By
From the * Infection and Immunity Group, Department of Biology, National University of Ireland,
Maynooth, County Kildare, Ireland; and the Department of Veterinary Pathology, University College
Dublin, Dublin, Ireland
Using a murine respiratory challenge model we have previously demonstrated a role for Th1
cells in natural immunity against Bordetella pertussis, but could not rule out a role for antibody. Here we have demonstrated that B. pertussis respiratory infection of mice with targeted disruptions of the genes for the IFN- receptor resulted in an atypical disseminated disease which was
lethal in a proportion of animals, and was characterized by pyogranulomatous inflammation
and postnecrotic scarring in the livers, mesenteric lymph nodes and kidneys. Viable virulent
bacteria were detected in the blood and livers of diseased animals. An examination of the
course of infection in the lung of IFN-
receptor-deficient, IL-4-deficient and wild-type mice
demonstrated that lack of functional IFN-
or IL-4, cytokines that are considered to play major
roles in regulating the development of Th1 and Th2 cells, respectively, did not affect the kinetics
of bacterial elimination from the lung. In contrast, B cell-deficient mice developed a persistent infection and failed to clear the bacteria after aerosol inoculation. These findings demonstrate
an absolute requirement for B cells or their products in the resolution of a primary infection
with B. pertussis, but also define a critical role for IFN-
in containing bacteria to the mucosal
site of infection.
Respiratory infection with the gram-negative coccobacillus Bordetella pertussis results in whooping cough, a
major cause of morbidity and mortality in human infants. It
is well known that during colonization of the respiratory
tract this bacterium can specifically adhere to ciliated epithelium, however a number of studies have suggested that
B. pertussis may also exploit an intracellular niche during
infection. Persistence of B. pertussis within murine and rabbit alveolar macrophages has been described (1, 2), and it has
recently been reported that B. pertussis can invade and survive within human macrophages (3). Although controversial, these studies suggest that intracellular localization
may be an important mechanism in the disease process.
Recovery from a primary B. pertussis infection provides
long lasting protective immunity against subsequent disease, however it is not clear which components of the immune response confer protection or contribute to bacterial
clearance. Passive and active immunization studies in mice
have shown that antibody can induce varying degrees of protection against either aerosol or intracerebral challenge (6,
7). In contrast, clinical trials of an acellular pertussis vaccine
consisting of chemically detoxified pertussis toxin (PT)1
and filamentous hemagglutinin (FHA), failed to demonstrate a correlation between serum antibody responses to
PT or FHA and protection (8). Furthermore, studies using
a murine respiratory infection model have demonstrated a
role for B. pertussis-specific CD4+ T cells that secrete IL-2
and IFN- The development of mouse strains with gene-targeted
disruptions in key components of the immune response has
provided insights into the mechanisms of antimicrobial defense and pathology (13). Studies with gene knockout mice
have highlighted the central role of T cells in immunity
against the intracellular pathogens Listeria monocytogenes and
Mycobacterium tuberculosis (13, 14). In the present study we
have employed mice with targeted disruption of the genes
for IL-4 (IL-4 Mice.
All mice used were commercially obtained (B+K Universal Ltd., Hull, UK) and bred and maintained according to the
guidelines of the Irish Department of Health. The IFN- Aerosol Infection.
Respiratory infection of mice was initiated
by aerosol challenge by a modification of the method described
by Sato et al. (18). Phase 1 B. pertussis Wellcome 28 (W28; a kind
gift from Keith Redhead, National Institute for Biological Standards and Control, South Mimms, Herts, UK) was grown under
agitation conditions at 37°C in Stainer-Scholte liquid medium.
Bacteria from a 48-h culture were resuspended at a concentration
of ~2 × 1010 CFU/ml in physiological saline containing 1%
casein. The challenge inoculum was administered to mice as an
aerosol over a period of 15 min by means of a nebulizer as previously described (19). Groups of three or four mice were killed at
various times after aerosol challenge to assess the number of viable
B. pertussis in the lungs, livers, or peripheral blood.
Enumeration of Viable Bacteria.
Lungs or livers were removed
aseptically from infected mice and homogenized in 1 ml of sterile
physiological saline with 1% casein on ice. 100 µl of undiluted
homogenate or of serially diluted homogenate from individual
lungs or livers were spotted in triplicate onto Bordet-Gengou
agar plates and the number of CFU was estimated after 5 d of incubation at 37°C. Results are reported as the mean viable B. pertussis for either individual lungs or livers from at least three mice
per time point per experimental group. The limit of detection
was ~log10 0.5 CFU per organ. In experiments on disseminating
infection, blood was aseptically sampled from a peripheral vein
and assessed for the presence of viable B. pertussis. Samples of
whole blood or peripheral blood mononuclear cells, isolated by
metrizamide gradient centrifugation and lysis of red blood cells as
previously described (20), were plated on Bordet-Gengou agar
and the mean CFU/ml determined.
Bacterial Antigens.
Heat killed B. pertussis was prepared by incubation of cells at 80°C for 30 min. Inactivation was confirmed by
demonstrating that bacteria could not be cultured from these preparations. Genetically detoxified recombinant PT (PT-9K/129G),
native FHA and pertactin prepared from B. pertussis were kindly
provided by Rino Rappuoli (Chiron, Sienna, Italy). All antigen
preparations were of clinical grade, endotoxin free and produced
according to good manufacturing practice.
Analysis of B. pertussis-specific Antibody Production.
The levels of
serum antibody to B. pertussis were determined by ELISA using
B. pertussis sonicated extract (5 µg/ml) to coat the plates. Bound
antibodies were detected using alkaline phosphatase-conjugated anti-mouse IgG (Sigma Chem. Co. Poole, Dorset, UK). Mouse
IgG subclasses were determined using alkaline phosphatase conjugated anti-mouse IgG1 (clone G1-65), IgG2a (clone R19-15),
IgG2b (R12-3), or IgG3 (R40-8L) purchased from PharMingen
(San Diego, CA). Antibody levels are expressed as the mean endpoint titers calculated by regression of the straight part of a curve
of OD vs. serum dilution to a cutoff of two standard deviations
above background control values.
T Cell Proliferation Assays.
Spleen cells from naive and infected mice were resuspended at 2 × 106/ml in RPMI-1640 medium (Gibco, Paisley, UK) supplemented with 8% heat-inactivated
FCS (endotoxin content 0.071 ng/ml; Gibco), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were cultured for 4 d with
heat killed B. pertussis (105, 106, or 107/ml), heat-inactivated PT
(0.2-5.0 µg/ml), FHA (0.2-5.0 µg/ml), pertactin (0.2-5.0 µg/ml),
or medium alone and [3H]thymidine (0.5 µCi/well; specific activity 2.0 Ci/mmol) was added for the final 4 h of culture as described (9). Results are given as the mean cpm of [3H]thymidine
incorporation for triplicate cultures for groups of at least three
mice, after subtraction of the background responses with medium
alone.
Analysis of Cytokine Production.
Spleen cells from infected mice
were cultured with B. pertussis antigens as described for the proliferation assay. Supernatants were removed after 24 h to determine
IL-2 and after 72 h to determine IFN- Pathology.
Killed animals from 6 experiments (60 IFN- Respiratory infection with B. pertussis proved lethal in a number of IFN-
Examination of 60 B. pertussis-infected IFN-
In addition to areas of pyogranulomatous inflammation,
infiltrates of macrophages and neutrophils were present in the
alveolar septae and lumens of the lungs. Areas of consolidation were common in the lungs of each of 15 B. pertussis
infected IFN- Carriage of B. pertussis to secondary non pulmonary foci
was via the blood (Table I). Viable bacteria could be recovered from the blood of aerosol infected IFN- Table I.
Disseminating B. pertussis infection in IFN- (Th1 cells) in pulmonary clearance (9).
Adoptive transfer experiments demonstrated that CD4+ T
cells from convalescent mice were capable of mediating B. pertussis clearance from nude or sublethally irradiated mice
in the absence of a detectable serum antibody response (9).
However this study, as well as the results of passive immunization experiments reported by others (6, 7, 12), did not
rule out a role for CD4+ T cells in providing help for a
protective antibody response against B. pertussis.
/
), the IFN-
receptor (IFN-
R
/
), or
the immunoglobulin µ-chain (Ig
/
) to elucidate the role
of cytokines and the adaptive immune response to clearance of a primary B. pertussis respiratory infection. We
demonstrate that in addition to the known requirement for
CD4+ T cells, clearance of this bacterium is dependent on
B cells. Studies addressing the contribution of different cytokines demonstrated that functional IFN-
or IL-4 were
not essential for bacterial clearance from the lungs, but revealed that IFN-
was critical in preventing bacterial dissemination.
R
/
mice, in which IFN-
is nonfunctional (15), were used with the
kind permission of Dr. M. Aguet (University of Zurich, Switzerland). These mice were generated from the wild-type 129Sv/Ev
(H-2b) strain (15), which were employed as the control mice in
these experiments. The IL-4
/
mice (IL-4T strain) (16) were
used with the kind permission of Dr. Werner Muller (University
of Cologne, Germany), and the Ig
/
(µMT strain) (17) were
used with the kind permission of Dr. Klaus Rajewsky (University
of Cologne, Germany). The Ig
/
and IL-4
/
mice, were generated from wild-type C57BL/6 ( H-2b) mice, which were employed as control mice in these experiments. Unless otherwise
stated, all mice were 8-12 wk old at the initiation of experiments.
, IL-4, and IL-5 concentrations as previously described (9). In brief, IL-2 release was assessed by testing the ability of culture supernatants to support the
proliferation of the IL-2-dependent cell line CTLL-2 and the
concentrations of murine IL-4, IL-5, and IFN-
were determined
by immunoassay using commercially available antibodies (PharMingen). Concentrations were determined by comparing either
the proliferation or the OD for test samples with a standard curve
for recombinant cytokines of known potency and concentration.
R
/
,
28 Ig
/
, 28 IL-4
/
, 28 C57BL/6, and 54 129Sv/Ev mice) were
examined for signs of pathology. Furthermore in two separate
challenge experiments necropsies were specifically performed on
15 B. pertussis infected IFN-
R
/
mice, 4 wild-type 129Sv/Ev
mice and 4 uninfected IFN-
R
/
mice after euthanasia with
pentobarbitone sodium. Liver, lungs, spleens, kidneys, and brains
were placed in 10% neutral buffered formalin. After fixation, the
tissues were dehydrated and embedded in paraffin. Sections were
stained with hematoxylin and eosin (H & E) for histopathological
examination. For demonstration of B. pertussis antigen, paraffin
sections were stained using a commercial B. pertussis-specific antiserum (Difco Laboratories, Detroit, MI) at a dilution of 1/200 using an avidin-biotin technique (Vectastain Elite ABC; Vector
Laboratories, Burlingame, CA). Sections were counterstained with
hematoxylin.
Disseminating Infection in IFN-R
/
Mice after B. pertussis
Respiratory Challenge.
R
/
mice, particularly in mice younger than 8 wk and those that received a high challenge inoculum (Fig. 1 and data not shown).
Death appeared to result from organ failure associated with
disseminating disease; B. pertussis infected IFN-
R
/
mice
showed abnormal pathology not observed in the wild-type 129Sv/Ev mice. Furthermore, IFN-
R
/
mice that survived the challenge frequently showed overt lesions, visible
macroscopically, in the liver, lungs, kidneys, spleen, and
mesentery. All wild-type 129Sv/Ev mice (54 mice, 6 experiments) challenged with the same inoculum of bacteria
survived the infection and lesions were confined to the
lungs. All Ig
/
and IL-4
/
or C57BL/6 mice survived
more than 20 wk after B. pertussis challenge.
Fig. 1.
Survival of IFN-R
/
mice after B. pertussis challenge. In
one experiment groups of 24 IFN-
R
/
(
) or 24 wild-type 129Sv/Ev
(
) mice were aerosol challenged with B. pertussis which resulted in an
inoculum of 6 × 104 CFU/lung, determined from a sample group killed
2 h after challenge. In a separate experiment 6 IFN-
R
/
mice (
) received a higher dose challenge, which resulted in 1 × 106 CFU/lung 2 h
after challenge. Six wild-type 129Sv/Ev mice (
) exposed to the higher
dose of bacteria all survived the challenge (displayed offset for clarity).
[View Larger Version of this Image (13K GIF file)]
R
/
mice
killed between 7-100 d after challenge (6 experiments), revealed evidence of gross pathological changes outside the
lungs in 80% of animals. The most common examination
features were multiple pale, firm nodules up to 10 mm in
diameter projecting above the capsular surface of the liver.
Similar nodular lesions were visible in the kidneys, spleens,
and mesentery, but these were smaller than those in the
livers. In two separate experiments histopathological examination was performed on 15 B. pertussis infected IFN-
R
/
24 d after challenge and atypical liver pathology was observed in 100% of animals. The histological appearance of the
lesions was of pyogranulomatous inflammation and postnecrotic scarring. Aggregates of neutrophils and macrophages
adjoined the areas of necrosis and infiltrates of lymphocytes
and plasma cells (Fig. 2, a and b). Fibroblastic proliferation
and collagenization were prominent particularly in the livers. Localized infiltrates of neutrophils, macrophages and
lymphoid cells observed in the brains of a minority of surviving IFN-
R
/
mice were largely confined to the leptomeninges (Fig. 2 c and data not shown).
Fig. 2.
Pathological findings in IFN-R
/
mice after
respiratory infection with B. pertussis. Mice were challenged
with 2 × 1010 CFU/ml giving 6 × 104 CFU/lung 2 h later. (A)
Liver: aggregates of neutrophils
and macrophages with reactive
changes in adjoining hepatocytes.
(B) Mesentery: macrophages and
neutrophils surround a central
area of necrosis (necrotic pyogranuloma). (C) Brain: neutrophils, macrophages and fibrin deposits in the leptomeninges. (D)
Lung: granular basophilic debris
(arrows) in the alveolar spaces with
macrophage and neutrophil infiltration primarily in alveolar walls.
(A-D, hematoxylin and eosin [H
& E] staining) (E) Lung: B. pertussis
antigen in the lumen of alveoli
and a bronchiole (arrow). Anti-B.
pertussis, haematoxylin counterstain. (F) Mesentry: intracytoplasmic deposits of B. pertussis antigen in macrophages in an area of
pyogranulomatous inflammation.
Anti-B. pertussis, haematoxylin
counterstain. Results are representative of 15 mice from two
challenge experiments. Similar
pathological lesions were observed
in tissue from all mice examined,
except for the brain, where lesions
were visible in only 3 out of 7 mice
examined. Original magnifications: (A, C-F) ×400; (B) ×200.
[View Larger Versions of these Images (157 + 157 + 144 + 152 + 153 + 154K GIF file)]
R
/
mice examined. Granular deposits of
basophilic debris observed in the alveoli and bronchioles
correlated with the distribution of B. pertussis antigen (Fig.
2, d and e). Deposits of B. pertussis antigen were also evident in macrophages in areas of pyogranulomatous inflammation in mesenteric lymph nodes (Fig. 2 f ).
R
/
mice between days 3 and 11, and from the liver between
days 7 and 56 after challenge, but viable bacteria could not
be cultured from sites outside the lung beyond day 56. Analysis of the CFU counts from fractionated blood samples demonstrated that viable B. pertussis were associated
with the mononuclear cell fraction (data not shown). No
evidence of bacterial dissemination was observed in B. pertussis infected Ig
/
and IL-4
/
or C57BL/6 mice (Table
I) and all animals survived the challenge. Furthermore,
there was no evidence of pathological changes in uninfected
IFN-
R
/
mice or outside the lungs of B. pertussis infected
129Sv/Ev, IL-4
/
, Ig
/
or C57BL/6 mice. A minority of
B. pertussis infected Ig
/
mice (3/28) displayed lung consolidation and granulomas, limited to a single lobe.
R
/
mice
Mice
B. persussis (log10 CFU)
Lungs
Blood
Liver
IFN-
R
/
6.6 (0.2)
3.0 (2.5)
2.0 (1.3)
129Sv/Ev
5.7 (0.4)
Ig
/
5.8 (0.4)
C57BL/6
4.9 (0.3)
B. pertussis colony counts recovered from lungs (CFU/lung) and whole
blood (CFU/ml) 10 d after challenge and from liver (CFU/liver) 24 d
after aerosol challenge with 2 × 1010 CFU/ml B. pertussis (time points
reflect peak bacterial recovery from tissue, detectable in the blood of infected IFN- R
/
from days 3-11 and in the liver from days 7-56 after
challenge). Results are mean (SE) values for 4 mice tested individually
in triplicate and are representative of two experiments. The lower detection limit was 0.5 log10 CFU per organ or per ml blood.
*
Undetectable bacteria.
To examine the role of Th1 and Th2 cytokines on
the course of infection in the lungs, the kinetics of bacterial
clearance was monitored by performing CFU counts on
the lungs of wild-type and gene disrupted mice at intervals
after challenge. Although IFN-R
/
mice displayed an
atypical disease which proved lethal in ~30% of infected
mice, the kinetics of bacterial clearance from the lungs of
surviving animals was not significantly different from the
wild-type 129Sv/Ev mice (Fig. 3). There was no significant difference in the kinetics of bacterial clearance from
the lungs of IL-4
/
and the wild-type C57BL/6 mice
(Fig. 3). We have previously reported that MHC and non-MHC differences between mouse strains influence murine
survival in the B. pertussis intracerebral challenge model (11). Here we show that unlike BALB/c (H-2d) mice
which reproducibly clear a respiratory challenge within 40 d (9), C57BL/6 and 129Sv/Ev (both H-2b) do not completely clear a respiratory challenge until up to 100 d after
challenge (Fig. 3).
Aerosol infection of Ig/
mice resulted in a persistent
chronic lung infection and a failure to clear the bacteria
from the lungs (Fig. 3 C). However B cell knockout mice
were not overwhelmed by the infection and survived to
the termination of the experiment 20 wk after challenge.
The pattern of infection was similar to that seen in athymic
nu/nu BALB/c mice (9), thus demonstrating a requirement
for B cells as well as T cells in resolution of B. pertussis respiratory infection.
The contribution of antigen-specific B cells and
specific Ig to the resolution of B. pertussis infection of humans and experimental animals is controversial (6). The
development of the specific antibody response after aerosol
challenge was characterized in normal mice and in mice
with targeted gene disruptions. The results shown in Table
II confirm that Ig/
mice, which lack mature B cells, fail to
mount an IgG antibody response. In contrast, wild-type
C57BL/6 and 129Sv/Ev mice, as well as the IL-4
/
and
IFN-
R
/
gene knockout mice develop B. pertussis-specific serum antibody by day 24 after challenge, which increases in titer until at least day 100. Higher antibody titers
were observed early after infection in IFN-
R
/
mice,
when compared with the wild-type strain. Conversely IL-4
/
mice produced lower titers of specific antibody than
that observed in wild-type C57BL/6 mice at early time
points. An examination of the antibody isotypes revealed
that the predominant IgG subclass detected was IgG2a,
with little or no IgG1, except in IFN-
R
/
mice which
displayed higher titers of IgG2b and lower levels of IgG2a
(Fig. 4). This pattern was consistent between analyses on
serum samples recovered 24, 43, and 100 d after challenge.
|
Cell-mediated Immune Responses in B. pertussis-infected Gene Knockout Mice.
The development of systemic cell-mediated immune responses in B. pertussis infected animals is
thought to be central to clearance of the bacteria from the
lungs (9). Positive spleen cell proliferative responses to
heat killed B. pertussis could be detected in gene knockout
and wild-type mice at day 14 after aerosol challenge (data not
shown). Although relatively high background responses were
observed with medium alone, this has previously been reported during B. pertussis infection of mice and children
(21, 22) and may reflect activation of cells in vivo. The
proliferative response to the putative protective antigens
showed some variation; responses to PT were strongest in
IFN-R
/
mice and were detectable in this group from
day 14, but were detectable in all animals by day 43 (Fig. 5 A).
Unlike the C57BL/6 wild-type mice, responses to FHA
could not be detected in IL-4
/
mice until 24 d after challenge. Responses to pertactin could not be detected from
any mice before day 24. The B. pertussis-specific proliferative responses of Ig
/
mice were compromised compared
with the wild-type C57BL/6 strain. Proliferative responses
to B. pertussis and component antigens were detected on
day 43 (Fig. 5 A), but this had declined by day 100 and
positive proliferative responses could only be detected in
Ig
/
mice at time points beyond 100 d against whole bacterial preparations at high concentrations. Responses to the
putative protective antigens PT, FHA and pertactin could
not be restored by the addition of MHC-matched APC to
the cultures (data not shown).
An examination of the pattern of cytokines induced by
B. pertussis infection of gene knockout mice and their controls revealed that spleen cell preparations from all mice secreted IL-2 in an antigen-specific manner by day 14, and
this response was generally strongest at day 43 after challenge (Fig. 5 B). Levels of IL-2 remained high 100 d after
infection in all except Ig/
mice. The production of IFN-
by spleen cells from infected animals was low at day 14 after challenge. However by day 24 and beyond antigen-specific IFN-
production could be detected in all mice (Fig.
5 C). The reason for the lower level of IFN-
production by spleen cells from IFN-
R
/
mice compared with the
wild-type strain is not clear, but may reflect a lack of positive feedback on Th1 cells and negative regulation on the
Th2 population. There was no evidence of bacterial outgrowth from spleen cell cultures derived from infected
IFN-
R
/
mice and IFN-
could not be detected from
unstimulated control cultures of these cells (Fig. 5 C).
The Ig/
strain showed less robust IFN-
production
with appreciable levels only reached by day 43, and only in
response to whole bacteria and not the component bacterial antigens tested. The levels of the Th2 cytokine IL-4
were also tested, but as previously reported (9) no significant IL-4 was detected after a single in vitro stimulation of
bulk spleen preparations (data not shown). In contrast, the
Th2 cytokine IL-5 was detected in the supernatant of antigen-stimulated spleen cells from B. pertussis infected IFN-
R
/
mice, but not from the wild-type or other strains
examined.
The results of this study provide direct evidence of an
obligatory role for B cells and IFN- in the protective immune response against B. pertussis. Using a murine respiratory infection model and mice with disruptions targeted to
the genes for IL-4, the IFN-
receptor, or for the immunoglobulin µ-chain (B cell knockouts) we demonstrate atypical disease resulting from disseminating infection in the
absence of functional IFN-
and a persistent infection, confined to the lungs, in the absence of antibody and B cells.
We have previously shown that effective immunization
against B. pertussis respiratory infection in mice is dependent on the induction of cell mediated immunity (9).
Respiratory challenge of athymic (nu/nu) BALB/c mice results in a persistent infection demonstrating an essential role
for T cells in the clearance of a primary infection (9). Furthermore, adoptive transfer studies have shown that MHC
class II-restricted CD4+ cells but not CD8+ T cells mediate protection against subsequent infection (9). These studies suggested a role for Th1 cells in protective immunity against B. pertussis, but interestingly also suggested that
primed B cells and macrophages contribute to effective
bacterial elimination. The present study demonstrates an
essential role for B cells in bacterial clearance, with Ig/
mice failing to clear a respiratory challenge, and indicates that both CD4+ T cells and B cells are required for complete elimination of B. pertussis from the lungs. It is not
clear which aspect of B cell function is central to the protective mechanism. The T cell proliferative and cytokine
responses against B. pertussis and in particular against the
soluble bacterial components are less robust than in wild-type control mice, however these responses are detectable
but do not correspond with a decline in bacterial load.
These findings suggest that while defective antigen presentation to T cells may contribute to the lack of clearance in
Ig
/
mice, failure to mount an antibody response may also
contribute to bacterial persistence. Virulent B. pertussis is
also known to exhibit varying degrees of resistance to the
classical complement pathway of lysis by virtue of the recently described products of the brk locus (23). Taken together these data suggest that the lack of specific opsonizing
antibody may render Ig
/
mice unable to clear the bacterium. It should also be noted that bacterial numbers in the
lungs of B cell knockout mice increase and then plateau at
day 21. The observation that these mice are not overwhelmed by infection but survive beyond 100 d after challenge strongly supports a role for other nonhumoral immune mechanisms in limiting pulmonary infection.
The demonstration of B. pertussis in the liver and the
early bacteremia, together with the pathological changes in
the liver and other organs of aerosol infected IFN-R
/
mice indicate an important role for IFN-
in the strict localization of B. pertussis infection to the lungs of immunocompetent animals. Although the full significance of the
brain pathology observed in certain IFN-
R
/
mice is not
clear, cases of encephalitis have been associated with whooping cough in children (24). The probable hematogenic dissemination of B. pertussis in the absence of functional IFN-
may be of significance to the reported isolation of B. pertussis from blood culture of a patient with Wegener's granulomatosis (25), and the ability of B. holmesii, B. hinzii, and B. bronchiseptica to cause bacteremia or sepsis (26). The reduced ability of the immature immune system to secrete
IFN-
and other cytokines may explain the greater susceptibility of human infants and neonatal mice to B. pertussis
infection. Although neonatal mice were not examined in
the present study, the results of preliminary experiments did
demonstrate that in comparison with adult mice, younger
IFN-
R
/
mice were more susceptible to the lethal effects of bacterial dissemination.
Interestingly viable virulent bacteria were isolated from
the livers of IFN-R
/
mice early after infection and immunohistochemical analysis of mesenteric lymph nodes demonstrated intracytoplasmic bacterial aggregates within macrophages. We found evidence of disseminating infection in
IFN-
R
/
mice before the development of B. pertussis-specific antibody or T cell responses in wild-type or knockout mice. This suggests that IFN-
produced by cells of the
nonadaptive immune response such as NK cells or
T cells
plays an important role in containing B. pertussis to the lung
during the initial period of infection, perhaps through the
killing of bacteria exploiting an intracellular niche within
macrophages (29). Atypical disease has previously been reported in disease models of obligate and facultative intracellular bacteria using gene knockout mice. Experimental L. monocytogenes infection of TCR
/
mice results in large
abscess-like liver lesions not seen in normal control mice
(30), and disseminated tuberculosis is observed in IFN-
gene disrupted mice infected either intravenously or aerogenically (31), highlighting the key role of IFN-
in another important respiratory disease.
Whole B. pertussis and its components are known to
elicit NO production by murine macrophages under a variety of conditions (32, 33). It has been reported that IFN-
can augment bacterial killing and NO production by B. pertussis infected macrophages in vitro (28, 32). Furthermore, IFN-
R
/
mice have an impaired ability to produce reactive nitrogen intermediates (34, 35 and unpublished observations). Taken together with the findings of
the present study, these observations suggest that NO or
reactive nitrogen intermediates may play a role in the destruction of an intracellular reservoir of B. pertussis and the
prevention of disseminated disease. However IFN-
R
/
mice show unimpaired bacterial clearance from the lungs
suggesting that this is not an essential mechanism of pulmonary elimination during B. pertussis infection and that
TNF-
, TNF-
, or Th2 derived cytokines may compensate for certain of the functions of IFN-
in immunity to
B. pertussis in the knockout animals.
It has been suggested that both Th1 and Th2 cells must
interact for optimal mucosal protection against B. pertussis
(36). Our demonstration of similar kinetics of bacterial clearance from the lungs of IFN-R
/
or IL-4
/
and wild-type
mice demonstrates that there is a degree of redundancy in
the cytokines or the Th subpopulations that mediate bacterial elimination from the lungs. However, IFN-
, whether
T cell derived or from other cell types, appears necessary to
contain infection within the lungs. It is likely that the early
production of IFN-
by cells of the innate immune system
in response to B. pertussis infection favors the subsequent
induction of a Th1 type response. This may explain the
high level of protection observed in convalescent mice or
in animals immunized with pertussis vaccines formulated with IL-12 or with vaccines which include components
that induce endogenous IL-12 production (19). Thus IFN-
may function to activate the antimicrobial activity of macrophages against an intracellular reservoir of bacteria. However this does not exclude the obvious role for CD4+ T
cells in providing help for antibody responses of a functionally relevant isotype (37). Our demonstration of reduced
levels of B. pertussis-specific IgG2a in infected IFN-
R
/
mice compared with the wild-type strain is consistent with
the latter possibility.
Our findings may also provide an explanation for the
failure to demonstrate a serological correlate of protection
induced with acellular pertussis vaccines, that have recently
been shown to confer a high level of protection against disease in children. (38, 39). These vaccines induce potent,
but short lived antibody responses (38, 39) and B. pertussis-specific T cells with a Th0 or mixed Th1/Th2 cytokine
profile (22). We have previously shown that infection preferentially induces Th1 cells and that these cells appear to
play a critical role in protection against B. pertussis (9, 21).
In the present study, the production of Th2 cytokines and
the lack of functional IFN- did not affect the clearance of
bacteria from the lung during a primary B. pertussis infection, but did result in dissemination of this bacterium. In
conclusion, our findings support a model of bacterial clearance from the respiratory tract which requires both B cells
and CD4+ T cells to resolve infection and indicate that
functional IFN-
plays an essential role in confining B. pertussis to the mucosal site of the lung.
Address correspondence to Dr. Kingston Mills, Infection and Immunity Group, National University of Ireland Maynooth, Co. Kildare, Ireland. Tel.: (+353) 1-708-3838; Fax: (+353) 1-708-3845; E-mail: kmills{at}may.ie
.
1 Abbreviations used in this paper: FHA, and filamentous hemagglutinin; IFN-This work was supported by a grant from the Wellcome Trust (no. 039583).
1. | Cheers, C., and D.F. Gray. 1969. Macrophage behaviour during the complaisant phase of murine pertussis. Immunol. 17: 875-887 [Medline]. |
2. | Saukonen, E., C. Cabellos, M. Burroughs, S. Prasad, and E. Tuomanen. 1991. Integrin mediated localization of Bordetella pertussis within macrophages: role in pulmonary colonization. J. Exp. Med. 173: 1143-1149 [Abstract]. |
3. | Masure, H.R.. 1993. The adenylate cyclase toxin contributes to the survival of Bordetella pertussis within human macrophages. Microb Pathog. 14: 253-260 [Medline]. |
4. | Bromberg, K., G. Tannis, and P. Steiner. 1991. Detection of Bordetella pertussis associated with the macrophages of children with human immunodeficiency virus. Infect. Immun. 59: 4715-4719 [Medline]. |
5. | Ewanowich, C.A., A.R. Melton, A.A. Weiss, R.K. Sherburne, and M.S. Peppler. 1989. Invasion of Hela 229 cells by virulent Bordetella pertussis. Infect. Immun. 57: 2698-2704 [Medline]. |
6. | Halperin, S.A., T.B. Issekutz, and A. Kasina. 1991. Modulation of B. pertussis infection with monoclonal antibodies to pertussis toxin. J. Infect. Dis. 163: 355-361 [Medline]. |
7. | Sato, H., and Y. Sato. 1984. Bordetella pertussis infection in mice: correlation of specific antibodies against two antigens, pertussis toxin and filamentous hemagglutinin with mouse protection in an intracerebral or aerosol challenge system. Infect. Immun. 46: 415-421 [Medline]. |
8. | Ad hoc group for the study of pertussis vaccines. . 1988. Placebo-controlled trial of two acellular pertussis vaccines in Sweden-protective efficacy and adverse events. Lancet. i: 955-960 . |
9. | Mills, K.H.G., A. Barnard, J. Watkins, and K. Redhead. 1993. Cell mediated immunity to Bordetella pertussis: role of Th1 cells in bacterial clearance in a murine respiratory infection model. Infect. Immun 61: 399-410 [Abstract]. |
10. | Redhead, K., A.L. Barnard, J. Watkins, and K.H.G. Mills. 1993. Effective immunization against Bordetella pertussis is dependent on induction of cell mediated immunity. Infect. Immun. 61: 3190-3198 [Abstract]. |
11. | Barnard, A., B.P. Mahon, J. Watkins, K. Redhead, and K.H.G. Mills. 1996. Th1/Th2 cell dichotomy in acquired immunity to Bordetella pertussis: variables in the in vivo priming and in vitro cytokine detection techniques affect the classification of T cell subsets as Th1, Th2 or Th0. Immunol. 87: 372-380 [Medline]. |
12. | Kaufmann, S.H.E.. 1994. Bacterial and protozoan infections in genetically disrupted mice. Curr. Opin. Immunol. 6: 518-525 [Medline]. |
13. | Shahin, R.D., J. Hamel, M.F. Leef, and B.R. Brodeur. 1994. Analysis of protective and nonprotective monoclonal antibodies specific for Bordetella pertussis lipooligosaccharide. Infect. Immun. 62: 722-725 [Abstract]. |
14. |
Flynn, J.L.,
J. Chan,
K.J. Triebold,
D.K. Dalton,
T.A. Stewart, and
B.R. Bloom.
1993.
An essential role for Interferon-![]() |
15. |
Huang, S.,
W. Hendriks,
A. Althage,
S. Hemmi,
H. Bleuthmann,
R. Kamijo,
J. Vilcek,
R.M. Zinkernagel, and
M. Aguet.
1993.
Immune response in mice that lack the IFN-![]() |
16. | Kuhn, R., K. Rajewsky, and W. Muller. 1991. Generation and analysis of interleukin-4 deficient mice. Science (Wash. DC). 254: 707-710 [Medline]. |
17. | Kitamura, D., J. Roes, R. Kuhn, and K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene. Nature (Lond.) 350: 423-426 [Medline]. |
18. | Sato, Y., K. Izumiya, H. Sato, J.L. Cowell, and C.R. Manclark. 1980. Aerosol infection of mice with Bordetella pertussis. Infect. Immun. 29: 261-266 [Medline]. |
19. | Mahon, B.P., M. Ryan, F. Griffin, and K.H.G. Mills. 1996. Interleukin-12 is produced by macrophages in response to live or killed Bordetella pertussis and enhances the efficacy of an acellular pertussis vaccine by promoting induction of Th1 cells. Infect. Immun 64: 5295-5301 [Abstract]. |
20. | Mills, K.H.G. 1996. Induction and detection of T cell responses. In Methods in Molecular Medicine: Vaccine Protocols. A. Robinson, G. Farrar, C. Wiblin, editors. Humana Press Inc., Totowa, NJ. 197-221. |
21. | Ryan, M., G. Murphy, L. Gothefors, L. Nilsson, J. Storsaeter, and K.H.G. Mills. 1997. Bordetella pertussis respiratory infection in children is associated with preferential activation of type 1 Th cells. J. Infect. Dis. 175: 1246-1250 [Medline]. |
22. | Ryan, M., L. Gothefors, J. Storsaeter, and K.H.G. Mills. 1997. B. pertussis-specific Th1/Th2 cells generated after respiratory infection or immunization with an acellular vaccine: comparison of the T cell cytokine profiles in infants and mice. Dev. Biol. Stand. 89: 251-259 . |
23. | Fernandez, R.C., and A.A. Weiss. 1994. Cloning and sequencing of a Bordetella pertussis serum resistance locus. Infect Immun. 62: 4727-4738 [Abstract]. |
24. | Cherry J.D., P.A. Brunnell, G.S. Golden, and D.T. Karzon. 1988. Report of the task force on pertussis and pertussis immunization. Pediatrics. 81(Suppl.):939-984. |
25. | Janda, W.M., E. Santos, J. Stevens, D. Craig, L. Terrile, and P.C. Schreckenberger. 1994. Unexpected isolation of B. pertussis from blood culture. J. Clin. Microbiol. 32: 2851-2853 [Abstract]. |
26. | Weyant, R.S., D.G. Hollis, R.E. Weaver, M.F. Amin, A.G. Steigerwalt, S.P. O'Connor, A.M. Whitney, M.I. Daneshvar, C.W. Moss, and D.J. Brenner. 1995. Bordetella holmesii sp. nov., a new gram negative species associated with septicemia. J. Clin. Microbiol. 33: 1-7 [Abstract]. |
27. | Cookson, B.T., P. Vandamme, L.C. Carlson, A.M. Larson, J.V.L. Sheffield, K. Kersters, and D.H. Spach. 1994. Bacteremia caused by a novel Bordetella species, B. hinzii. J. Clin. Microbiol. 32: 2569-2571 [Abstract]. |
28. | Bauwens, J.E., D.H. Spach, T.W. Schwacker, M.M. Mustafa, and R.A. Bowden. 1992. Bordetella bronchiseptica pneumonia and bacteremia following bone marrow transplantation. J. Clin. Microbiol. 30: 2471-2475 [Abstract]. |
29. | Torre, D., G. Ferrario, G. Bonetta, L. Perversi, R. Tambini, and F. Speranza. 1994. Effects of recombinant human gamma interferon on intracellular survival of Bordetella pertussis in human phagocytic cells. FEMS Immunol. Med. Microbiol. 9: 183-188 [Medline]. |
30. |
Mombaerts, P.,
J. Arnold,
F. Russ,
S. Tonegawa, and
S.H.E. Kaufmann.
1993.
Different roles of ![]() ![]() ![]() ![]() |
31. |
Cooper, A.M.,
D.K. Dalton,
T.A. Stewart,
J.P. Griffin,
D.G. Russell, and
I.M. Orme.
1993.
Disseminated tuberculosis in
interferon-![]() |
32. | Torre, D., G. Ferrario, G. Bonetta, L. Perversi, and F. Speranza. 1996. In vitro and in vivo induction of nitric oxide by murine macrophages stimulated with Bordetella pertussis. FEMS Immunol. Med. Microbiol. 13: 95-99 [Medline]. |
33. | Heiss, L.N., J.R. Lancaster, J.A. Corbett, and W.E. Goldman. 1994. Epithelial autotoxicity of nitric oxide: role in the respiratory cytopathology of pertussis. Proc. Natl. Acad. Sci. USA 91: 267-270 [Abstract]. |
34. |
Matthys, P.,
G. Froyen,
L. Verdot,
S. Huang,
H. Sobis,
J. van
Damme,
B. Vray,
M. Aguet, and
Alfons Billiau.
1995.
IFN-![]() |
35. |
Kamijo, R.,
J. Le,
S. Huang,
M. Aguet,
M. Bosland, and
J. Vilcek.
1993.
Generation of nitric oxide and induction of
major histocompatibility complex class II antigen from mice
lacking the interferon-![]() |
36. | Daugelat, S., and S.H.E. Kaufmann. 1996. Role of Th1 and Th2 cells in bacterial infections. In Th1 and Th2 Cells in Health and Disease. Chem. Immunol. S. Romagnani, editor. Karger, Basel. 63:66-97. |
37. | Mahon, B.P., K. Katrak, A. Nomoto, A.J. Macadam, P.D. Minor, and K.H.G. Mills. 1995. Poliovirus-specific CD4+ Th1 clones with both cytotoxic and helper activity mediate protective humoral immunity against a lethal poliovirus infection in transgenic mice expressing the human poliovirus receptor. J. Exp. Med. 181: 1285-1292 [Abstract]. |
38. |
Greco, D.,
S. Salmaso,
P. Mastrantonio,
M. Giuliano,
A.E. Tozzi,
A. Anemona,
M.L. Cioti, and
Degli Atti, A. Giammanco,
P. Panel, W.C. Blackwelder, D.L. Klein, S.G.F. Wassilak,
and The Progetto Pertosse working group.
1996.
A controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis.
N. Engl. J. Med.
334:
341-348
|
39. |
Gustafsson, L.,
H.O. Hallander,
P. Olin,
E. Reizenstein, and
J.A. Storsaeter.
1996.
A controlled trial of a two-component
acellular, a five-component acellular, and a whole-cell pertussis vaccine.
N. Engl. J. Med.
334:
349-355
|