By
From the Division of Cell Biology, TVW Telethon Institute for Child Health Research, West Perth, Western Australia 6872
A key rate-limiting step in the adaptive immune response at peripheral challenge sites is the transmission of antigen signals to T cells in regional lymph nodes. Recent evidence suggests that specialized dendritic cells (DC) fulfill this surveillance function in the resting state, but their relatively slow turnover in most peripheral tissues brings into question their effectiveness in signaling the arrival of highly pathogenic sources of antigen which require immediate mobilization of the full range of host defenses for maintenance of homeostasis. However, the present report demonstrates that recruitment of a wave of DC into the respiratory tract mucosa is a universal feature of the acute cellular response to local challenge with bacterial, viral, and soluble protein antigens. Consistent with this finding, we also demonstrate that freshly isolated respiratory mucosal DC respond in vitro to a variety of CC chemokines as well as complementary cleavage products and N-formyl-methionyl-leucine-phenylalanine. This suggests that rapid amplification of specific antigen surveillance at peripheral challenge sites is an integral feature of the innate immune response at mucosal surfaces, and serves as an "early warning system" to alert the adaptive immune system to incoming pathogens.
The host response to invading pathogens is classically
viewed as a two-tiered system, comprising a series of
innate (inflammatory) and acquired (adaptive) immune mechanisms which operate over distinct time scales (1). Thus,
the first line of defense is provided by the rapid recruitment
of phagocytic granulocytes (usually neutrophils) into sites
of tissue injury in response to locally produced chemotactic factors. This is followed up to 48 h later by a second wave
of mononuclear cells containing large numbers of macrophages which are effective in both the uptake of persisting antigen and its subsequent presentation to the T cell
system. In this classical scheme, the adaptive immune system serves as an optional backup to innate host defenses,
and is called upon only in situations where significant
amounts of antigen persist at the challenge site beyond the
time frame of the acute inflammatory response.
While this scheme appears inherently economical, it could
equally be viewed as flawed in at least one respect; viz, the
delayed recruitment of adaptive immune mechanisms into
the host response provides a temporal window for the establishment and spread of incoming pathogens. This danger
would appear to be greatest in the case of pathogens not
previously encountered by the host, i.e., for which neither
antibody nor T-effector memory cells are available. Under
such circumstances, host survival may ultimately be determined by the rapidity with which naive T cells are primed against antigens displayed by the pathogen at the challenge
site, a process which occurs initially in local draining lymph
nodes.
Recent evidence suggest that dendritic cells (DC) function as first-line sentinels in immune surveillance of peripheral tissues (2), including mucosal surfaces such as those in
the lung and airways (3, 4). These DC migrate into peripheral tissues from a circulating monocytelike precursor pool,
and differentiate locally to a stage in which they are specialized for acquisition and processing of antigen, but remain unable to effectively present the antigen locally to T cells (2).
This latter function, in particular a unique capacity for potent activation of naive T cells, is acquired after their migration to regional lymph nodes (2).
Thus, individual DC present a "snapshot"of the antigens
encountered during their transient sojourn through their
respective peripheral tissue sites, presumably including those
antigens derived from incoming pathogens.
The effectiveness of such a surveillance system in the
context of infectious disease is presumably a direct function
of the DC traffic (i.e., cell number/unit time) between
peripheral tissue sites and their respective regional lymph
nodes. In relatively quiescent tissues such as skin and muscle, mean DC transit times are estimated to normally be in
the order of weeks (5). However, DC turnover is considerably more rapid at the main mucosal surfaces in direct
contact with the outside environment (viz, the gastrointestinal and respiratory tracts) where resident populations are
renewed every 3-4 d (7, 8).
Experimental Animals.
Specified pathogen-free adult PVG rats
were supplied by the Animal Resource Centre (Murdoch, Western Australia). All animal experimentation was carried out with
the prior approval of the Institute for Child Health Animal Ethics
and Experimentation Committee which complies with conditions set down by the Australian National Health and Medical
Research Council.
Bacterial Models.
(a) Moraxella catarrhalis was grown in Mueller
Hinton broth, washed extensively with saline, and suspended at
~109 CFU/ml. The suspension was heated at 60°C for 1 h and
passed through a 26 gauge needle several times to break up any
bacterial clumps. Rats were exposed by aerosol to the suspension
for 1 h using a Tri-R inhalation exposure apparatus (Tri-R Instruments, New York). (b) Bordetella pertussis (Welcome strain 28;
provided by Dr. P. Novotny, Kent, England) was grown on
Charcoal agar, washed in sterile PBS, and ~109 organisms in 50 µl were deposited directly onto the tracheal surface of adult rats
by intratracheal intubation.
Viral Model.
Sendai virus was provided by Dr. Jane Allan
(University of Western Australia, Perth, Western Australia) and
grown for 3 d in the allantoic cavity of eggs. Allantoic fluid was
stored at Antigen Sensitization.
For antigen sensitization experiments,
animals were intraperitoneally primed with 100 µg of ovalbumin
(OVA; Sigma Chemical Co., St. Louis, MO) in 0.5 ml PBS containing 10 mg aluminium hydroxide (Wyeth Amphojel). 14 d
later, the animals were challenged with a 30 min aerosol of 1%
(wt/vol) OVA in PBS.
Immunohistochemical Analysis.
Tracheas were removed and
immediately fixed in cold ethanol for 30 min. The tissue was then
rehydrated in PBS, embedded in 100% OCT, and frozen in liquid nitrogen-cooled iso-pentane. Tangential sections 8-10 µm
were cut on a cryostat and immunostained as detailed in reference
4. Eosinophils were identified by cytochemical staining (4). Sections were counterstained with hematoxylin, dehydrated, and
mounted. Primary antibodies used were Ox6 (rat MHC class II;
9), Ox12 (rat kappa light chains on B cells; 9), R73 (rat TcR DC Chemotaxis.
The assay system was based upon that described in 14, except that 6.5 mm Costar Transwells (Costar Corp.,
Cambridge, MA) with polycarbonate membranes (3-µm pore
size) were used. Briefly, DC were enriched to 75-82% purity
from collagenase digests of respiratory tract tissue by flow cytometry gating for Ox6+, Ox12
70°C until used. Adult rats were inoculated intranasally
with 103 HAU in 50 µl of virus containing allantoic fluid. Control animals were similarly inoculated with virus-free allantoic
fluid. Infection of airway epithelium was confirmed by staining
with mAb WS16 against the nucleoprotein antigen of Sendai virus (provided by Dr. A. Portner, St. Jude Children's Research
Hospital, Memphis, TN). There was no influx of DC or T cells
into the airway epithelium of control animals.
;
10), ED2 (rat macrophage; 11), and RP3 (rat neutrophils; 12).
DC identification criteria were pleiomorphic morphology, together with positive staining with Ox6 and negative staining for
Ox12, R73, and ED2, as detailed previously (4, 13).
, and ED2
-cells, as previously described (13). 600 µl medium (RPMI plus 2.5% fetal calf serum)
containing putative chemoattractant was placed in the lower
chamber, and 105 enriched DC in 100 µl medium were placed in
the insert; after incubation for 1 h at 37°C, the top surface of the
inserts was washed free of cells and the insert was fixed in cold
ethanol for 10 min. The polycarbonate membranes were excised,
and the contralateral surface immunostained with MoAb Ox6;
migrating cells were observed under a ×25 objective and enumerated as mean number cells/high power field.
8 M; Sigma Chemical Co.), rat MCP-1,
GRO/KC, GRO
(all at 100 ng/ml), rat RANTES (200 ng/
ml), and murine eotaxin (100 ng/ml) from Peprotech (London,
UK), human MCP-4 (100 ng/ml; Glaxo Wellcome, Geneva, Switzerland), and human IL-8 (100 ng/ml; Genzyme Corp., Boston, MA). The human IL-8 used here was shown to chemoattract rat
neutrophils in this assay, in preliminary experiments.
The present study sought to ascertain whether a similar DC response occurred after challenge of the airways with live infectious organisms (both viral and bacterial pathogens) to which animals had not been previously exposed, and an inert protein antigen (OVA) to which the animals were presensitized by parenteral immunization.
In principle, groups of normal adult rats were subjected to local airways challenge with aerosol or a liquid bolus delivered directly onto the airway surface, containing either heat-killed or live bacteria (M. catarrhalis or B. pertussis, respectively), live parainfluenza type1 virus (Sendai), or OVA; for the latter stimulus, comparisons were made between OVA-preprimed and naive animals. Animals were killed in groups at strategic (generally daily) intervals, and tracheal tissue samples cryopreserved for subsequent immunohistochemical analysis of cell populations within the airway epithelium, using methods detailed previously (4).
The time frame of interest in the present studies was the
duration of the host acute cellular inflammatory responses
triggered by the various stimuli, and the latter was defined
in each model by a series of initial trials. Representative
data are shown in Fig. 1. The acute cellular response to microbial agents depicted in A-C of Fig. 1 demonstrate the
transient influx of PMN, which is the hallmark of the acute
host response to this class of stimuli. These cells are enumerated in tangential sections through the tracheal epithelium after immunostaining with the PMN-specific mAb RP3
(12). The PMN response is most rapid following challenge with proinflammatory bacterial cell wall extract (Fig. 1 A).
In the case of intranasally delivered live virus, we were able
to identify the time of onset of the PMN response (starting
on day 3) as occurring within 24 h of the first detectable
expression of viral protein within infected airway epithelial
cells (Fig. 1 C, arrow). It was of interest to note the intensity
of the T cell chemotactic responses, which were initiated in
both systems which used live pathogens (see particularly
the virus model in Fig. 1 C), in contrast to the inflammatory response to bacterial cell wall products which did not
include a significant T cell component.
These responses contrast with that of animals challenged with an inert protein antigen to which they are presensitised (Fig. 1 D); the acute phase of this latter response was characterized by the influx of eosinophils as opposed to PMN, and a relatively small number of T cells. No cellular response was seen in unprimed animals. Macrophage influx was not a significant feature of the initial cellular response in any of these models.
However, the feature common to all these challenge models is the transient waves of DC which are recruited into the epithelium coincident with the early phase of respective cellular inflammatory responses. The most intense response is that triggered by high-level local stimulation with pro-inflammatory bacterial cell wall extract, in which intraepithelial airway DC density transiently increases to levels up to threefold those of resting tissue (Fig. 1 A), before their migration on to regional lymph nodes (13). The more subtle stimuli, such as infection with live virus and bacteria and even inhalation of inert nominal antigen, also clearly evoke qualitatively similar responses, involving the transient build up of intraepithelial DC numbers in challenged airway tissue to levels that are two- to threefold those of resting tissues.
We have additionally screened a broad panel of chemotactic agents for capacity to attract respiratory mucosal DC
in vitro (Fig. 2). Consistent with findings in Fig. 1 indicating
rapid in vivo recruitment of DC in response to bacterial
stimuli, complement cleavage products, and f MLP exhibit
potent chemoattractant activity in vitro. Additionally, MCP-1,
-4, RANTES, and eotaxin, all of which are members of the
CC chemokine family, exhibited significant chemoattractant activity, whereas the CXC chemokines IL-8, GRO/KC,
and GRO/, were inactive. These findings are comparable to those recently reported using putative DC cultured from
human blood in the presence of GM-CSF and IL-4, which
also responded to a broad spectrum of chemotactic stimuli
(14); however, the blood-derived DC were unresponsive
to MCP-1, which may reflect subtle species differences in
receptor expression, or possibly variations related to the degree of maturation of respective DC populations at the
time of assay.
These findings indicate that current perceptions of the scope of innate host defense responses need to be broadened to include the rapid activation of an in-built "early warning system" at sites of inflammation to facilitate the most rapid possible flow of information on the presence of previously unencountered antigens to the adaptive immune system. This mechanism fulfills an essential prediction of the model proposed recently (1, 15) for the evolution of the immune system, viz, that DC may be evolution's answer to the problem posed by organisms that evade primitive (innate) first-line defense systems.
Address correspondence to Andrew S. McWilliam or Patrick G. Holt, Division of Cell Biology, TVW Telethon Institute for Child Health Research, PO Box 855, West Perth, Western Australia 6872. T.N.C. Well's present address is Geneva Biomedical Research Institute, Glaxo Wellcome Research & Development, Geneva.
Received for publication 10 June 1996
This work was supported by Glaxo Wellcome, the Raine Foundation of Western Australia, and the National Health and Medical Research Council of Australia.1. | Janeway, C.A.. 1992. The immune response evolved to discriminate infectious nonself from noninfectious self. Immunol. Today. 13: 11-16 [Medline] . |
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