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*
Department of Pediatrics and the Immunology Program, Stanford University School of Medicine, Stanford, CA 94305;
Department of Molecular Biology, Immunex Corporation, Seattle, WA 98101; and Departments of
Medicine and
§
Comparative Medicine, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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6–12 h later by a late phase response, in which there is the onset
of progressive inflammation of the lung parenchyma and airway with
eosinophils and mononuclear leukocytes (4) and the
recurrence of bronchoconstriction. The recruitment of leukocytes to the
lungs of allergic individuals is believed to be primarily mediated by
CC-chemokines, a number of which, including macrophage inflammatory
protein (MIP)4-1
,
monocyte chemoattractant protein (MCP)-1, MCP-3, MCP-4, eotaxin, and
RANTES, are expressed in increased amounts in the lungs of asthmatic
patients (2) and in rodents with experimentally induced
asthma (3, 5). The pleiotropic functions of CC-chemokines suggest that they not only play a critical role in the recruitment of inflammatory cells during the late phase response (6), but potentially may also directly enhance Ag-driven T cell activation, cytokine production and differentiation (7, 8), IgE synthesis by B cells (9), and dendritic cell Ag presentation (6). In addition to their role in inflammation and adaptive immune responses, CC-chemokines are also important in the normal homeostasis of lymphocytes via their effects on their circulation and homing between lymphoid and nonlymphoid organs (6, 10). The presence of CCRs on nonhematopoietic cells, such as smooth muscle cells (11, 12), also raises the possibility that CC-chemokines by exert effect in tissues that are not directly related to their role in innate and adaptive immune responses.
The functional importance of CC-chemokines in eliciting inflammation
and airway hyperreactivity in rodent asthma models was demonstrated in
studies in which CC-chemokine function was systemically blocked using
neutralizing Abs, amino-terminal modified CC-chemokines that act as
receptor antagonists (5) or by selective gene targeting
(13, 14). Although there is growing evidence for the
biological importance of locally elaborated CC-chemokines in the lung
in allergen-induced asthma, it remains unclear whether selective
blockade of CC-chemokines in the lung compartment has potential as a
therapy for the inflammatory and physiologic abnormalities in this
disease. Broad spectrum chemokine/CCR antagonists have been
proposed to be more advantageous than selective antagonists
(15). This view (6, 16) is supported by a
recent study that demonstrated a distinct temporal sequence of the
pulmonary expression and role of the individual chemokines MCP-1,
eotaxin and MIP-1 following repeated aeroallergen challenge of
sensitized mice (5). It is also plausible that the
relative importance of a particular CC-chemokine in asthma may be
influenced by the context of provocation of acute attacks. For
example, because respiratory syncytial virus infection of airway
epithelial cells induces their production of CC-chemokines, such as
RANTES (17, 18), it is possible that RANTES might play a
more important role in asthmatic airway hyperreactivity and
inflammation triggered by respiratory viral infection than by allergen
alone.
Viral-derived CC-chemokine inhibitor (vCCI) is a 35-kDa secreted virulence protein encoded in the genome of pox viruses that enhances their evasion of host immune responses. It is the only CC-chemokine inhibitor known to bind specifically and generically to CC-chemokines (human and rodent) with high affinity and to completely inhibit their biological activity; the mechanism is one of competitive inhibition of CC-chemokine receptors (19). The dissociation constant of vCCI is in the subnanomolar range for all 15 different CC-chemokines that have been tested, and the affinity of CC-chemokines for vCCI is often higher than that for their native CCRs (19), arguing for an unusually effective inhibition. Although vCCI has no apparent amino acid or structural homology to known mammalian or other eukaryotic proteins, including CCRs (20), its broad high-affinity CC-chemokine binding suggested that it might be particularly useful dissecting the biological role of CC-chemokines in inflammatory diseases and as a clinical anti-inflammatory agent. To examine the potential for local and generic blockade of CC-chemokines to treat asthma, we determined the effect of the intranasal (i.n.) administration of a vCCI to the respiratory tract in a murine model of allergen-induced asthma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The entire coding region of the 32-kDa vCCI protein from the cowpoxvirus genome was fused to a segment encoding the Fc region of human IgG1, expressed and purified as previously reported (19).
Allergen-induced pulmonary and peritoneal disease
Female BALB/cJ mice, 8–12 wk of age, were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained under specific pathogen-free conditions. The dose of crystalline OVA (Pierce, Rockland, IL) given was 100 µg unless specified. In the standard protocol, OVA-treated mice received single i.p. injections of alum-precipitated OVA on days 1 and 14, and i.n. OVA in normal saline on days 14, 24, and 25 (21). Two hours before each i.n. dose of OVA, mice were briefly anesthetized by i.p. injection with 100 µmg of ketamine and 1.5 mg of xylazine to facilitate pulmonary aspiration, and given 50 µmg of vCCI or purified human IgG1 (Sigma, St. Louis, MO) in 50 µml of normal saline by the i.n. route. Unsensitized mice, which were treated in parallel, received alum alone for the two i.p. injections and normal saline for all four i.n. administrations (21). In certain experiments, as indicated, mice were sensitized on day 14 by i.p. injection with a mixture of OVA and KLH (50 µmg each) in alum instead of OVA alone, and were then treated as described above. For experiments evaluating the effect of i.n. vCCI treatment on extrapulmonary inflammation, mice were treated with OVA according to the standard protocol on days 1, 14, and 24, and on day 25 were treated with either 50 µg of vCCI or hIgG1. One hour later, mice were challenged by i.p. injection with either 100 µg of OVA in PBS (pH 7.4) or with PBS alone; mice in both of these groups received a final i.n. challenge dose of OVA concurrently.
Bronchoalveolar and peritoneal lavage
Lavage was performed following the euthanasia of mice on day 26. Bronchoalveolar lavage (BAL) of the right lung for leukocytes was performed as previously described (21). Peritoneal lavage was performed with 5 ml of PBS, 0.1% BSA, 0.5 mM EDTA. Microscope slides of cells obtained by BAL or peritoneal lavage were prepared by cytocentrifugation and stained with Diff-Quik (Fischer Scientific, Pittsburgh, PA). Differential cell counts were performed by counting a least 300 cells per slide.
Lung histology
Lung tissue from euthanized mice was fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were coded and evaluated by a morphologist (D.H.L.) who was blinded to the treatment groups.
Pulmonary function testing
Mice were anesthetized with pentobarbital 24 h after the
final dose of i.n. OVA, and then were mechanically ventilated in a
plethysmograph to determine resistance (R) and dynamic compliance
(Cdyn) (21). For each animal, pulmonary function was
determined basally and following i.v. injection of 120 µmg/kg of
acetyl-ß-methacholine (Mch; Sigma, St. Louis, MO in normal saline.
This dose results in an 40–60% reduction in dynamic compliance and
a 300–450% increase in resistance in OVA-sensitized/challenged
BALB/cJ mice (21). The methods used were similar to those
previously described (21), except that the chest was not
opened and a commercial plethysmograph, transducer, and amplifier
system was used for data collection (Buxco, Sharon, CT). Enhanced
pause (Penh) was assessed by whole body plethysmography (Buxco) of
conscious, unrestrained mice (22).
OVA-specific IgE, KLH-specific IgG1 and total IgE ELISAs
OVA-specific and total IgE determinations were performed as previously described using TMB substrate (Kierkegaard & Perry, Gaithersburg, MD) for development (23). Pooled plasma from BALB/c mice immunized twice with alum-precipitated OVA was used as an internal standard, and was arbitrarily assigned an OVA-specific IgE titer of 10 U/ml. KLH-specific IgG1 ELISAs were performed as for OVA-specific IgE ELISA, except that KLH (Pierce) was used to coat wells and the IgE mAb was replaced by peroxidase-conjugated goat anti-mouse IgG1 Ab (Southern Biotechnology Associates, Birmingham, AL).
OVA-specific T cell responses in vitro
Bronchial lymph node cells were isolated and cultured as
previously described (24) with medium alone or with OVA
(100 µmg) for 96 h (24). IL-4, IL-5, and IFN-
content in cell culture supernatants was determined by ELISA
(PharMingen, San Diego, CA).
Statistical analysis
Statistical significance was determined using either the one-way ANOVA or the two-tailed, unpaired Student’s t test as appropriate. Differences between means were considered significant when p values were less than 0.05.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A murine asthma model was used in which the combination of OVA
administered twice i.p. and three times i.n. results in disease that
faithfully mimics cardinal features of human allergen-induced asthma,
with intense eosinophilic and mononuclear inflammation of the lung,
marked airway hyperreactivity, high levels of circulating IgE, and
expression of Th2 cytokines by lung-associated lymph node cells
(21, 23). A similar murine OVA-induced asthma model
induces the pulmonary expression of a number of
CC-chemokines, including eotaxin, RANTES MCP-1, MCP-5, and
MIP-1 (5). To evaluate the effect of the blockade of
local intrapulmonary chemokines on experimental allergen-induced
asthma, vCCI in the form of a dimeric fusion protein with the human
IgG1 Fc domain, or an equivalent amount of purified human IgG1 (hIgG1),
was administered i.n. 2 h before each of three i.n. doses of OVA
on days 14, 24, and 25. vCCI treatment significantly reduced the number
of total leukocytes and eosinophils in BAL fluid obtained on day 26
compared with mice that received an equivalent amount of hIgG1 (Fig. 1
A). The noneosinophil
leukocytes of the vCCI-treated and hIgG1-treated groups were >95%
lymphocytes and mononuclear phagocytes (data not shown), in agreement
with previous results (21). In contrast, OVA
sensitization/challenge resulted in intense inflammation of the lung
parenchyma in mice that received hIgG1, including in the perivascular
and peribronchiolar regions (Fig. 2B), in agreement with
previous results (21). vCCI administration dramatically
reduced this inflammation, particularly in the peribronchial region
(Fig. 2A), which had a histological appearance similar to
that of tissue from unsensitized mice (Fig. 2C).
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B). In contrast, it did not affect the recruitment of
lymphocytes, eosinophils and monocytes into the peritoneal
cavity of mice subsequently challenged with OVA i.p. (Fig. 1C). Together, these results indicated that the
vCCI-mediated inhibition of CC-chemokine function by i.n.
administration was highly effective for the local blockade of
eosinophil and mononuclear phagocyte lung inflammation, while sparing
effects on inflammatory reactions in the peritoneum. Blockade of airway hyperreactivity by vCCI
Asthma is characterized by airway hyperreactivity, in which there
is an exaggerated increase in airway resistance (R), a measure of the
amount of pressure to achieve a given airflow, and a decrease in
dynamic compliance (Cdyn), a measure of how distensable the lungs are
at end-expiration, in response to provocative stimuli, such as Mch
(21, 25). We found that local intrapulmonary
administration of vCCI was strikingly effective in preventing the
increased airway hyperreactivity characteristic of the late phase
response (21). In mice receiving vCCI there was both lower
R (Fig. 3, A and B)
and higher Cdyn (Fig. 3, C and D) after Mch
provocation than in mice that received hIgG1. The effect on R was
particularly striking in that vCCI treatment almost completely reduced
the level of this physiologic parameter to the normal values of
unsensitized mice. vCCI treatment also increased basal Cdyn compared
with that of hIgG1-treated mice (Fig. 3C), although this did
not quite achieve statistical significance (p =
0.06 by Student’s t test). We did not observe any effect of
vCCI treatment on R or Cdyn of unsensitized mice (data not shown),
suggesting that locally elaborated CC-chemokines in the airway or
adjacent tissues may have a minimal effect on normal pulmonary
physiological function, at least in the short term. Finally, vCCI
abrogated by 50% the increases in Penh, a physiological measurement
that correlates with airway obstruction and increased R
(22), in sensitized mice in response to Mch challenge
compared with mice treated with hIgG1 (data not shown). This indicated
that these beneficial effects on pulmonary function applied to
conscious, spontaneously breathing animals.
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Respiratory tract administration of vCCI does not influence systemic immune responses
Previous studies have found that CC-chemokines, in addition to
their chemotactic effects, can augment in vitro lymphocyte functions
that are critical for allergic disease, such as the production of IgE
by B cells (9) and the production of IL-4 by T cells
(8). To determine whether vCCI administration by the i.n.
route altered in vivo Ag-specific responses involved in allergic
disease, we analyzed the circulating levels of total and OVA-specific
IgE at day 26 of the standard protocol. Mice that received
alum-precipitated OVA by the i.p. route had an 25-fold increase in
total IgE levels compared with sham immunized mice in agreement with
previous results (21, 23) and vCCI administration had no
influence on this increase (Fig. 4A). In addition, the levels
of KLH-specific IgG1 in hIgG1-treated mice (43 ± 11 U/ml;
mean ± SEM, n = 6) were not significantly
different (p = 0.1 by Student’s t
test) from those in vCCI-treated mice (125 ± 42 U/ml,
n = 6) following KLH given with OVA i.p. on day 14.
Because >90% of protein allergen-specific IgE production by B cells
and their plasma cell derivatives is dependent on IL-4 production by
CD4 T cells (27), these results also suggest that vCCI
treatment did not inhibit CD4 T cell activation, expansion, or Th2
differentiation in vivo. This was supported by studies demonstrating
that OVA-stimulated bronchial lymph node T cells from vCCI-treated mice
produced similar amounts of IL-4, IL-5, and IFN- (Fig. 4B) and proliferated equally, based on the incorporation of
[3H]thymidine (data not shown), as cells from
hIgG1-treated mice. Thus, vCCI treatment did not alter T cell-dependent
immune responses in vivo, including those associated with the local
lymphoid tissue draining the lung, and appeared to act locally within
the airway and adjacent parenchymal tissue to inhibit asthmatic
inflammation and airway hyperreactivity.
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Acknowledgments |
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Footnotes |
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2 K.D. and Y.X. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. David B. Lewis, Division of Immunology and Transplantation Biology, Room H-307, Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5208.
4 Abbreviations used in this paper: MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; i.n., intranasal; vCCI, viral-derived CC-chemokine inhibitor; BAL, bronchoalveolar lavage fluid; R, resistance; Cdyn, dynamic compliance; Penh, enhanced pause; Mch, methacholine; h, human.
Received for publication May 9, 2000. Accepted for publication June 28, 2000.
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selectively enhance immunoglobulin (IgE) and IgG4 production by human B cells. J. Exp. Med. 183:2397.This article has been cited by other articles:
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