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*
Department of Biology, University of York, York, United Kingdom; and
Department Roche Genetics, Hoffmann-La Roche, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing
CD4+ T cells in a focal response around challenge larvae.
IFN- can promote production of TNF and can synergize with this
cytokine in its actions on responder cells. We have examined whether
TNF plays a role in lung phase immunity to schistosomes using mice with
a disrupted gene for TNFRI (TNFRI-/-). The most dramatic
finding was that the schistosome vaccine elicited no protection
whatsoever in these mice. However, this could not be attributed to a
lack of responder cells, because more lymphocytes were lavaged from the
airways of TNFRI-/- than wild-type mice. Furthermore,
CD4+ T cells were equally represented in airway populations
from the two groups and produced IFN- upon Ag stimulation in vitro.
In contrast, pulmonary macrophage function was defective in
TNFRI-/- mice, as indicated by a failure to up-regulate
inducible NO synthase mRNA. Histopathological analysis revealed that
focal infiltrates were of similar size and cell composition in the two
groups but that more parasites were free of foci in the
TNFRI-/- mice. These animals had a greatly impaired IgG
response to schistosomes, which may explain their lack of residual
protection due to Ab in a situation where cell-mediated immunity is
disabled. We suggest that the absence of protective immunity could
result from a retarded build-up of leukocytes around migrating lung
worms and/or a deficit in accessory cell function within a focus, both
of which would permit parasite escape. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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upon antigenic stimulation in vitro.
Administration of cytokine-neutralizing Ab to vaccinated mice after
challenge results in 90% abrogation of immunity, revealing the crucial
role of IFN- in the effector response (2). Furthermore,
vaccination of mice with a disrupted IFN- receptor gene
(IFN-R-/-) elicits only low levels of
protection (3), most probably mediated by Ab (P. S.
Coulson, unpublished data).
IFN- has a well-characterized role in the priming of
macrophages/monocytes to produce TNF in response to LPS stimulation
(4). These two cytokines can act synergistically in host
responses to infectious agents and are also involved in leukocyte
recruitment to sites of inflammation. A possible link between IFN-
and TNF in the schistosome vaccine model is provided by the observation
of a lower overall level of TNF-
mRNA in the lungs of poorly
protected IFN-R-/- mice after challenge
compared with highly protected C57BL/6 mice (3). Direct
killing of schistosome larvae by cytotoxic agents such as NO
(3) is one suggested effector mechanism in which both
IFN- and TNF- could participate. The two cytokines synergize in
the production of inducible NO synthase (iNOS)3
and NO will mediate cytotoxic killing of parasites in vitro
(5). Although increased iNOS mRNA has been demonstrated in
the lungs of C57BL/6 mice after challenge (6, 7), a
significant level of protection is elicited by vaccination of mice with
a disrupted iNOS gene (7, 8), suggesting that NO is not a
major factor in challenge parasite elimination. An alternative effector
mechanism, in which both IFN- and TNF could participate, might
involve the aggregation of leukocytes into a tight focus that simply
blocks parasite migration through the vascular bed of the lungs.
However, the adhesive interactions so far examined between pulmonary
leukocytes (ICAM-1/LFA-1 and CD2/CD48) do not appear to influence the
cohesiveness of effector foci or be important for protection
(9).
The biological activities of TNF are mediated by two structurally
related, but functionally distinct receptors, TNFRI (p55) and TNFRII
(p75), which are coexpressed on most cell types. The derivation of mice
with disrupted genes for the two receptors has enabled their respective
functions in vivo to be dissected (10, 11, 12). The
proinflammatory activities of TNF- are mediated primarily via TNFRI
(10, 12). In contrast, the role of TNFRII is less well
defined, but it appears to be involved in lymphocyte proliferation and
the induction of apoptosis by a novel pathway (4). We have
investigated the involvement of TNF in the pulmonary inflammatory
response to schistosome larvae using mice with a disrupted TNFRI gene.
When these animals were vaccinated with attenuated parasites, we
observed a complete absence of protective immunity. This could not be
attributed to a lack of responder T cells in the lungs after challenge,
or a switch to Th2 cytokine production. Although effector foci formed
in TNFRI-/- mice, more parasites were free of
surrounding infiltrates than in comparable wild-type (WT) animals, and
iNOS mRNA was not up-regulated, suggesting that defects in macrophage
function might explain the lack of protection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice of the 129 strain with a targeted disruption of the gene for the 55-kDa TNFR (TNFRI-/-; Ref. 10) were obtained from Hoffmann-LaRoche (Basel, Switzerland) and backcrossed six times to the C57BL/6 background. Homozygous mutants and WT mice from the sixth cross were bred in isolators at the University of York and maintained in laminar flow cabinets.
Parasite exposure regimens and experimental design
Mice were exposed to a Puerto Rican isolate of S. mansoni, routinely maintained in albino Biomphalaria glabrata and outbred MF1 mice. Groups of test mice were vaccinated with 500 radiation-attenuated cercariae via the shaved abdomen (13). They were challenged 5 wk later, together with control groups of age-matched naive mice, by tail skin exposure to 200 normal cercariae. The level of protection was determined 5 wk later by a comparison of adult worm burdens in test and control groups. Pulmonary immune responses were investigated in additional groups of vaccinated mice at 14 days postchallenge, the time of peak inflammation in the lungs (14).
Analysis of pulmonary leukocytes, cytokines, and iNOS expression
Airway leukocytes were recovered from the perfused lungs by bronchoalveolar lavage (BAL) and characterized as previously described (13). Individual BAL cell suspensions were adjusted to 1 ml in Glasgow MEM, containing 10% FCS (Globefarm, Esher, Surrey, U.K.), 200 U/ml penicillin, and 100 µg/ml streptomycin (Sigma, St. Louis, MO). Total cell number was estimated by hemocytometer, and analyses were performed on aliquots of fresh cells using an EPICS XL flow cytometer (Coulter Electronics, Hialeah, FL). CD4+ T lymphocytes were phenotyped using fluorochrome-conjugated rat mAb (clone YTS 191.1; Caltag, Burlingame, CA).
Ag-stimulated production of cytokines by the remaining BAL leukocytes
was measured after culture in 96-well plates (Nunclon, Life
Technologies, Paisley, Scotland) with or without 50 µg/ml soluble
18-h schistosomular Ag (SSP; Ref. 15), for 72 h at
37°C, 5% CO2/95% air. The amounts of IFN-
and IL-4 in culture supernatants were measured by double Ab ELISAs as
described in detail elsewhere (3, 14).
RT-PCR analysis of total RNA extracted from whole lung was conducted for iNOS and the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase, using the sense and antisense primers previously described (7). Semiquantitative measurement of enzyme PCR product was performed on slot-blots by hybridizating end-labeled oligonucleotide probes, and bound radioactivity was estimated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Histopathology
Lungs were excised 14 days postchallenge and processed as previously described (3). A total of 140 consecutive sections was scanned from a single lobe for each of five vaccinated and challenged TNFRI-/- and WT mice (1400 sections in all). The number of parasites in each sequence of the sections was recorded and any association with a focus was noted. The cellular composition of foci was determined, and each was photographed in the section displaying the largest profile for estimation of maximum dimension.
Serum Ab assays
Serum samples were collected on the day before challenge and again at day 14 postchallenge. The levels of Ag-specific IgG isotypes were determined by ELISA according to a previously described protocol (16). Briefly, microtiter plates were coated overnight at 4°C with 2.5 µg/ml 18 h SSP in carbonate buffer (pH 9.6). Plates were washed and probed with 1:150 dilution of the test sera for 1.5 h. The plates were then probed with HRP-conjugated rabbit Abs to mouse IgG and IgG1, IgG2a, IgG2b, or IgG3 isotypes (Zymed, San Francisco, CA). Ab binding was assessed following addition of peroxidase substrate (Sigma) and measurement of color development at 490 nm.
Statistical analysis
Sample groups were composed of five mice at all times in all experiments, and results are expressed as the mean ± SE unless the samples were pooled. Data were analyzed for statistical significance using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether TNF is required for the protective response
induced by the radiation-attenuated schistosome vaccine, a series of
vaccination and challenge experiments was performed using mice
genetically deficient in TNFRI. Exposure of WT mice to attenuated
cercariae resulted in the anticipated significant reduction in
challenge worm burden relative to control animals
(p < 0.02; Fig. 1
); this amounted to a mean of 53.4%
protection in three experiments. In striking contrast, no significant
reduction in worm burden was observed after challenge of vaccinated
TNFRI-/- mice compared with each control group
(p > 0.25); the mean protection of the three
vaccinated groups was -1.2%.
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Vaccination of mice with attenuated cercariae results in the
recruitment of leukocytes to the pulmonary parenchyma and airways,
culminating in a persistent CD4+ T cell
population that participates in the effector response (1).
Because TNF is considered an important cytokine in the tissue
recruitment of leukocytes, we examined whether there was a reduced cell
infiltration into the lungs of TNFRI-/- mice,
which might explain their lack of immunity. When airway leukocytes were
recovered at day 14 postchallenge by BAL for flow cytometric analysis,
the total cell yield from TNFRI-/- mice was
double that from their WT counterparts (Fig. 2). In both groups of mice, macrophages
and lymphocytes were the dominant cell population, with granulocytes
(eosinophils) a minor component. The cell increment in
TNFRI-/- mice was evenly distributed between
the three leukocyte subpopulations, showing that the overall increase
was not due to differential recruitment. CD4+ T
cells represented 64% of total lymphocytes in the
TNFRI-/- mice vs 67% in the WT animals.
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In other gene-disrupted mice (e.g.,
IFN-R-/-), where we have observed low levels
of protection after exposure to the radiation-attenuated vaccine, there
was a marked shift to a Th2 cytokine profile in the Ag-stimulated BAL
leukocyte cultures. We therefore compared the cytokines produced by BAL
cells following recovery from WT and TNFRI-/-
mice 14 days after challenge. In cultures from WT mice, Ag-stimulated
IFN- production greatly exceeded that of IL-4 (Fig. 3); there was also a low level of
endogenous IFN- release in the absence of added Ag. Cultures from
TNFRI-/- mice showed, if anything, a stronger
Th1 bias, with higher IFN- and lower IL-4 levels than WT animals.
Indeed, when cytokine production was expressed per
105 lymphocytes in culture to take into account
the differences in cell content of the BAL fluid from the two groups,
the pattern of Th1 dominance was reinforced. Thus, the ratio of
IFN-:IL-4 in culture supernatants from WT mice was 4.9:1, whereas
that from TNFRI-/- mice was 14.5:1.
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Challenge parasites in the lungs of vaccinated C57BL/6 mice
attract a focal inflammation that brings about their elimination
(1). We therefore undertook a histopathological study of
lungs from TNFRI-/- and WT mice at day 14
postchallenge to determine whether focus formation was impaired. Fewer
foci were detected in TNFRI-/- than WT mice,
and the mean maximum dimension of each focus was also smaller in the
former animals (Table I). In contrast,
more parasites were found in TNFRI-/- than WT
mice but fewer of these were actually associated with foci in the
former animals. The pulmonary cellular infiltrates in both groups of
mice had a predominantly mononuclear cell composition, indicative of a
Th1-biased response (data not shown).
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Because T cell function is apparently normal in
TNFRI-/- mice, the lesion responsible for the
lack of protection may be present in the accessory cells involved in
the effector response. Elevated production of NO is a feature of
pulmonary macrophage activation in C57BL/6 mice after vaccination and
challenge (7). Furthermore, TNF-mediated synergism with
IFN- for NO production by macrophages is thought to act via TNFRI
signaling (19). We therefore measured the level of iNOS
mRNA in whole lungs as an indicator of the functional state of
macrophages after challenge. In WT animals, there was a significant
increase (2.5-fold) in iNOS mRNA at day 14 compared with the naive
level (Fig. 4). In contrast, although
iNOS mRNA expression was evident in naive
TNFRI-/- mice, no iNOS mRNA up-regulation was
observed in mice at day 14 postchallenge.
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In C57BL/6 mice after a single exposure to the attenuated
schistosome vaccine, the pulmonary effector mechanism is dependent on
CD4+ T cells, with little evidence for the
involvement of B cells (17). However,
TNFRI-/- mice have defects in the
differentiation of follicular dendritic cell networks and the
development of germinal centers in lymphoid tissue, which affect Ab
responses (18). A plausible explanation for the lack of
protection against schistosomes in such mice is that Ab is an
unsuspected and essential component of the effector
mechanism. We compared the levels of specific Ab in the serum
of vaccinated WT and TNFRI-/- mice
immediately before challenge and at the height of the effector response
14 days later. A significant primary IgG response was observed in WT
animals following vaccination, with IgG1, IgG2a, and IgG2b production
(Fig. 5) but no IgG3 production (data not shown); the
total specific IgG and the levels of the three IgG isotypes were
boosted by challenge exposure. In marked contrast, the total specific
IgG level in TNFRI-/- mice at challenge was
approximately one-third that of WT counterparts, and the same was true
for all three isotypes. Furthermore, exposure of
TNFRI-/- mice to a normal parasite challenge
had no immunostimulatory effect. Indeed, with the exception of
IgG2a, the Ab levels were lower than after the primary exposure.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in protection against
schistosomes was first highlighted by in vivo cytokine neutralization
experiments (2, 23). We have now demonstrated the
requirement for a second cytokine, TNF. Given the involvement of TNF in
the IFN--mediated immunity both to pathogenic bacteria
(24) and protozoa (25), this result is
perhaps not surprising. However, what is unusual is the absolute nature
of the requirement, because protection was completely absent when
TNFRI-/- mice were vaccinated and challenged.
Such a complete loss of function rarely occurs when gene-disrupted mice
are used to probe immune responses because of the degree of redundancy
among immune effector mechanisms (cf. vaccination of
IFN-R-/- and IL-12
-/- mice; Refs. 3 and
26). Protection is also absent in mice deprived of both
the p55 and p75 receptors for TNF (mean of 0% protection in two
experiments; our unpublished data), but signaling through TNFRI is
clearly the key. It is possible that the lack of protection in the absence of TNFRI signaling is due to TNF action at a site distant from the lungs. The role of TNF in the development of secondary lymphoid organs (reviewed in Ref. 27) would come into this category. Thus, primary specific Ab responses after exposure to attenuated schistosomes were poor in TNFRI-/- mice compared with controls. Furthermore, secondary responses to challenge larvae were negligible. This could be taken as evidence for a hitherto unsuspected role for Ab in the pulmonary effector response of mice that were vaccinated once. Two recent studies have addressed this question by vaccination and challenge of µMT mice lacking functional B cells (17, 28). Both studies reported that significant protection was induced in these mice, although, overall, somewhat less than in compatible WTs. In one study (28), restitution of the immunity to the WT level was achieved by administration of immune serum to vaccinated µMT mice, a result that implies a role for Ab- and cell-mediated mechanisms in WT animals that were vaccinated once.
In addition, we need to consider a local role for TNF in the effector
mechanism. The cytokine has been implicated in the up-regulation of
adhesion molecule expression on vascular endothelial cells and the
production of several chemokines at sites of delayed-type
hypersensitivity reactions (29). Thus, in the absence of
signaling via TNFRI, we might anticipate reduced cell infiltration into
the lungs. However, analysis of airway leukocyte populations after
challenge revealed elevated cell numbers rather than impaired
recruitment. Clearly, the failure to eliminate challenge parasites in
the lungs of vaccinated TNFRI-/- mice is not
due to a reduced frequency of responder cells. This is emphasized by
the high level of IFN- and low level of IL-4 in Ag-stimulated BAL
cultures from both WT and TNFRI-/- mice. The
Th1 bias of the elicited immune response is thus maintained in the
absence of signaling via TNFRI, in contrast to the marked switch to a
Th2 cytokine profile, which we have previously observed in the lungs of
IFN-R-/- and
IL-12-/- mice (3, 26). (This Th1
bias in the response of TNFRI-/- mice coupled
with their defective germinal centers also means that, unlike the
foregoing gene-disrupted mice, they fail to mount the alternative Th2
response that leads to a degree of protection mediated by Ab.)
The elevation in leukocyte numbers we observed in the lungs is similar to the situation reported following infection with L. major (30). Here, nonhealing skin lesions developed in TNFRI-/- mice, with marked lymphocyte infiltration of the epidermis and severe edema, in the absence of parasite-containing macrophages. It was subsequently suggested that the failure of such lesions to resolve was due to the absence of lymphocyte apoptosis, normally signaled via TNFRI (31). By analogy, the elevated lymphocyte numbers in the lungs, reported in the present study, could reflect diminished apoptosis and cell clearance.
Elimination of challenge parasites in the lungs of vaccinated WT mice
requires the formation of a focal aggregate of cells around a migrating
intravascular larva (32). Early ultrastructural
observations suggested that qualitative aspects of this focal response
were crucial to its success, with a predominantly mononuclear cell
composition of paramount importance (33). This was borne
out by subsequent observations in IFN-R-/-
mice where challenge elicits a more intense cellular infiltrate in
which eosinophils predominate, although protection (i.e., effectiveness
of foci) is greatly diminished (3). The precise way in
which the effector focus achieves parasite elimination has not been
satisfactorily explained. There are two contending hypotheses, direct
cytotoxic killing by agents such as NO (34), and simple
physical blocking of migration until the parasite expires
(35). The failure of nonprotected
TNFRI-/- mice to up-regulate the iNOS message
in the lungs after challenge argues for NO involvement in the effector
response. Conversely, the induction of protection in
iNOS-/- mice (7, 8), which still
mount a dominant Th1 response (8), and the
insusceptibility of lung stage larvae (1–2 wk old) to
NO-mediated killing (36), argue against NO involvement in
the effector response.
Our histopathological observations in the present study reveal that foci still develop in the lungs after challenge of vaccinated mice. Superficially, their cellular composition is normal, yet they are obviously completely ineffective at eliminating parasites. One hypothesis we can offer is temporal; we have previously shown that in a C57BL/6 mouse it takes more than 2 but less than 4 days for an effector focus to form around an embolized larva in the lungs (37). This timing is crucial because it is virtually identical with the first transit time of a larva through the lung vascular bed (38). Thus, a small delay in cellular aggregation around larvae due to the absence of TNFRI signaling would allow their escape. We are currently undertaking a histopathological investigation of the kinetics of focus formation after i.v. challenge of vaccinated WT and TNFRII-/- mice with lung schistosomula. That a focus would form at all in the above circumstances requires explanation. However, "empty" foci are quite frequent events in the lungs of vaccinated C57BL/6 mice after challenge. We have always assumed, in the absence of a discriminatory test, that they represent either an inflammatory infiltrate in which a larva has died and disintegrated, or perhaps more likely a "footprint" left by the larva as it sheds Ag during passage through the pulmonary vasculature (this traverse is a very tight squeeze that brings the larval tegumental surface into close and prolonged contact with capillary endothelium; Ref. 39).
It is not possible to draw precise parallels between microbial infections that are more widely disseminated in the lungs and schistosome larvae, each of which must serve virtually as a point source of Ag. However, analogous situations of defective granuloma formation have been reported after exposure of TNF-/- mice to heat-killed Corynebacterium parvum (40) and TNFRI-/- mice to live Mycobacterium avium (41, 42). In the former animals, although little or no initial response was observed, at later times a vigorous but disorganized inflammation developed, leading to death. In the latter, granuloma formation was delayed by 2 wk and the inflammatory lesions were less compact and malorganized than in WT mice (41). The above studies suggest that TNF is required to maintain the integrity and organization of granulomatous responses.
TNF has been implicated in several aspects of accessory cell function
in immune responses. This is evidenced by a lack of germinal centers,
due to defective follicular dendritic cell networks, in
TNFRI-/- mice (18). TNF is
also required for dendritic cell migration from peripheral tissue to
lymphoid organs, best exemplified by epidermal Langerhans cells in a
process that involves altered expression of adhesion molecules
(42). The failure to up-regulate iNOS mRNA expression in
the lungs of TNFRI-/- mice in the present study
is also indicative of defective macrophage function. Although we have
found altered adhesion molecule expression on
CD4+ T cells recovered from the lungs of
IFN-R-/- mice, we were unable to make a link
between the reduced homotypic adhesion of such cells and the minimal
immunity displayed by these mice (9). We are currently
investigating whether there are any phenotypic or functional
differences in dendritic cells and macrophages from the lungs of
TNFRI-/- vs WT mice, which might explain why
effector foci in the former animals fail to eliminate challenge
parasites.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Street, Department of Biology, P.O. Box 373, University of York, York YO10 5YW U.K. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible NO synthase; WT, wild type; BAL, bronchoalveolar lavage; SSP, soluble schistosomular Ag preparation.
Received for publication April 9, 1999. Accepted for publication July 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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modifies pulmonary inflammatory responses and abrogates immunity to Schistosoma mansoni in mice vaccinated with attenuated cercariae. J. Immunol. 149:3654.[Abstract]
receptor gene exposed to the irradiated Schistosoma mansoni vaccine. Immunology 87:275.[Medline]
is required for the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561.[Medline]
) in leishmaniasis. I. TNF mediates host protection against cutaneous leishmaniasis. Immunology 69:570.[Medline]
-dependent effector mechanisms. J. Immunol. 162:345. and interleukin 1ß for migration. Immunology 92:388.[Medline]
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