(Received for publication, November 21, 1995; and in revised form, February 9, 1996)
From the
GRO proteins are -chemokine cytokines that attract
neutrophils and stimulate the growth of a variety of cells. Previously,
we observed that rabbit alveolar macrophages transcribe the genes for
at least two GRO homologues. In order to study the role of GRO
cytokines in lung inflammation, we cloned the predominant rabbit GRO
cDNA (RabGRO) from alveolar macrophages, expressed bioactive
recombinant protein (rRabGRO) in Escherichia coli, and
developed a sensitive and specific enzyme-linked immunosorbent assay
for RabGRO protein. We found that rabbit AM express and secrete GRO in vitro in response to both exogenous (e.g. lipopolysaccharide, heat-killed Staphylococcus aureus,
and crystalline silica) and endogenous inflammatory stimuli (e.g. tumor necrosis factor-
) as determined by both
radioimmunoprecipitation and enzyme-linked immunosorbent assay.
Biologically significant amounts of GRO are present in vivo in
the bronchoalveolar lavage fluid of rabbits with E. coli pneumonia; by in situ hybridization, GRO mRNA is
detectable in infiltrating pulmonary leukocytes and bronchial
epithelial cells. These results indicate that GRO chemokines are likely
to be important mediators of the inflammatory response that accompanies
acute infectious processes in the lungs.
Human GRO-, -
, and -
are members of the
-chemokine family of proteins, which also includes interleukin-8
(IL-8)(
)(1) . Like most
-chemokines, GRO
proteins are potent neutrophil chemoattractants and
activators(2) . In addition to their effects on neutrophils,
these cytokines have many other biological activities, including
regulatory effects on melanoma cell growth(3) , fibroblast
collagen production(4) , monocyte activation and adhesion to
endothelial cells(5, 6) , myelopoiesis(7) ,
and angiogenesis(8, 9) .
It is likely that GRO
proteins are relevant in lung inflammation, as human alveolar
macrophages (AM) express GRO mRNA and protein in response to
stimulation with lipopolysaccharide (LPS)(10) , toxic shock
syndrome toxin-1(11) , and tumor necrosis factor-
(TNF-
)(12) . Although these in vitro studies
suggest that GRO chemokines are mediators of the lung response to both
exogenous and endogenous inflammatory stimuli, demonstration of their
biological relevance in vivo is lacking.
Rabbits, like
humans and in contrast to rodents(1) , produce a spectrum of
-chemokines that includes IL-8 (13) and
GRO(6, 14, 15) . We reported previously the
molecular cloning of two distinct rabbit GRO cDNA homologues from
LPS-stimulated AM (RabGRO and rabbit permeability factor-2 or
RPF2)(15) . Of the two transcripts, RabGRO was the predominant
AM-derived GRO homologue. We now report the expression in Escherichia coli of bioactive recombinant RabGRO protein
(rRabGRO) and the development of a sensitive and specific RabGRO
immunoassay. Using this assay, we have found that rabbit AM secrete
native GRO protein in vitro in response to stimulation with a
spectrum of biologically relevant agonists. We also report that
biologically significant quantities of GRO protein are present in the
bronchoalveolar lavage fluid (BALF) of rabbits with acute bacterial
pneumonia and identify the sites of GRO mRNA expression in the lungs.
These data suggest that GRO chemokines make important contributions to
acute inflammatory responses in the lungs.
Figure 1:
Purification of rRabGRO-fp from
bacterial lysates. A, SDS-PAGE gel of material eluting from a
Ni affinity column with buffer at pH 2.0. EK, enterokinase. Lanes 1, 2, and 13,
molecular weight markers; lane 3, crude bacterial extract; lane 4, crude bacterial supernatant; lane 5,
pass-through fraction at neutral pH containing unbound bacterial
supernatant proteins; lanes 6-12, sequential elutions at
pH 2.0. Lanes 6-10 contain visible rRabGRO-fp of the
expected size (14 kDa, arrow); B, separation of
rRabGRO-fp/EK reaction products by reverse-phase HPLC using a C4 column
and a 5-95% CH
CN elution gradient (%B).
Recombinant RabGRO eluted from the column in approximately 40%
CH
CN (32 min).
Figure 2:
Rabbit and human neutrophil chemotactic
activity of rRabGRO and recombinant human GRO-. A, chemotaxis induced by rRabGRO for rabbit and human neutrophils; B, recombinant human GRO-
(rHumGRO
)
chemotactic activity for human and rabbit neutrophils. Values are means
± S.E. for four experiments. The number of rabbit and human
neutrophils migrating with PBS was 2.88 ± 0.48 and 6.13 ±
0.44. For zymosan-activated rabbit and human serum, respectively, the
values were 28.25 ± 2.69 for rabbit and 56.00 ± 2.04 for
human neutrophils.
To assess in vivo bioactivity, rRabGRO was instilled endobronchially into a rabbit. This produced a brisk influx of neutrophils into the lung (45% neutrophils in the BALF cell count from the treated lung at 4 h). For comparison, the BALF from the untreated lung in the same rabbit and from the lungs of a rabbit treated with sterile 0.9% NaCl alone contained only 6 and 5% neutrophils, respectively.
Figure 3:
Radioimmunoprecipitation of GRO protein
from the supernatants of adherent rabbit AM. Adherent rabbit AM
stimulated for 19 h with crystalline SiO (100 µg/ml),
Al
O
(100 µg/ml), LPS (10 µg/ml), human
TNF-
(500 U/ml), heat-inactivated (hi-) human TNF-
(500
units/ml), ConA (5 µg/ml), HKSA (1
10
bacteria/ml), or NaCl (0.9%). The autoradiogram of radiolabeled
proteins in the supernatants, resolved by SDS-PAGE, is shown on top, and the corresponding analysis by phosphorimaging is
shown below. The ``+'' lanes denote incubation with goat
anti-rRabGRO IgG and the ``-'' lanes refer to
incubation with nonimmune goat IgG. The vertical axis is the
relative intensity of the signal from the major band (7 kDa, the
expected size for RabGRO protein) in each lane compared with the
background radioactivity of the gel.
Because adherence alone appeared to be a stimulus for GRO
protein production in the RIP studies, we measured GRO levels by ELISA
using the supernatants of rabbit AM cultured in polypropylene tubes
which were periodically agitated. These results showed significant
time- and stimulus-dependent differences in the accumulation of GRO
protein in the supernatants of relatively nonadherent AM (Fig. 4). In response to stimulation by HKSA and LPS,
significant amounts of extracellular GRO were detected after only 2 h
of incubation. For HKSA, GRO increased steadily and was maximal by 20 h
of incubation. For LPS, maximal amounts of GRO were detected at 4 h of
incubation and the concentration failed to increase further at 20 h.
Incubation with SiO caused the secretion of relatively
small amounts of GRO protein during the first 6 h, but relatively high
concentrations of GRO were present in the supernatants at 20 h.
Stimulation with ConA and Al
O
did not result in
significant secretion of GRO when compared with incubation with 0.9%
NaCl (negative control). Likewise, human TNF-
and rabbit IFN-
(both intact and heat-inactivated) failed to stimulate increased
amounts of GRO in the supernatants of nonadherent AM (data for
heat-inactivated proteins not shown).
Figure 4:
Rabbit GRO protein levels in stimulated
nonadherent AM supernatants. Measurement of GRO protein (ng/ml) by
immunoassay in the supernatants of rabbit AM incubated with crystalline
SiO (100 µg/ml), Al
O
(100
µg/ml), ConA (5 µg/ml), HKSA (1
10
bacteria/ml), human TNF-
(500 units/ml), LPS (10 µg/ml),
rabbit IFN-
(500 units/ml), or 0.9% NaCl for the times
indicated.
Figure 5:
Localization of GRO mRNA in lung tissue in E. coli pneumonia by in situ hybridization. Lung
tissue specimens (9-µm sections) from a rabbit treated with the
endobronchial instillation of 10 colony-stimulating
factor/ml E. coli at 4 h. A and C, hybridization with the RabGRO antisense probe; B and D, hybridization with RabGRO sense probe (negative control).
The large arrowheads indicate bronchial epithelial cells and small arrowheads refer to parenchymal inflammatory leukocytes.
Specimens were counterstained with hematoxylin and eosin
(magnification:
450).
Alveolar macrophages are important cellular mediators of
pulmonary inflammation, in part due to their ability to elaborate a
variety of leukocyte chemotactic factors(20) . Among these
factors, IL-8 is thought by many investigators to be the major
neutrophil chemoattractant in the lung(21) . However, studies
using specific antisera to neutralize IL-8 in the supernatants of
activated human AM (22) and BALF from patients with adult
respiratory distress syndrome (23) suggest that a significant
proportion of AM-derived neutrophil chemotactic activity is due to
other non-IL-8 chemoattractants. Possible candidates include the GRO
subgroup of -chemokines, which are closely related to IL-8 and are
also potent neutrophil chemoattractants and activators(2) . In
this study, our goals were: 1) to express recombinant rabbit GRO
protein and develop a specific and sensitive ELISA; 2) to investigate
GRO protein expression by rabbit AM in response to stimulation with
both endogenous and exogenous inflammatory agents; and 3) to determine
whether GRO protein and mRNA can be detected in the lung in vivo during the acute inflammatory response that accompanies bacterial
pneumonia.
In order to generate the species-specific reagents
necessary to measure rabbit GRO protein, we first produced rRabGRO as a
fusion protein in E. coli. We used a prokaryotic expression
system for producing the recombinant protein because GRO proteins have
been shown not to undergo post-translational sulfation, glycosylation,
or phosphorylation(24) . RabGRO amino acid sequence alignment
with all reported full-length GRO homologues demonstrated identities
ranging from 41% for 9E3 (chicken GRO homologue) to 78% for human
GRO- (Table 2). Given the high degree of identity with human
GRO proteins, it is perhaps surprising that rRabGRO had little
chemotactic activity for human neutrophils. However, this confirms
previous observations by other investigators that
I-labeled human GRO-
does not bind to rabbit
neutrophils (25) and further underscores the necessity of using
species-specific reagents when studying
-chemokines(26) .
The rRabGRO-fp proved to be a good immunogen for raising specific
goat anti-rabbit GRO polyclonal antibodies, as Western analysis
confirmed that the goat antibodies did not cross-react with either
rabbit IL-8 or MCP-1. The antibodies did show a small amount of
cross-reactivity for recombinant human GRO-, which is not
surprising given the high degree of homology between these proteins (Table 2). As RabGRO and RPF2 are nearly identical at the mature
protein level (93% identity), the anti-rRabGRO antibodies are likely to
cross-react with RPF2 as well.
We have also shown that rabbit AM
express and secrete GRO protein in response to stimulation by LPS,
TNF-, SiO
, Al
O
, and HKSA.
Adherence and/or culture conditions alone also appear to stimulate GRO
expression. Under nonadherent conditions, LPS, HKSA, and SiO
were the most potent inducers of GRO protein secretion, whereas
human TNF-
was a weak stimulus. By contrast, human TNF-
was a
potent stimulus for GRO protein production in adherent AM. Cell
adherence has been shown to induce the expression of both TNF-
and
IL-1
in AM(27, 28) , and it is possible that
increased expression of these cytokines in adherent AM may in turn
mediate increased GRO protein expression by these cells. The increase
in GRO secretion by rabbit AM in response to the tested stimuli is in
agreement with studies performed with AM from other species. In human
AM, GRO expression has been described in response to
LPS(10, 12) , TNF-
(12) , and S.
aureus(11) . In rat AM, the GRO homologue KC (CINC-1) is
induced by LPS, and MIP-2 expression increases following stimulation
with TNF-
, LPS, and SiO
(29) . Incubation with
rabbit IFN-
did not cause rabbit GRO expression under nonadherent
conditions. It is possible that IFN-
may actually inhibit GRO
protein secretion by AM, as the measured concentrations were less than
those observed with 0.9% NaCl alone. Although this finding needs to be
confirmed in further studies, it is consistent with a prior report that
IFN-
inhibits KC gene expression in mouse peritoneal
macrophages(30) .
The data also show that GRO protein is present in the lungs of rabbits with Gram-negative bacterial pneumonia. All of the rabbits with E. coli pneumonia had elevated concentrations of GRO in BALF, and the amounts correspond to biologically significant levels based on the chemotactic activity of the recombinant GRO protein that we measured in vitro. Moreover, the amount of GRO protein present in the rabbit BALF is roughly equivalent to the levels of IL-8 present in the BALF of patients with the adult respiratory distress syndrome(23) . While the in vitro data suggest that AM are probably a significant source of GRO protein in the lung, many other lung cell types can produce GRO chemokines, and each may be an important contributor to the expression of GRO in the lungs, depending on the in vivo circumstances. In rabbits with Gram-negative bacterial pneumonia, the in situ hybridization studies indicate that airway epithelial cells and tissue inflammatory cells are significant sources of GRO gene expression within 4 h of bacterial entry into the lungs. These results agree with studies by Becker and associates who showed increased GRO mRNA expression in human airway epithelial cells in response to LPS-stimulation (12) and with studies by Xing et al. showing localization of GRO mRNA expression in parenchymal inflammatory cells in LPS-treated rat lung tissue(31) . In contrast, Rogivue and colleagues (32) have reported in bovine pneumonic pasteurellosis that GRO protein is detectable in type II alveolar epithelial cells and mesothelial cells, but not in bronchial epithelial cells or pleural fibroblasts. Lung fibroblast cell lines (33) and endothelial cells (6) also have been shown to express GRO homologues in vitro, but we did not find evidence of GRO production by these cell types in vivo in bacterial pneumonia.
Using a cDNA
library prepared from LPS-stimulated rabbit AM and Northern analysis,
we have shown previously that LPS-stimulated rabbit AM express the
genes for two GRO homologues, RabGRO and RPF2(15) . The
expression of more than one GRO subtype is not unique to rabbits and
has been shown to occur in humans(34, 35) ,
mice(35) , and rats (36, 37) as well. Our data
and that of others suggest that as in humans (34) , the
predominant GRO subtype in rabbits varies by cell type and stimulus. In
peritoneal exudates produced by the intraperitoneal injection of
zymosan, Jose and colleagues found only RPF2(14) , while in
aortic endothelial cells stimulated with minimally modified low density
lipoprotein, Schwartz et al. found a GRO homologue that shares
only 66% identity with RabGRO(6) . This variation in GRO
subtype expression suggests that these cytokines are under specific
regulatory control and may have distinct biological roles depending on
the cell type and stimulus. However, as the effects of GRO-,
-
, and -
on neutrophil chemotaxis and activation are
essentially equivalent(2) , it is likely that any GRO
subtype-specific bioactivities involve cells other than neutrophils.
In conclusion, we have found that rabbit AM produce two GRO
homologues that are distinct from a GRO chemokine produced by rabbit
endothelial cells. We have expressed the predominant GRO homologue
produced by rabbit AM, developed a specific immunoassay, and used this
to show that rabbit AM produce GRO protein in response to both
exogenous (LPS, HKSA, and SiO) and endogenous (TNF-
)
inflammatory stimuli. Finally, we have shown that GRO mRNA and
biologically significant levels of GRO protein are present in the lungs
of rabbits with E. coli pneumonia. The relative contribution
of GRO proteins (particularly in comparison to other
-chemokines)
to the inflammation accompanying lung injury needs to be clarified. In
rabbit models of lung reperfusion injury(38) , acid
aspiration(39) , and bacterial pneumonia(40) , the
closely related
-chemokine IL-8 has been shown to have a prominent
role in mediating pulmonary neutrophil infiltration and tissue injury.
However, rats pretreated with neutralizing antibodies to GRO homologues
have significantly decreased BALF neutrophil accumulation in response
to the intratracheal instillation of LPS(41) . Apart from their
potential role in recruiting neutrophils to the lungs, GRO and other
-chemokines have bioactivities that may be important in other
aspects of pulmonary inflammation, such as mediating chemotaxis and/or
activation of leukocytes other than
neutrophils(6, 42, 43, 44) ,
collagen turnover(4) , and vascular proliferation(8) .
The exact roles of GRO chemokines and related proteins in acute and
chronic inflammatory processes in the lung warrant further study.