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Infection and Immunity, April 1999, p. 1789-1797, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid Local Expression of Interleukin-12, Tumor
Necrosis Factor Alpha, and Gamma Interferon after Cutaneous
Francisella tularensis Infection in Tularemia-Immune
Mice
Stephan
Stenmark,1
Dan
Sunnemark,2
Anders
Bucht,3,4 and
Anders
Sjöstedt1,5,*
Department of Infectious Diseases, University
of Umeå,1 and Department of
Biomedicine4 and Department of
Microbiology,5 Defence Research Establishment,
Umeå, and Microbiology and Tumorbiology
Centre2 and Department of
Rheumatology,3 Karolinska Institute,
Stockholm, Sweden
Received 18 August 1998/Returned for modification 8 October
1998/Accepted 5 January 1999
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ABSTRACT |
Francisella tularensis LVS is an effective live vaccine
strain used for cutaneous vaccination against tularemia in man. In mice, injection of LVS causes invasive disease and subsequent development of immunity that is characterized by effective control of
otherwise lethal doses of the organism. In the present investigation, it is shown that LVS-immune mice controlled an intradermal infection much more effectively than did naive mice; bacterial counts in skin
samples were 1.5 to 2.0 log10 lower 24 h after
injection and 6 log10 lower 72 h after injection in
immune mice. Moreover, in contrast to naive mice, no bacteria were
demonstrated in samples from livers and spleens of immune mice. By
immunohistochemistry, skin samples from immune mice showed an intense
staining for interleukin-12 (IL-12) and a moderate staining for tumor
necrosis factor alpha (TNF-
) at 24 h postinoculation, after
which staining for both cytokines faded. In naive mice, the staining
for IL-12 was weak at all time points and no staining for TNF- was
observed. No staining for gamma interferon (IFN-
) was observed in
any group before 72 h. At that time point, skin samples from
immune mice showed moderate staining and skin samples from naive mice
showed weak staining. Reverse transcriptase PCR showed an induction of mRNA of the three cytokines in the skin within the first day after injection. A quantitative analysis demonstrated higher IFN- and TNF- mRNA levels in immune mice at 24 h postinoculation. In
conclusion, immunization with F. tularensis LVS conferred a
capability to respond to cutaneous reinfection, with rapid local
expression of IL-12, TNF-, and IFN-, and this expression was
paralleled by containment and mitigation of the infection. The cytokine
response may be part of a local barrier function of the skin, important to host protection against tularemia.
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INTRODUCTION |
Tularemia is caused by
Francisella tularensis, a facultative intracellular
bacterium. The disease is endemic in rodents and lagomorphs, which are
believed to be the main sources for the spread of tularemia to humans.
In man, tularemia presents in different ways, depending on the route of
entry (reviewed in reference 26). Inhalation of
infected dust leads to the respiratory form of the disease, while
transmission by arthropods or direct contact with an infected animal
causes the ulceroglandular form of the disease. The respiratory form of
the disease is more severe, and in the United States, where the highly
virulent type A strains are prevalent, the fatality rate before the use
of antibiotics was 20 to 30%, considerably higher than the same figure
for ulceroglandular tularemia, <10%. Irrespective of host species,
the resulting diseases have many features in common, and experimental
infection in the mouse is generally held to be an appropriate model for
understanding the host-parasite interaction of human infection.
Work on the murine model of tularemia has disclosed mechanisms of host
resistance that are similar to those relevant for intracellular bacteria in general. During the first few days of a primary infection, neutrophils play a critical role, as evidenced by a dramatic
exacerbation of infection following their depletion or the prevention
of their recruitment (3, 23). Later, an immunospecific
T-cell response develops which is crucial for the eradication of
F. tularensis (4, 30).
Immunization against F. tularensis affords effective
protection. For example, only a few cases of human reinfection have
been reported (26). Similarly, mice given a sublethal
inoculum of the live vaccine strain F. tularensis LVS
develop long-lasting immunity and can survive reinfection with up to
100 LD50 doses (23, 24).
Work on other models of intracellular infections, e.g., murine
listeriosis, the prototypic model of intracellular bacterial infection,
has revealed a critical requirement for certain cytokines in innate and
acquired host resistance. Requirements for resistance to tularemia are
similar. At an early stage of murine tularemia, interleukin-12 (IL-12)
and tumor necrosis factor alpha (TNF-) are expressed by infected
mononuclear phagocytes (9), and these cytokines are believed
to trigger the production of gamma interferon (IFN-) by NK cells.
The latter cytokine mediates the activation of mononuclear phagocytes
and is vital for them to kill F. tularensis (7).
In support of a critical role for these cytokines, neutralization of
the biological effects of IFN- and TNF- during the early phase of
primary tularemia leads to lethal exacerbation of infection (1, 6,
14, 24). These cytokines also play important roles after
reinfection (24). Although the requirement of IL-12 for host
resistance against tularemia has not been established, there is ample
evidence from other models of intracellular infections that IL-12
drives differentiation of the protective Th1 immune response, i.e., the
differentiation into IFN--secreting T cells (10, 15, 16,
27-29). Knowledge about mechanisms of host resistance to
F. tularensis derives from experimental infection in normal and immunodeficient mice, with the focus on lymphoid organs. There is,
however, little information on the possible local presence of cytokines
and other immunoregulators within anatomic barriers, such as skin,
penetrated by F. tularensis.
Host protective mechanisms in the skin need to be effective, since they
often constitute a first line of defense and the skin is a principal
target of infections caused by highly infectious agents. The murine
model of tularemia is a striking example of the effectiveness of
cutaneous immune mechanisms, since a thousand- to a million-fold-more
bacteria are required to generate a lethal infection by the dermal
route versus other routes of inoculation, e.g., intranasal,
intravenous, or intraperitoneal (8). Thus, experimental
tularemia may serve as a model to elucidate (i) cutaneous immune
mechanisms that confer the host with a means to locally control and
contain infection and (ii) the very effective host resistance
mechanisms expressed in an immune individual. Such a characterization
is of special relevance to understand how host resistance against
F. tularensis is triggered in humans, since vaccination with
the attenuated strain F. tularensis LVS is administered by
scarification and the ulceroglandular form of infection is the most
prevalent variant.
In the present study, bacterial replication in target organs and its
temporal association with expression of IL-12, TNF-, and IFN-
during primary infection and reinfection in the skin were monitored.
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MATERIALS AND METHODS |
Animals.
Female 5- to 6-week-old BALB/cJBom mice were
purchased from Bomholtgård, Ry, Denmark, and used in experiments at
approximately 8 weeks of age. The mice were housed at the Animal
Facility, Defence Research Establishment, Umeå, Sweden, under
conventional conditions and given food and water ad libitum.
Bacteria.
The vaccine strain F. tularensis LVS
(ATCC 29684) was supplied by the U.S. Army Medical Research Institute
of Infectious Diseases, Fort Detrick, Frederick, Md. Bacteria were
grown on modified Thayer-Martin agar (21) at 37°C to the
logarithmic phase, suspended at a density of 3 × 109
organisms per ml in saline with the addition of 10% (wt/vol) glycerol,
and stored in 200-µl aliquots at 
70°C. For each experiment, inocula were prepared from frozen stocks and bacteria were diluted in
sterile saline to the required concentration. Bacterial counts were
retrospectively assessed after injection.
Inoculation and enumeration of bacteria.
Approximately 2 cm2 of the skin of the upper thorax was shaved 2 days
before inoculation. Mice were challenged with an intradermal inoculation of 3.8 × 105 F. tularensis LVS
and killed by decapitation after 1, 3, 5, 8, and 15 days of infection.
One square centimeter of the skin at the site of inoculation was
excised and put into tubes with saline after a brief wash in 70%
ethanol. Spleen, liver, and draining lymph nodes were also collected.
Samples from each of the four organs were homogenized, and the number
of F. tularensis LVS was calculated by plating 10-fold
serial dilutions. Mice that received a secondary challenge had been
given a subcutaneous inoculation at another dermal site of
104 CFU of F. tularensis LVS 5 to 6 weeks
earlier. Colonies were counted after 3 days of incubation at 37°C.
mRNA preparation and cDNA synthesis.
Mice were intradermally
inoculated with 3 × 105 F. tularensis LVS
and killed at 1, 6, 12, 24, 48, and 72 h postinfection. One square
centimeter of the skin, including the site of inoculation in the
center, was excised and immediately frozen in liquid nitrogen and
stored at 70°C. RNA was isolated from frozen samples of skin, lymph
nodes, spleens, and livers by using a guanidine
isothiocyanate-phenol-chloroform single-step method (2). The
optical density at 260 nm was used to estimate the concentration of
total mRNA, yields being in the order of 10 to 100 µg per sample.
For cDNA synthesis, approximately 5 µg of total mRNA was incubated
with 2.5 µg of random hexamers (Promega, Madison, Wis.) for 5 min at
94°C. After the solution was cooled on ice, the total volume was
adjusted to 50 µl in reverse transcriptase buffer (Gibco BRL, Grand
Island, N.Y.) with the addition of 0.6 mM of each deoxynucleoside triphosphatase (Pharmacia, Uppsala, Sweden), 40 U of RNasin (Promega), and 20 U of Superscript (Gibco BRL). After incubation at 37°C for 60 min, 42°C for 30 min, and 70°C for 5 min, a 1.0-µl portion of
each sample was subjected to PCR amplification. Bands were visualized
by ethidium bromide staining of agarose gels after electrophoresis.
PCR procedure.
One microliter of cDNA was added to a PCR mix
containing (at a final concentration) 200 µM deoxynucleoside
triphosphate mix, Taq reaction buffer (Advanced
Biotechnologies, London, United Kingdom), 0.4 µM each primer, 1.5 mM
MgCl2, and 1 U of thermostable Taq polymerase
(Advanced Biotechnologies) in a total reaction volume of 25 µl. The
reaction mixtures were subjected to 25 cycles (2-microglobulin), 35 cycles (TNF-, IFN-), or 40 cycles (IL-12p40) of amplification in a DNA thermal cycler 4800 (Perkin-Elmer, Norwalk, Conn.). An amplification cycle consisted of
denaturation for 30 s at 94°C, primer annealing to the template
at 65°C for 60 s, and primer extension at 72°C for 45 s.
After amplification, 5 µl of each reaction mixture was subjected to
electrophoresis in a 2% agarose gel and the amplified gene products
were visualized by UV light after ethidium bromide staining. A 1-kb
ladder (Gibco BRL) was used as a size marker.
PCR primers for TNF- (354 bp) and IFN- (365 bp) were purchased
from Clontech (Palo Alto, Calif.). The primers for murine 2-microglobulin (300 bp) and IL-12p40 (396 bp) have been
published elsewhere (5, 31).
Competitive PCR.
To quantify the cDNA levels for -actin,
TNF-, and IFN-, a competitive PCR from Clontech was used
(12). IL-12p40 mRNA was quantitated by use of a
competitive fragment purified from the plasmid pMUS (13).
An initial PCR with 10-fold serial dilutions of the
-actin, TNF-
,
IL-12p40, or IFN-
competitor fragments was followed by
a PCR with
twofold serial dilutions. The PCR procedures for
-actin,
IFN-
,
and TNF-
were as previously described (
12). The amount
of
cDNA was determined by identifying the dilution of the competitor
fragment showing the same intensity after amplification as that
of the
amplicon resulting from the sample cDNA. The number of
cycles was
chosen such that amplicons accumulated in a constant
ratio, although
their original concentrations varied up to 100-fold.
The samples were
subjected to 35 cycles of amplification, each
consisting of
denaturation for 30 s at 94°C, primer annealing
to the template
at 60°C for 30 s, and primer extension at 72°C
for 45
s.
Immunohistochemistry.
Skin biopsy specimens excised from the
thorax of infected mice and draining lymph nodes were prepared for
immunocytochemical staining by snap freezing in liquid propane. Tissues
were placed in OCT compound (Tissue Tek), and samples were stored at
70°C until sectioned.
Immunohistochemistry for cytokine expression in infectious foci was
performed as previously described (
25). Rat anti-mouse
IL-12
(10 µg/ml), TNF-
(15 µg/ml), and IFN-
(5 µg/ml) were all
from Pharmingen, San Diego, Calif. Secondary antibody was biotinylated
rabbit anti-rat IgG (adsorbed against mouse antiserum) from Vector
Lab
Inc., Berlingame, Calif. Primary antibody was visualized with
a
peroxidase-labeled antibody. Microscopy was performed with a
Leitz
DRMBE microscope. No staining was visualized after incubation
with
isotype-matched irrelevant antibodies (rat IgG1
clone R3-34,
rat
IgG2a
clone R35-95). To determine the frequency of
cytokine-expressing
cells, the number of peroxidase-stained epidermal
cells per tissue
section was determined, a commonly used method for
quantitation
(
11,
19,
25). The slides were enumerated in a
blinded fashion
by two observers. The score was recorded as 1+ if 1 to
5 cells
were stained in each visual field, 2+ for 5 to 10 cells, and 3+
for >10 cells. For each organ, the average score of 15 to 30 visual
fields was calculated. The term moderate expression was used to
denote
mean arbitrary scores exceeding 0.5, and intense denoted
scores
exceeding 1.5.
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RESULTS |
Growth of F. tularensis LVS after primary infection and
reinfection.
The growth of F. tularensis LVS was
followed for 2 weeks after intradermal inoculation of a sublethal dose,
3.8 × 105 organisms, approximately 0.1 LD50. Immune mice had been immunized by an intradermal
inoculation 5 weeks before reinfection. It was demonstrated in a
previous publication that enhanced nonspecific antibacterial resistance
had completely waned 3 weeks postinoculation (24). It was
therefore assumed that resistance to reinfection at this time
represented specific immunity.
After primary infection, bacterial numbers increased during the first 2 days at the inoculation site and then started to decline
but were not
eradicated until 14 days after challenge (Fig.
1).
By contrast, bacterial numbers
declined within 1 day of infection
at the inoculation site in immune
mice and all bacteria were cleared
within 8 days (Fig.
1). In draining
lymph nodes, significantly
lower bacterial numbers were detected in
samples from immune mice
throughout the course of infection (Fig.
1).
No bacteria were
present in the livers and spleens of immune mice. By
contrast,
bacteria replicated in the latter organs in nonimmune mice,
commencing
on the second day, and were not eradicated until 2 weeks
postchallenge
(Fig.
1). In other experiments, clearance was often
complete at
2 weeks postinoculation and always complete at 3 weeks
postinoculation.
The kinetics of bacterial growth in the organs was
similar in
repeated experiments.
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FIG. 1.
Growth curves of F. tularensis LVS in the
skin, lymph nodes, livers, and spleens of immune (diamonds) and naive
(open circles) mice. Mice received a sublethal intradermal inoculum,
3.8 × 105 CFU, of F. tularensis LVS, and
bacterial numbers were determined over 15 days. Immune mice had
received a sublethal intradermal inoculum 5 weeks before
secondary challenge. The line represents the detection limit of the
assay. The means for five mice per group and time point are shown.
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To assess the longevity of this memory immune response, control of
bacterial growth was followed after reinfection in mice
immunized 1 and
3 months before rechallenge. As shown in Table
1, by contrast to naive mice, no group of
immune mice exhibited
dissemination of bacteria, and the immune mice
had much lower
bacterial numbers in skin
4.5 to 5.5 log
10than did the naive
mice. Approximately 10-fold-lower
bacterial numbers were present
in skin samples of 1 month-immune mice
compared to those in the
3 months-immune group (
P < 0.05). The memory immune response was
monitored up to 6 months in
other experiments. On no occasion
were bacteria observed in samples
from liver and spleen. Although
immunization in the reported
experiments was intradermal, similar
levels of resistance to
reinfection were noted after intravenous
immunization 1 month prior to
intradermal reinfection (data not
shown).
Cytokine expression in skin.
Protein expression in skin
lesions was determined by immunoperoxidase labeling of cryostat
sections. In naive mice, staining for IL-12 at the three time points
was weak, with scores of 0.15, 0.45, and 0.3 (Table
2). By contrast, IL-12 staining was
already intense in the epidermal area at 24 h postchallenge, with
a mean average arbitrary score of 2.2 out of a maximum of 3.0. Intense staining (mean score, 1.05) was also observed at 48 h but had disappeared at 72 h. Staining for TNF- was weak or absent
throughout the experiment in naive mice, whereas moderate staining for
TNF- (mean score, 0.6) was observed at 24 h postchallenge in
immune mice. Later, the TNF- staining faded, with mean scores of 0.5 and 0.2 at 48 and 72 h, respectively. No IFN- expression was visible at 24 or 48 h, and weak staining (mean score, 0.45) was observed in naive mice at 72 h postinoculation, whereas immune animals displayed moderate to intense staining (mean score, 1.6) at the
latter time point. Virtually no staining of any cytokine was observed
after the injection of saline. Representative examples of the
immunohistochemistry are shown in Fig. 2
to 4. An
identical experiment was performed, and onset of cytokine expression
occurred in the two groups at the time points indicated above.
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TABLE 2.
Immunohistochemical analyses of TNF-, IFN-, and
IL-12 expression in skin of mice infected with
F. tularensis LVSa
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FIG. 2.
Expression of TNF- in skin of infected mice. Samples
are from naive (A) and immune (B) mice. For both types of samples,
stained section from noninfected tissue (a), 24 h after
inoculation (b), 48 h postchallenge (c), and 72 h
postinoculation (d) are shown. Controls included staining with
irrelevant primary antibodies and the absence of cross-reactivity of
the secondary labeled antibodies with the primary antibodies of
mismatched isotypes.
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FIG. 3.
Expression of IFN- in skin of infected mice. Samples
are from naive (A) and immune (B) mice. For both types of samples,
stained section from noninfected tissue (a), 24 h after
inoculation (b), 48 h postchallenge (c) and 72 h
postinoculation (d) are shown. Controls included staining with
irrelevant primary antibodies and the absence of cross-reactivity of
the secondary labeled antibodies with the primary antibodies of
mismatched isotypes.
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FIG. 4.
Expression of IL-12 in skin of infected mice. Samples
are from naive (A) and immune (B) mice. For both samples, stained
section from noninfected tissue (a), 24 h after inoculation (b),
48 h postchallenge (c), and 72 h postinoculation (d) are
shown. Controls included staining with irrelevant primary antibodies
and the absence of cross-reactivity of the secondary labeled antibodies
with the primary antibodies of mismatched isotypes.
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Kinetic analysis of cytokine gene expression in skin during primary
and secondary tularemia.
PCR-assisted amplification of cDNA
prepared before and after challenge with F. tularensis LVS
was used to qualitatively assess the expression of cytokine mRNA and to
correlate it to the observed cytokine expression. The number of cycles
used for amplification was such that constitutive expression in skin
was barely discernible or not discernible at all (data not shown).
RT-PCR revealed the presence of TNF-
and IFN-
mRNAs at
24 h and at all later time points after either primary infection
or reinfection (Fig.
5). Weak expression
of IL-12p40 was seen
at 48 and 72 h in naive mice and at 6, 24, 48, and 72 h, but not
at 12 h, in immune mice (Fig.
5). The
experiment was performed
three times, and similar results were
observed.
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FIG. 5.
PCR-assisted amplification of cytokine cDNA from skin
samples after challenge with F. tularensis LVS. The number
of cycles used for amplification was such that constitutive expression
in skin was barely discernible or not discernible at all. Samples were
taken at indicated time points after intradermal challenge of 5 × 105 CFU of F. tularensis LVS.
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Competitive PCR for assessment of mRNA levels in skin after primary
infection and reinfection.
The PCR analyses used in the initial
experiments did not allow any definite conclusions about the mRNA
levels expressed. To make a quantification, competitive PCR was used
and cytokine cDNA was amplified in the presence of various dilutions of
competitor DNA for IL-12p40, IFN-, TNF-, or -actin. The
amounts of cDNA from the samples were equalized with -actin
competitor DNA.
The kinetic analysis had revealed that IFN-
and TNF-
mRNAs were
observed from 24 h on, and therefore samples from these
time
points were analyzed by competitive PCR. After determination
of the
relevant 10-fold dilution, a more exact quantitation was
obtained by
serial twofold dilutions in the appropriate range.
The use of this
competitive PCR indicated that TNF-
and IFN-
mRNA levels were 5- and 30-fold higher, respectively, in immune
mice than in naive mice at
24 h postinoculation. At 48 h, TNF-
mRNA levels were
10-fold higher and IFN-
mRNA levels were 20-fold
higher in naive
mice. The 10-fold titrations of TNF-
and IFN-
mRNAs are shown in
Fig.
6. These results corroborated the
kinetic
PCR analysis which had indicated that higher mRNA levels of
TNF-
and IFN-
were present in immune mice at 24 h
postinoculation
and in naive mice at 48 h (Fig.
5).
Similar differences were observed
in two experiments. IL-12p40 mRNA
levels were also quantitated.
Although higher mRNA levels were observed
in immune mice at 24
h in the experiment shown in Fig.
5, two
repeated experiments
showed slightly higher levels in naive mice at
this time point.
Thus, no consistent differences with regard to IL-12
mRNA levels
could be determined between the two groups. Variations in
the
IL-12p40 mRNA levels can also be seen in Fig.
5. A visible amplicon
was present at 6 and 24 h but not at 12 h.
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FIG. 6.
Competitive PCR analysis of TNF- and IFN- cDNA
levels in skin samples taken at 24 and 48 h postchallenge. The
concentration of cDNA was determined by identifying the dilution of the
competitor fragment showing the same intensity after amplification as
that of the amplicon of the sample cDNA. For each cytokine, the
competitor fragment yielded a larger fragment (indicated with an
arrow). The IFN- competitor DNA ranged, in 10-fold dilutions, from
10 19 to 1023 mol, and the TNF- DNA
ranged from 1018 to 1022 mol.
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DISCUSSION |
We demonstrate here the capability of immune mice to respond
rapidly to infection through the skin with expression of IL-12 and
TNF-, cytokines known to be crucial for host protection as well as
effective in containing the spread of bacteria and in killing them. By
contrast, naive mice could not mount rapid control of infection at the
site of inoculation and did not prevent systemic dissemination.
Moreover, TNF- and IL-12 staining in skin samples from these naive
mice were weak or not observed at all. The ability to prevent any
spread of bacteria to liver and spleen appeared to be a hallmark of the
acquired immune response and was still present 6 months after
immunization. Notably, though, a slight but significant waning of
memory immunity was observed in the skin in 3 month-immune mice
compared to 1 month-immune mice.
The onset of IL-12 expression in immune mice occurred within 1 day. As
recruitment of immune cells requires at least one day and as many as
several days, it is possible that the source of IL-12 was a resident
cell population. Indeed, several cell types present in the skin can
produce cytokines, e.g., keratinocytes and dendritic cells. Dendritic
cells have been identified as an important producer of IL-12 and can
direct the development of Th1 T cells (16). The mechanism
triggering the rapid onset of expression is unknown.
The rapid IL-12 secretion is probably beneficial for the host to
protect against F. tularensis infection. For example, in an
in vitro model of listeriosis, it has been shown that IL-12 together
with TNF- stimulates the release of the macrophage-activating agent
IFN- by NK cells, enhancing bacterial killing (29). IL-12 also drives differentiation of Th1 T cells (10, 22). A
previous communication showed that nonspecific killing of, e.g.,
Listeria monocytogenes, occurs after a challenge with
F. tularensis (24). However, this mechanism was
not operative after the first two weeks of infection (24).
Therefore, it is not likely that such a nonspecific mechanism has any
relation to the early induction of IL-12 that was operative 4 to 5 weeks postimmunization. It should be noted that virtually no staining
for IL-12 was observed in samples from immune mice after the injection
of saline.
There was a marked waning of IL-12 expression in immune mice between 1 and 3 days postinoculation. One explanation may be that the
concentration of Francisella-specific, complement-activating antibodies increases at infectious foci after the first day of infection as part of the inflammatory response. Since a recent study
showed that suppression of IL-12 secretion occurs after signaling via
the CR3 receptor (17), such an increase can lead to
activation of complement, binding to the CR3 receptor, and thereby to
suppression of IL-12 secretion.
Expression of IFN- was not observed before the third day of
reinfection. This lack of expression in immune mice during the first 2 days, when effective control of infection was observed, casts some
doubt on its role in the memory immune response. In a previous study,
it was found that neutralization of IFN- after reinfection affected
bacterial killing only when the inocula of F. tularensis
were very high, thereby demonstrating the possibility of
IFN--independent killing (24). In view of this, the late appearance of IFN- may not be critical for host protection.
Alternatively, biological effects may be present below the levels
detected by the immunohistochemical technique used.
The quantitative reverse transcriptase PCR indicated the presence of
higher mRNA levels of TNF- and IFN- in immune mice at 24 h
postchallenge, reflected by higher expression of the two cytokines in
these mice. However, the much-higher expression of IL-12 present in
skin samples from the immune group was not been preceded by higher mRNA
levels of IL-12p40. The reason for this discrepancy may be related to
the complex transcriptional and translational control of the cytokine.
It is possible that posttranscriptional or posttranslational events
affect expression of the IL-12 dimer, hence explaining the observed
discrepancy between mRNA levels of IL-12p40 and protein expression.
The skin is particularly relevant to immunity to tularemia, as the
ulceroglandular form of the disease is the most common and inherent
capability to control infection is much greater in the skin than in the
internal organs. The present results demonstrate that potent mechanisms
capable of expressing IL-12, IFN-, and TNF- reside in the skin.
This is of definite interest in relation to the rapid killing and
containment of bacteria occurring in immune mice, since previous
reports have documented a critical need for TNF- and IFN- to
protect against primary (14, 24) as well as secondary
F. tularensis infection (24). As previously discussed, IL-12 is critically required for host resistance against infections caused by intracellular bacteria (10, 22, 28). Thus, each of the three cytokines probably plays an important role in
the control of F. tularensis infection, and the early appearance of the cytokines at the site of inoculation is likely to
benefit host protection.
Several experimental models of intracellular infection have provided
evidence that the skin has a unique potential to effectively express
protective immunity and to direct an immune response that subsequently
affords systemic protection. For example, cutaneous infection with
Leishmania donovani resulted in the development of a
prominent local and systemic Th1 immune response and no detectable visceral parasitism, whereas intravenous inoculation resulted in a
delayed Th1 immune response, as evidenced by minimal IL-12 mRNA
expression and a progressive visceral parasite burden (18). In experimental schistosomiasis, the anamnestic immune response of the
skin was already characterized by marked inflammation and increased
tissue expression of ICAM-1 and mRNA for iNOS at 8 h after
infection (20). In contrast, inflammation and expression of
ICAM-1 and iNOS mRNA were minimal in naive controls up to 72 h
postinfection. Together, these findings indicate that expression of
memory immunity in the skin is characterized by rapid recruitment of
inflammatory cells, early and prominent expression of Th1 cytokines, increased expression of adhesion molecules, and early activation of the
pathway generating nitric oxide. The present finding, demonstrating early local production of IL-12, TNF-, and IFN-, is an additional piece of evidence supporting the role of the skin as an essential barrier protecting against infection.
The cutaneous form of experimental L. donovani infection
results in the generation of a Th1 cell response and no progression of
disease. By contrast, intravenous inoculation of Leishmania is followed by progressive visceral disease (18). In
experimental tularemia, effective protection results regardless of the
route of immunization, and control of infection occurs with similar kinetics, regardless of whether inoculation is cutaneous or parenteral. Thus, in contrast to Leishmania infection, the control of
F. tularensis infection is not dependent on the route of
immunization. This result is probably related to the fact that F. tularensis without exception induces a Th1 immune response,
whereas Leishmania infection and other parasitic diseases
under certain circumstances result in the expansion of Th2 T cells and
thereby disease progression.
Previous studies have focused mainly on the requirement of cytokines
and cell subsets for host protection after parenteral routes of
F. tularensis challenge. However, there is no direct evidence that these requirements are similar to those that are operative after cutaneous infection. The present study has provided evidence that helps elucidate which mechanisms are involved. To further
clarify the process, study to identify the cellular source of the
rapidly secreted IL-12, IFN-, and TNF- is under way.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Swedish Medical
Research Council (project no. 9485), Västerbotten Läns
Landsting, and the Medical Faculty, Umeå University.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Defence Research Establishment, S-901 82 Umeå, Sweden. Phone: 46-90-106665. Fax: 46-90-106806. E-mail:
sjostedt{at}ume.foa.se.
Editor:
R. N. Moore
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Infection and Immunity, April 1999, p. 1789-1797, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
