/ml). Final gel analysis demonstrated a single band by gel electrophoresis.
Bronchoalveolar lavage (BAL) and OVA treatment
OVA/alum was prepared as described previously (18). Briefly, alum precipitate was prepared by the addition of 10N NaOH (Sigma, St. Louis, MO) to a 10% solution of AlKSO4 (Sigma) until precipitate formed. This precipitate was then extensively washed with sterile PBS and stored at 4°C. On the day of the injections, OVA (grade V; Sigma) in PBS was bound to the alum precipitate (OVA/alum) by mixing at 4°C. Mice were primed i.p. with 10 µg OVA/alum on day 0 and boosted on day 7. On day 14, mice were aerosolized with OVA in PBS using a Passport aerosol compressor (Invacare, Elyria, OH) connected to a 3-ft3 box for 20 min. Previous studies have shown that the aerosolization delivers 3–5 µg of OVA to each mouse (18).
Mice were sacrificed at the indicated timepoints following OVA aerosolization, and BALs were performed by intratracheal insertion of a needle. The BAL was harvested in 3 ml of incomplete RPMI 1640 and stored frozen until analysis. BALs were concentrated by precipitating proteins with cold acetone for 1 h at -20°C. The samples were centrifuged, and the pellets were resuspended in PBS. Protein concentration was determined by the Bradford method using Bio-Rad solutions (Bio-Rad, Richmond, CA).
N. brasiliensis and Aspergillus treatment
BALB/c mice were given 500 N. brasiliensis worms in 50 µl of PBS s.c. in the flank on day 0. Lungs were harvested on days 8 and 10, and total RNA was isolated for Northern blot analysis.
BALB/c mice were treated with 50 µg of Aspergillus extract in 50 µl of PBS intranasally on day 0, and lungs were harvested 6 h after Ag challenge. Total RNA was prepared and analyzed by Northern blot.
Western blotting
A total of 10 µg of protein was loaded in 18% acrylamide denaturing gels (Novex, San Diego, CA). The procedure was performed following conventional methods (14). Rabbit polyclonal affinity-purified antiserum (Genemed, South San Francisco, CA) was used as the primary Ab for Lungkine detection. Anti-rabbit IgG, HRP, was used as secondary Ab; Super Signal (Pierce, Rockford, IL) solutions were used to develop the blot.
Cell isolation
Thioglycolate medium (1 ml) (Difco Laboratories, Detroit, MI) was injected i.p. in 6- to 8-wk-old BALB/c mice. Mice were sacrificed at 24 h postinjection, and peritoneal lavages were performed with 5 ml of cold PBS. The cells obtained were treated with RBC lysing buffer (Sigma) and counted.
Transwell chemotaxis assay
Chemotaxis was performed as described previously (19). Briefly, the cells and chemokines used in this assay were resuspended in DMEM containing 1% low endotoxin BSA (Sigma) at pH 6.95. Transwell plates of 3-µm pore size (Corning Costar, Cambridge, MA) were coated with Sigmacote (Sigma) and loaded with 600 µl of medium or with different chemokine dilutions in duplicate (lower chamber). Cells were resuspended at 2 x 107 cells/ml, and 100 µl of this suspension was placed in the inserts (upper chamber). After 2 h of incubation at 37°C and 5% CO2, 50 µl of 70 mM EDTA was added in the lower chamber to release adherent cells from the membrane and the bottom of the plate. Inserts were removed, and 10,000 beads (Dynospheres Uniform Microspheres, mean diameter of 15 µm; Bangs Laboratories, Fishers, IN) were added per well. The cells in the lower chamber were collected along with the starting cell population, stained with Gr-1-FITC (stains granulocytes) (PharMingen, San Diego, CA) and F4/80-PE (stains macrophages) (Caltag, Burlingame, CA) and analyzed by flow cytometry in a FACScan (Becton Dickinson, Milpitas, CA). A total of 10,000 events were collected per analysis. The ratio of beads to cells was determined, allowing us to calculate the number of cells that had migrated to the bottom well.
In vivo chemotaxis
A total of 10 µg of purified recombinant protein, either murine Lungkine or murine keratinocyte-derived-chemokine (KC) (R&D Systems, Minneapolis, MN), LPS (endotoxin-matched control), or PBS (negative control), was injected i.p. into 8-wk-old C3H/HeJ and BALB/C mice. Mice were sacrificed at 3 and 24 h postinjection. Cold endotoxin-free PBS (5 ml) was used to wash the peritoneum and collect cells. The recovered cells were counted. A total of 500,000 cells from each mouse were stained with Gr-1- (PharMingen), F4/80-, and CD4+- and CD8+- (Caltag) conjugated Abs and analyzed by flow cytometry as described above. Results are expressed as the percentage of a specific cell population with respect to the number of total cells counted using a Coulter counter (Coulter, Hialeah, FL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially sought to isolate the mouse homologue of MIP-3. To
achieve this, we analyzed several cDNA libraries by Southern blot with
human MIP-3; a RAG-1-/- lung cDNA library yielded a
strong signal. This cDNA library was screened with a human MIP-3
probe. Several positive clones were isolated and sequenced.
Unexpectedly, one 1-kb positive clone comprised most of the complete
open reading frame (ORF) and the 3' untranslated region (UTR) sequence
of a new ELR-CXC chemokine that had not been described before. At that
time, no human or mouse expressed sequence tag (EST) encoding Lungkine
was present in the GenBank database of ESTs (dbEST).
A panel of cDNA libraries was analyzed by Southern blot using the 1-kb
Lungkine cDNA probe. The libraries that gave a positive signal were:
mouse RAG 1-/- lung, mouse normal lung, and N.
brasiliensis-infected lung cDNA libraries (data not shown). The
highest abundance of Lungkine cDNA was detected in the N.
brasiliensis-infected lung cDNA library. Isolation of the
full-length cDNA was conducted by screening the latter library. One of
the positive clones obtained in the screening, which was 
1 kb in
size, contained a 5'UTR sequence, the complete ORF, and the 3'UTR
sequence. (This sequence has been deposited in the GenBank database and
assigned accession no. AF082859). In addition, three mRNA instability
sequences (ATTTA) were present in the 3'UTR, characteristic of cytokine
mRNAs (data not shown).
Murine Lungkine cDNA displays a 166-aa ORF (Fig. 1
). The processing of a predicted 25-aa
leader sequence (http://www.cbs.dtu.dk/services/SignalP/) results
in a mature protein of 141 aa with an unusually short N terminus and an
extremely long C-terminal tail that protrudes beyond the chemokine
fold. The function of this extended tail is unknown; however, it has
been shown (20) that severe truncations of the long C terminus of
Lymphotactin abolish its chemotactic activity, possibly by
destabilizing the protein structure. It is likely that the long
C-terminal tail of Lungkine could also be involved in stabilizing the
structure of the protein.
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with 26%, and rat
cytokine-induced neutrophil chemoattractant-2ß (CINC-2ß)
with 22%. Chromosomal localization
The initial experiment resulted in specific labeling of the middle portion of a medium-sized chromosome, which was believed to be chromosome 5 on the basis of 4',6-diamidino-2-phenylindole staining. A second experiment was conducted in which a probe specific for the telomeric region of chromosome 5 was cohybridized with a Lungkine cDNA probe. This experiment resulted in the specific labeling of the telomere and the mid portion of chromosome 5. Measurements of 10 specifically hybridized chromosomes 5 demonstrated that the Lungkine gene is located at a position that is 50% of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome 5. A total of 80 metaphase cells were analyzed, with 67 exhibiting specific labeling (data not shown). Therefore, we conclude that the Lungkine gene is located in mouse chromosome 5.
Most CXC chemokine genes are located in human chromosome 4 (4q12–21) and mouse chromosome 5. The clustering of this gene subfamily strongly suggests that all have diverged from a common ancestral gene.
Lungkine is a chemokine specifically expressed in the lung
The results obtained from Southern blot analyses of the cDNA
libraries suggested that Lungkine had a very restricted pattern of
expression (data not shown). To confirm this, a mouse tissue Northern
blot and a cell line Northern blot were hybridized with a Lungkine cDNA
probe. All of the mouse cell lines tested, including 3D.1 (thymus
epithelium), MC9 (mast cell), A3.2 (NK T cell hybridoma), HT-2
(T cell clone), L cells (fibroblast), a prothymocyte T cell hybridoma,
a prethymocyte T cell hybridoma, A20-2J (B cell lymphoma), Lewis lung
carcinoma cells, as well as the BW5147 T cell thymoma, gave no signal
(data not shown). Conversely, the tissue blot showed an intense signal
on lung RNA samples (Fig. 2A),
a weak signal on fetal lung tissue, and a faint signal in the heart.
However, other cDNA libraries derived from the total heart or aorta
showed no signal for Lungkine (data not shown). The thymus and Con
A-stimulated spleen, lymph nodes, brain, kidney, and fetal liver were
negative for Lungkine expression. These results agree with the signals
detected using a murine tissue Northern blot (Clontech) (Fig. 2B). Until now, all other chemokines reported expressed in
the lung were also expressed elsewhere. For example, IFN-inducible T
cell chemoattractant (I-TAC) is expressed not only in the
lung but also in the pancreas, thymus, spleen, and brain (21).
Fractalkine is expressed in the lung, kidney, skeletal muscle, heart,
brain, and testis (22). Monocyte chemoattractant protein-5
(MCP-5) is expressed in the lymph nodes, thymus, and lung (23). MCP-4
is constitutively expressed in the small intestine, colon, and lung
(24). Macrophage-derived chemokine (MDC) is detected in the
thymus, lung, and spleen (25). Other examples include MCP-2 (26),
pulmonary and activation-regulated chemokine (PARC) (27),
MIP-3 (7, 10, 11), etc. We conclude that the highly specific pattern
of expression that Lungkine exhibits is unique.
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Based on the expression data obtained from mouse cDNA libraries
and tissue blot analyses, we sought to examine Lungkine mRNA regulation
in the inflamed lungs of various animal disease models: an asthma OVA
model, an N. brasiliensis infection model, and an
Aspergillus infection model. Lung and mediastinal lymph
nodes were obtained from OVA-sensitized BALB/c mice (see
Materials and Methods) at 3, 6, and 24 h after Ag
challenge. Total RNA was prepared, and Northern blot analyses were
performed. Fig. 3A shows that
mediastinal lymph nodes are not responsible for Lungkine mRNA
expression. Nevertheless, there was approximately a 2-fold increase in
Lungkine mRNA levels in the OVA lung samples 3 h after Ag
challenge. The same result was observed with the LPS-treated lung
samples at 3 h postinjection. Lungkine mRNA levels were
still up-regulated 24 h after the OVA challenge.
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B). We observed that
OVA-challenged mice as well as
Aspergillus-infected, LPS-treated, and
N. brasiliensis-infected mice showed a small but significant
increase (2-fold) in Lungkine mRNA expression, suggesting a role
for Lungkine in neutrophil trafficking during normal and inflammatory
conditions. Lungkine mRNA is expressed by bronchoepithelial cells
We sought to identify the cellular source of Lungkine. For this
purpose, we prepared sections from normal lungs and performed
immunohistochemical analyses. The latter detected the presence of
Lungkine protein in bronchoepithelial cells (Fig. 4C). The negative controls
used in this experiment included sections treated with a blocking mix
(peptide-Ab) (Fig. 4B) or without the primary Ab (Fig. 4A).
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A shows a fetal (day 16) lung
tissue section hybridized with a Lungkine antisense probe. The bright
punctate signal of the silver grains is very clear in Fig. 5A; however, this signal was absent when the control sense
probe was used (Fig. 5B).
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C) and OVA-challenged lung (24 h) sections from BALB/c
adult mice (Fig. 5D) showed a positive signal in the
epithelial cells surrounding the bronchi when hybridized with the
Lungkine antisense probe. In agreement with our Northern blot results,
a stronger signal was detected in the OVA-treated lungs compared with
the unstimulated lung sections. Lungkine is secreted into the bronchoalveolar space
The content of Lungkine protein present in the BAL of normal
BALB/c or OVA-sensitized and challenged mice was compared by Western
blot analysis (Fig. 6). Lungkine protein
was present in the BALs of both unstimulated and challenged mice.
After Ab removal, the same blots were incubated with rabbit preimmune
serum or with an anti-peptide Ab against another chemokine,
thymus-expressed chemokine (TECK) (as a control) (data not
shown). No signal was detected, confirming the specificity of the
anti-peptide Ab against Lungkine.
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These results, along with the immunohistochemistry and ISH analyses, confirm that Lungkine is produced by bronchoepithelial cells and indicate that it is released into the airways.
Lungkine is chemotactic for granulocytes
In vivo migration assay.
Because Lungkine is an ELR-CXC chemokine, it was likely to be a
neutrophil chemoattractant. To test this hypothesis, C3H/HeJ mice
(endotoxin-insensitive) were injected i.p. with either 10 µg of
Lungkine, with another CXC chemokine, KC (as a positive control), or
with PBS. The i.p. injection of Lungkine or KC induced an increase in
the number of Gr-1+ cells in the peritoneum
(15–20%, respectively) compared with mice injected with PBS (1%) at
3 h postinjection (Fig. 7A). By 24 h, the
percentage of Gr-1+ cells had decreased significantly
(2–4%). However, there was no increase in the percentage of
F4/80+ cells in the animals treated with either Lungkine or
KC. The percentage of T cells in the peritoneal cavity did not vary
significantly between KC, Lungkine, and PBS controls. Similar
fold increments were obtained when using BALB/c mice (data not shown).
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In vitro chemotaxis assay.
To confirm the results obtained with the in vivo migration assay, we
tested Lungkine in a transwell chemotaxis assay using a cell population
enriched in neutrophils (see Materials and Methods).
Lungkine did induce the migration of neutrophils (Fig. 7B).
At a concentration of 10-5 M, Lungkine induced the
migration of 65% of Gr-1+ cells; KC induced the migration
of 55%, of these cells. However, 10-6 M and
10-7 M concentrations of Lungkine did not induce
significant cell migration, whereas corresponding concentrations of KC
induced only a slightly better response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(7)
or pulmonary and activation-regulated chemokine (PARC)/DC-Ck-1 (27) are
expressed in the lung as well, they are also expressed elsewhere.
Lungkine is the only chemokine whose expression appears restricted to
the lung.
We discovered Lungkine while screening a mouse RAG-/-
lung cDNA library during a search for the mouse counterpart of
MIP-3. Although Lungkine shares sequence similarity at the amino
acid level with some human CXC chemokines (platelet basic protein,
ENA-78, IL-8, and others), the degree of sequence similarity is not
high enough to unequivocally assign a known human counterpart to
Lungkine. Furthermore, none of these chemokines exhibit the
characteristic long C-terminal tail of Lungkine. Alternatively, several
efforts to identify a human counterpart were attempted, so far yielding
negative results. In addition to the screenings of human genomic
libraries and of cDNA libraries, Northern blot analyses of
human lung cell lines yielded no solid evidence of a human counterpart
of Lungkine (data not shown). Finally, no human ESTs for a putative
human Lungkine homologue exist in the GenBank dbEST database, despite
the fact that the number of human ESTs it contains currently exceeds
the number of mouse ESTs by >3-fold. These results, along with the low
sequence identity with other known human ELR-CXC chemokines, strongly
suggest that human Lungkine, if it exists, will exhibit a highly
specific expression pattern. We are currently testing the hypothesis
that the expression of human Lungkine may be restricted to
certain human inflammatory lung conditions. At present, we conclude
that Lungkine represents a novel lung-specific chemokine for which a
human homologue has not yet been identified.
Lungkine was found to be expressed by lung epithelial cells (Fig. 5, C and D). The fact that Lungkine is produced by
this cell type and is up-regulated during inflammation suggests a role
for this chemokine in leukocyte recruitment to the airways upon Ag
challenge.
Similar to other ELR-CXC chemokines, Lungkine induces the in vivo and
in vitro migration of neutrophils (Fig. 7, A and
B). Neither T cells nor macrophages migrate to the
peritoneal cavity in response to Lungkine between 3 and 24 h after
i.p. injection. However, we cannot eliminate the possibility that
Lungkine could cause neutrophil activation. Lungkine, as shown
for IL-8 (12), could activate neutrophils and generate a T cell
migration (72 h after protein injection) by inducing the release of T
cell chemoattractants from neutrophils, indirectly affecting another
important cell type in asthma.
We hypothesize that Lungkine is involved (along with other chemokines and adhesion molecules) in the homing of neutrophils to protect the airways against exogenous Ags or pathogens. In cases in which certain Ags or pathogens were present in the airways, Lungkine levels would rise, increasing the number of neutrophils recruited to the airways.
ELR-CXC chemokines are known to bind to CXC chemokine receptor 2 (CXCR2) (with the exception of IL-8, which also binds to CXCR1), which is essentially expressed by neutrophils and, to a lesser extent, by T lymphocytes (28, 29). We have tested Lungkine binding to mouse CXCR2 but have been unable to demonstrate the binding of Lungkine to CXCR2 using either Ca2+ flux or binding assays (data not shown).
CXC chemokines have been described as key molecules in the regulation of angiogenesis (30). Whereas ELR-CXC chemokines promote angiogenesis, non-ELR-CXC chemokines suppress it. Lungkine could be involved in the angiogenic processes occurring during embryonic development; in addition its mRNA up-regulation in the adult stage may be necessary to support the growth and development of the lung. Experiments currently underway will aim to test this hypothesis.
Most of the literature has focused on the CC chemokines responsible for the eosinophilic and T lymphocyte infiltration observed during allergic airway inflammation. In contrast, not much information is available on the role of CXC chemokines and neutrophils in allergy and asthma. For example, in sudden-onset fatal asthma cases (also called "sudden asphyxic asthma"), patients have shown a larger number of neutrophils than eosinophils in the airway submucosa (31, 32). These data not only confer relevance to the neutrophilic infiltrate in asthma, but also suggest an important role for neutrophil chemoattractants in this disease.
These data, along with the suggested role of eosinophils and T cells as players in asthma (33, 34, 35), broaden our understanding of how chemokines orchestrate allergic airway inflammation. Given that Lungkine represents a new ELR-CXC chemokine that is specifically expressed by lung bronchoepithelial cells, it will be interesting to explore its role both in normal lung homeostasis and in pathological conditions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Albert Zlotnik, DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304.
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; KC, keratinocyte-derived chemokine; cRNA, complementary RNA; HPRT, hypoxanthine phosphoribosyltransferase; ISH, in situ hybridization; BAL, bronchoalveolar lavage; MCP, monocyte chemoattractant protein; CXCR, CXC chemokine receptor ORF, open reading frame; UTR, untranslated region; EST, expressed sequence tag; ENA, epithelial cell-derived neutrophil activating protein.
Received for publication October 28, 1998. Accepted for publication February 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-3ß. J. Immunol. 158:1033.[Abstract]
inducible protein-10, stimulate transendothelial chemotaxis of T lymphocytes. Eur. J. Immunol. 25:3482.[Medline]
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+ T cells or interferon (IFN)- in a murine model of allergen sensitization. J. Exp. Med. 187:721. chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3. J. Exp. Med. 187:2009./LD78 and chemotactic for T lymphocytes, but not for monocytes. J. Immunol. 159:1140.[Abstract]
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J. N. Vanderbilt, E. M. Mager, L. Allen, T. Sawa, J. Wiener-Kronish, R. Gonzalez, and L. G. Dobbs CXC Chemokines and Their Receptors Are Expressed in Type II Cells and Upregulated following Lung Injury Am. J. Respir. Cell Mol. Biol., December 1, 2003; 29(6): 661 - 668. [Abstract] [Full Text] [PDF]
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 T. Blom, A. Franzen, D. Heinegard, and R. Holmdahl Comment on "The Influence of the Proinflammatory Cytokine, Osteopontin, on Autoimmune Demyelinating Disease" Science, March 21, 2003; 299(5614): 1845a - 1845a. [Full Text] [PDF]
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E.-B. Haddad, K. McCluskie, M. A. Birrell, D. Dabrowski, M. Pecoraro, S. Underwood, B. Chen, G. T. De Sanctis, S. E. Webber, M. L. Foster, et al. Differential Effects of Ebselen on Neutrophil Recruitment, Chemokine, and Inflammatory Mediator Expression in a Rat Model of Lipopolysaccharide-Induced Pulmonary Inflammation J. Immunol., July 15, 2002; 169(2): 974 - 982. [Abstract] [Full Text] [PDF]
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 T. S. Olson and K. Ley Chemokines and chemokine receptors in leukocyte trafficking Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2002; 283(1): R7 - R28. [Abstract] [Full Text] [PDF]
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 S. W. Chensue Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions Clin. Microbiol. Rev., October 1, 2001; 14(4): 821 - 835. [Abstract] [Full Text] [PDF]
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A. Pardo, K. M. Smith, J. Abrams, R. Coffman, M. Bustos, T. K. McClanahan, J. Grein, E. E. Murphy, A. Zlotnik, and M. Selman CCL18/DC-CK-1/PARC up-regulation in hypersensitivity pneumonitis J. Leukoc. Biol., October 1, 2001; 70(4): 610 - 616. [Abstract] [Full Text] [PDF]
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P. Ye, P. B. Garvey, P. Zhang, S. Nelson, G. Bagby, W. R. Summer, P. Schwarzenberger, J. E. Shellito, and J. K. Kolls Interleukin-17 and Lung Host Defense against Klebsiella pneumoniae Infection Am. J. Respir. Cell Mol. Biol., September 1, 2001; 25(3): 335 - 340. [Abstract] [Full Text] [PDF]
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N. Chouchakova, J. Skokowa, U. Baumann, T. Tschernig, K. M. H. Philippens, B. Nieswandt, R. E. Schmidt, and J. E. Gessner Fc{{gamma}}RIII-Mediated Production of TNF-{{alpha}} Induces Immune Complex Alveolitis Independently of CXC Chemokine Generation J. Immunol., April 15, 2001; 166(8): 5193 - 5200. [Abstract] [Full Text] [PDF]
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 K. Tateda, T. A. Moore, M. W. Newstead, W. C. Tsai, X. Zeng, J. C. Deng, G. Chen, R. Reddy, K. Yamaguchi, and T. J. Standiford Chemokine-Dependent Neutrophil Recruitment in a Murine Model of Legionella Pneumonia: Potential Role of Neutrophils as Immunoregulatory Cells Infect. Immun., April 1, 2001; 69(4): 2017 - 2024. [Abstract] [Full Text] [PDF]
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S.-C. Chen, B. Mehrad, J. C. Deng, G. Vassileva, D. J. Manfra, D. N. Cook, M. T. Wiekowski, A. Zlotnik, T. J. Standiford, and S. A. Lira Impaired Pulmonary Host Defense in Mice Lacking Expression of the CXC Chemokine Lungkine J. Immunol., March 1, 2001; 166(5): 3362 - 3368. [Abstract] [Full Text] [PDF]
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 J. A. Wiley, A. Cerwenka, J. R. Harkema, R. W. Dutton, and A. G. Harmsen Production of Interferon-{{gamma}} by Influenza Hemagglutinin-Specific CD8 Effector T Cells Influences the Development of Pulmonary Immunopathology Am. J. Pathol., January 1, 2001; 158(1): 119 - 130. [Abstract] [Full Text] [PDF]
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