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Lymphotoxin- Double Knockout Mice to Systemic Candidiasis Through Impaired Recruitment of Neutrophils and Phagocytosis of Candida albicans
Departments of
*
Medicine and
Medical Microbiology, University Hospital Nijmegen, Nijmegen, The Netherlands; and
Laboratoire d’Étude des Mécanismes de la Régulation de la Recombinaison Génétique, Unite Mixte de Recherche, Commissariat a l’Energie Atomique/Centre National de la Recherche Scientifique, Fontenay aux Roses, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and lymphotoxin- (LT) are members of the TNF family, and
these cytokines play crucial roles in the defense against infection
with Candida albicans. The aim of the present study was
to investigate the role of endogenous TNF and LT during disseminated
candidiasis in TNF-/-LT-/- knockout mice.
The TNF- and LT-deficient animals had a significantly increased
mortality following C. albicans infection compared with
control mice, and this was due to a 10- to 1000-fold increased
outgrowth of the yeast in their organs. No differences between
TNF-/-LT-/- mice and
TNF+/+LT+/+ were observed when mice were
rendered neutropenic, suggesting that activation of neutrophils
mediates the beneficial effects of endogenous TNF and LT.
Histopathology of the organs, combined with neutrophil recruitment
experiments, showed a dramatic delay in the neutrophil recruitment at
the sites of Candida infection in the
TNF-/-LT-/- mice. Moreover, the neutrophils
of deficient animals were less potent to phagocytize
Candida blastospores than control neutrophils. In
contrast, the killing of Candida and the oxygen radical
production did not differ between neutrophils of
TNF-/-LT-/- and
TNF+/+LT+/+ mice. Peak circulating IL-6 was
significantly higher in TNF-/-LT-/- mice
during infection. Peritoneal macrophages of
TNF-/-LT-/- mice did not produce TNF, and
synthesized significantly lower amounts of IL-1, IL-1
, IL-6, and
macrophage-inflammatory protein-1 than macrophages of
TNF+/+LT+/+ animals did. In conclusion,
endogenous TNF and/or LT contribute to host resistance to disseminated
candidiasis, and their absence in
TNF-/-LT-/- mice renders the animals
susceptible through impaired recruitment of neutrophils and impaired
phagocytosis of C. albicans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF- and lymphotoxin- (LT)2are members of the TNF family of cytokines
(2). Although they have different structures, both TNF and
LT bind to the same TNF receptors, induce overlapping intracellular
pathways, and lead to similar biological effects (3). On
the one hand, these cytokines are considered to be potentially
deleterious mediators of the inflammatory responses that occur during
sepsis (4), and anti-TNF Abs protect against lethal
endotoxemia and i.v. Gram-negative infections in experimental models
(5, 6). On the other hand, endogenous TNF production is
necessary for the normal immune response against an invading
microorganism, as has been demonstrated in various experimental models
(7, 8, 9). TNF also has important roles in host defense
against disseminated candidiasis. It has been shown that mannoprotein
constituents from the Candida albicans cell wall are able to
induce the production of TNF both in vitro (10) and in
vivo (11), and neutralization of endogenous TNF by either
anti-TNF Abs or pharmacological agents has deleterious effects on
the course of experimental disseminated candidiasis (12, 13). The crucial role of TNF-like molecules during C.
albicans infection has been underlined recently by experiments in
mice lacking TNF receptors, which were found to be highly susceptible
to systemic candidiasis (14). However, the underlying
mechanisms responsible for the high susceptibility of these genetically
modified animals to Candida infection have not been
identified.
There are several lines of evidence that suggest that neutrophils are
the main cellular component of the immune system responsible for the
defense against C. albicans (15). The growth of
C. albicans in vitro is inhibited by polymorphonuclear
neutrophils (16), granulocytopenic mice are highly
susceptible to disseminated candidiasis (17), and the
stimulation of neutrophil function by IFN-
(18) or
granulocyte CSF (19) improves the outcome of experimental
infection with C. albicans. TNF is also able to strongly
potentiate the function of neutrophils, resulting in activation of
microbicidal mechanisms such as superoxide production, with an
increased ability of the cells to kill Candida (20, 21). It is therefore reasonable to hypothesize that the
increased susceptibility to disseminated candidiasis in genetically
manipulated mice lacking signals mediated by cytokines of the TNF
family may be mediated through impaired neutrophil function. The aim of
the present study was to establish the mechanisms through which
endogenous TNF and LT protect against C. albicans infection.
We assessed the course of disseminated candidiasis in TNF and LT
double knockout (TNF-/-LT-/-) mice, and we
investigated the mechanisms responsible for the impaired defense
against Candida in these genetically modified animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Homozygous TNF-/-LT-/- and heterozygous TNF+/-LT+/- mice were produced as previously described (22). Specific pathogen-free knockout mice and age- and weight-matched TNF+/+LT+/+ control mice (20–25 g, 6–8 wk old) were used. Mice were fed sterilized laboratory chow (Hope Farms, Woerden, The Netherlands) and water ad libitum.
C. albicans infection model
C. albicans UC 820, a clinical isolate well described earlier (23), has been used in all experiments. Mice were injected i.v. with C. albicans (104–106 CFU/mouse) in a 100 µl volume of sterile pyrogen-free PBS, as indicated. Survival was assessed daily for 30 days in groups of at least 20 animals per group. At the end of the observation period, the surviving mice were anesthetized with ether and killed by cervical dislocation. In addition, subgroups of five animals were killed on day 1, 3, or 7 of infection, and blood was collected on EDTA for plasma cytokine concentration measurements. To assess the outgrowth of the microorganisms, the liver, spleen, and left kidneys of the sacrificed animals were removed aseptically, weighed, and homogenized in sterile saline in a tissue grinder. The number of viable Candida cells in the tissues was determined by plating serial dilutions on Sabouraud dextrose agar plates, as previously described (24). The CFU were counted after 24 h of incubation at 37°C, and expressed as log CFU/g tissue. From the same animals, the right kidneys were fixed in Formalin (4%) and embedded in paraffin, and serial sections were examined microscopically after staining with a combination of periodic acid Schiff and hematoxylin-eosin. Circulating leukocytes were counted using a hemacytometer, and differentiation was assessed microscopically after Giemsa staining.
To examine the distribution and clearance of C. albicans in the early stages of infection, groups of mice were infected with 106 CFU of C. albicans i.v., and subgroups of five animals were sacrificed 1, 2, 4, 8, and 24 h postinfection. The distribution of C. albicans in the liver, spleen, kidneys, lungs, heart, brain, and blood was assessed as described above.
To investigate the role of polymorphonuclear neutrophils (PMN) in the Candida infection in TNF-/-LT-/- mice, groups of deficient and normal mice were rendered neutropenic by pretreatment with cyclophosphamide (Bristol-Myers Squibb, Weesp, The Netherlands): 150 mg/kg was administered s.c. 4 days before infection, followed by 100 mg/kg 1 day before infection, as well as 1, 3, 5, 7, and 9 days after i.v. injection of 104 CFU C. albicans. This treatment leads to a profound and prolonged neutropenia, as has been described previously (25, 26, 27). Daily differential counts in peripheral blood smears confirmed that granulocytes remained <100 x 106/L throughout the infection (data not shown). The outgrowth of the microorganisms in the organs at days 1 and 3 of infection, and the survival of mice during disseminated candidiasis in neutropenic mice was investigated as described above.
Recruitment of neutrophils
To investigate the recruitment of PMN at the site of Candida infection, groups of five TNF-/-LT-/- mice and wild-type littermates were injected i.p. with 107 CFU C. albicans. After 2 and 4 h, peritoneal cells were collected in sterile saline containing 0.38% sodium citrate, and the total cell number was counted in a hemacytometer. The percentage and the absolute numbers of neutrophils were determined in Giemsa-stained cytocentrifuge preparations.
Phagocytosis and killing of C. albicans by neutrophils
Exudate peritoneal PMN were obtained, and phagocytosis and killing were performed as previously described (18). Briefly, groups of five TNF-/-LT-/- mice and wild-type littermates were injected i.p. with 1 ml of 10% proteose peptone, and after 4 h the exudate cells were collected in separate sterile tubes, as described above (18). Peritoneal cells were washed, counted, and resuspended in RPMI 1640 medium (Flow Laboratories, Irvine, CA). The number of cells was adjusted to 5 x 105 PMN/ml. Four hours after injection of proteose peptone, the peritoneal cell population of control mice consisted of 86–97% PMN and 3–14% macrophages. In contrast, the TNF-/-LT-/- mice have a 5- to 8-fold higher population of resident peritoneal macrophages than controls (28), resulting in a substantial percentage of macrophages (40–60%) in the peritoneal exudate 4 h after proteose peptone. A decreased recruitment of PMN after proteose peptone in TNF-/-LT-/- mice (data not shown), similar to that observed after Candida administration, also contributed to this distorted neutrophil to macrophage ratio. Although macrophages are poor in killing C. albicans, and therefore unlikely to disturb the killing assay (18), to correct for this difference between TNF-/-LT-/- and TNF+/+LT+/+ mice, resident peritoneal macrophages from a separate group of TNF-/-LT-/- mice were collected, and added to the peritoneal exudate of TNF+/+LT+/+ mice, in concentrations adjusted to obtain similar PMN/macrophage proportions as in the knockout mice. Subsequently, 5 x 104 CFU C. albicans were added to the cell suspension (PMN to Candida ratio 10:1), and the mixtures were incubated at 37°C in 10 ml silicone-coated tubes (Sherwood Medical, Ballymoney, N. Ireland) under continuous slow rotation.
To assess phagocytosis, a 100 µl sample was taken from each tube after 5 or 15 min of incubation and centrifuged (500 x g, 10 min). The supernatant containing the uningested microorganisms was discarded, and the cells were washed twice in PBS. Light microscopy confirmed that the Candida blastospores were phagocytized, and that no extracellular or attached microorganisms were present. Lysis of the PMN was performed in water containing 0.01% BSA (Sigma, St. Louis, MO). Serial dilutions of the suspension containing the intracellular yeasts were plated on Sabouraud agar plates, and the C. albicans were counted after 24 h of incubation (18). To confirm the role of TNF in the phagocytosis process, PMN of TNF-/-LT-/- mice were coincubated with 1 ng/ml of exogenous murine rTNF (a kind gift of Dr. G. R. Adolf, Bender GmbH, Vienna, Austria), during the phagocytosis assay.
Killing of C. albicans by PMN was assessed in the same cell suspension. After the initial 15-min incubation time to allow for the phagocytosis of blastospores, the tubes were gently centrifuged as described above, and the uningested extracellular Candida cells were discarded. The cells were resuspended in RPMI 1640, and before incubation and after 1-, 2-, and 3-h incubation at 37°C under rotation, a 100 µl sample from each tube was taken, cells were lysed, and C. albicans CFU were enumerated after 24 h of culture on Sabouraud agar plates. Microscopic examination and repeated washes revealed that no C. albicans blastospores were clumped or remained attached to the tubes. The number of viable intracellular C. albicans colonies after each of the incubation periods represented the amounts of yeasts not killed by the cells. The percentage killing of Candida was defined as [1 - (CFU after incubation/CFU recovered at the start of incubation)] x 100.
Superoxide production
PMN from TNF-/-LT-/- and normal mice were collected 4 h after i.p. injection of proteose peptone, as described above. Luminol-enhanced chemoluminescence of proteose-peptone-elicited PMN was measured on a Victor 1420 counter (Wallac, Turku, Finland) at 20°C using white 96-well microplates (Costar, Cambridge, MA), as previously described (29). Each well contained 2 x 105 cells, 50 µM luminol, 4.5 U/ml horseradish peroxidase, and 50 ng/ml PMA in 200 µl of HBSS without phenol red (Life Technologies, Paisley, Scotland) supplemented with 0.25% human albumin (Behringwerke, Marburg, Germany). Reactions were started by adding PMA. Each experiment was performed in duplicate. HRP was added to the system to overcome peroxidase deficiency extracellularly. In previous experiments, we found that the addition of extra peroxidase did not affect superoxide production (measured as reduction of cytochrome c) of human neutrophils stimulated with PMA, but increased luminol-enhanced chemoluminescence 3- to 4-fold. Hence, solely the detection of superoxide is enhanced in the presence of extra peroxidase. The chemoluminescence was expressed as the total amount of superoxide produced during the assay period by integrating the area under the curve (in mV.s) per PMN.
Ex vivo cytokine production
Uninfected TNF-/-LT-/- mice and wild-type controls were sacrificed, and resident peritoneal macrophages were harvested by injecting 4 ml of sterile PBS containing 0.38% sodium citrate (18). After centrifugation and washing, the cells were resuspended in RPMI 1640 containing 1 mM pyruvate, 2 mM L-glutamine, 100 µg/ml gentamicin, and 2% fresh mouse plasma (culture medium). Cells were cultured in 96-well microtiter plates (Costar) at 105 cells/well, in a final volume of 200 µl. The cells were stimulated with either 1 ng/ml LPS (Escherichia coli serotype O55:B5; Sigma) or heat-killed (1 h, 100°C) C. albicans 107 CFU/ml. After 24 h of incubation at 37°C, the plates were centrifuged (500 x g, 10 min), and the supernatant was collected and stored at -80°C until cytokine assays were performed. To assess the cell-associated cytokines, 200 µl of culture medium was added to the remaining cells, and the membranes were disrupted by three freeze-thaw cycles. The samples were stored at -80°C until measurements.
Cytokine assays
IL-1, IL-1, and TNF- were determined by specific RIAs
(detection limit 20 pg/ml), as previously described (30).
IL-6 concentrations were measured by a commercial ELISA (CLB,
Amsterdam, The Netherlands; detection limit 16 pg/ml), according to the
instructions of the manufacturer. Macrophage-inflammatory protein-1
(MIP-1) concentrations were measured by a commercial ELISA (R&D
Systems, Abingdon, U.K.; detection limit 24 pg/ml), according to the
instructions of the manufacturer.
Statistical analysis
The differences between groups were analyzed by Mann-Whitney U test, and where appropriate by Kruskal-Wallis ANOVA test. Survival curves were analyzed by the Kaplan-Meyer log rank test. The level of significance between groups was set at p < 0.05. All experiments were performed at least twice, and the data are presented as cumulative results of all experiments performed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After infection of mice with 106 CFU
C. albicans, all of the
TNF-/-LT-/- and
TNF+/-LT+/- mice died,
whereas only 40% of the wild-type mice died (p
< 0.01). Similarly, after an infection with 5 x
104 CFU C. albicans, the mortality of
both homozygous and heterozygous knockout mice was significantly higher
when compared with the normal mice (p < 0.01)
(Fig. 1
). This was probably due to the
load of C. albicans in the organs, because the numbers of
CFU on days 1, 3, and 7 postinfection were significantly higher in the
organs of TNF-/-LT-/-
mice, and on day 7 also in the organs of
TNF+/-LT+/- mice, than in
those of
TNF+/+LT+/+ mice
(Fig. 2 and Table I). The distribution of C.
albicans to the liver, spleen, heart, lung, brain, and blood
during the initial 24 h of infection did not differ between the
TNF-/-LT-/-
and TNF+/+LT+/+ mice (data
not shown). The insert to Fig. 2 shows the numbers of C.
albicans in the kidneys of
TNF-/-LT-/- and
TNF+/+LT+/+ mice during the
first 24 h after infection, and demonstrates that the early
distribution of C. albicans to the organs is similar, and
that the differences observed at late time points are due to different
rates of outgrowth in the organs of the two mouse strains.
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). On day 7 after infection, the kidneys
of the TNF+/+LT+/+ mice
appeared almost completely healed. In contrast, very few infiltrating
neutrophils were present in the kidneys of
TNF-/-LT-/- mice on
days 1 and 3 after infection, with extensive growth of
Candida, which had formed numerous hyphae and pseudohyphae.
On day 7, despite the presence of large infiltrates of PMN,
Candida was present in large numbers in the centers of the
infiltrates in the kidneys of
TNF-/-LT-/- mice (Fig. 3). In contrast to the low numbers of PMN in the infected tissues of
TNF-/-LT-/- mice,
circulating concentrations of PMN tended to be higher in the
TNF-/-LT-/- than in
TNF+/+LT+/+ mice
on both day 1 (3.9 ± 1.5 vs 2.4 ± 0.9 x
109/L, p = 0.09) and day 3
(4.6 ± 1.9 vs 2.6 ± 1.1 x
109/L, p = 0.06) after infection,
suggesting that the PMN of the knockout mice remain in the circulation
rather than migrating to the infected tissues.
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and IL-1 were below the detection limit
at all time points. Peak IL-6 blood concentrations were observed 1 day
after the infection, with levels significantly higher in both
TNF-/-LT-/- (13.5
± 3.9 ng/ml) and
TNF+/-LT+/- (22.5 ±
6.5 ng/ml), than in
TNF+/+LT+/+ mice (3.8
± 3.1 ng/ml, p < 0.05). On days 3 and 7, the
circulating IL-6 concentrations had decreased considerably and did not
differ between groups (data not shown). To test whether these
differences in circulating cytokine concentrations were solely due to
the different fungal load in the
TNF-/-LT-/- and
TNF+/+LT+/+ mice, or to
differences in the cytokine production capacity of individual
cells from the two mouse strains, resident peritoneal macrophages
from
TNF-/-LT-/-
and TNF+/+LT+/+ mice were
stimulated with 1 ng/ml LPS or 107 CFU
heat-killed C. albicans in vitro for 24 h. The
production of TNF by control (+/+) macrophages in response to
Candida stimulation (Fig. 4B) was greater than that
after LPS stimulation (Fig. 4A), whereas LPS was a stronger
stimulus of the synthesis of IL-1 and IL-6 (Fig. 4, A and
B). IL-1 was induced in similar amounts by LPS (Fig. 4A) and C. albicans (Fig. 4B).
Macrophages obtained from
TNF-/-LT-/- mice did
not produce TNF, and moreover they synthesized significantly lower
amounts of IL-1, IL-1, and IL-6 than macrophages of
TNF+/+LT+/+ animals did
(Fig. 4), in agreement with earlier observations with LPS stimulation
in vitro (31). Moreover, macrophages of
TNF-/-LT-/-
also displayed an impaired capacity to synthesize chemokines, as their
production of MIP-1 was significantly lower than that in control
macrophages (204 ± 72 vs 713 ± 181 pg/ml, p
< 0.05).
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, the
outgrowth of Candida in the organs of neutropenic mice was
increased compared with those of nonneutropenic mice, but the fungal
load in the organs of cyclophosphamide-treated
TNF-/-LT-/- or
TNF+/+LT+/+ mice did not
differ at days 1 and 3 of infection, suggesting that the beneficial
effects of endogenous TNF and LT during systemic candidiasis are
mediated through activation of neutrophils. Likewise, survival during
disseminated candidiasis did not differ in neutropenic
TNF-/-LT-/- or
TNF+/+LT+/+ mice (50 vs
44%, p > 0.05).
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To investigate the recruitment of neutrophils at the site of a
C. albicans infection, groups of
TNF-/-LT-/- and
TNF+/+LT+/+ mice were
infected i.p. with 107 CFU C.
albicans, and exudate peritoneal neutrophils were harvested and
counted 2 and 4 h later. As shown in Fig. 5, there was significantly less
infiltration of neutrophils in the peritoneal cavity of
TNF-/-LT-/- than in
that of TNF+/+LT+/+ mice.
At 24 h after infection, a similar difference between the two
mouse strains was found (data not shown).
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Phagocytosis of C. albicans by PMN of
TNF-/-LT-/- mice
was significantly reduced compared with that by PMN of
TNF+/+LT+/+ mice
(p < 0.05 by Kruskal-Wallis ANOVA) (Fig. 6A). Addition of exogenous
murine rTNF to the neutrophils of
TNF-/-LT-/- mice
restored the normal phagocytic capacity (Fig. 6A). It has
been shown that exogenous TNF is able to increase the
Candida-killing capacities of neutrophils in vitro
(20). Neutrophils of
TNF-/-LT-/- mice tended
to show a decreased killing of C. albicans after 2 or 4
h of incubation compared with neutrophils of
TNF+/+LT+/+ mice (67
± 11% vs 82 ± 11% after 4 h), although these differences
were not significant (p > 0.05; Fig. 6B). Superoxide production by PMN of
TNF-/-LT-/- mice was
similar to that by PMN of
TNF+/+LT+/+ control mice
(0.15 ± 0.04 vs 0.17 ± 0.07 mV.s/PMN, p >
0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endogenous TNF is important for a proper activation of the host defense during infections such as bacterial peritonitis (9), Mycobacterium sp. (8, 32), and Listeria monocytogenes (7) infections. Because both TNF and LT interact with the same membrane receptors and have similar effects, the absence of one of these two cytokines may be partly compensated by actions exerted by the other one. To exclude the possible redundant effects of the two cytokines, we investigated the course of disseminated candidiasis in double knockout mice, lacking both functional TNF and LT. The results of the present study demonstrate a crucial role of TNF and/or LT in the defense against C. albicans infection, and this is in line with the deleterious effects of TNF inactivation in systemic candidiasis (12, 13). A recent study has also reported an increased susceptibility of TNFRp55-/- and TNFRp75-/- mice to C. albicans (14), and it has been suggested that the most important effects of TNF during Candida infection are mediated through TNFRp55, whereas TNFRp75 would have a secondary role (14). The crucial role of the signals mediated through TNF receptors for the defense against C. albicans is not an isolated phenomenon, as the TNFRp55-/- mice are highly susceptible to L. monocytogenes (33, 34), Mycobacterium tuberculosis (32), and Toxoplasma gondii (35), and the TNF-/- mice succumb more easily to Corynebacterium parvum infection (36).
In previous studies, the mechanisms mediating the increased susceptibility of TNF-/- or TNFR-/- mice to C. albicans infection have not been elucidated. The increased fungal burden in the TNF-/-LT-/- mice as early as 1 day after the infection may suggest that an altered distribution and/or clearance of Candida in the initial stages of the infection may have influenced the invasion of the organs, leading to differences in the initial organ burden. However, the clearance from the blood and the distribution of C. albicans to the liver, spleen, kidneys, lung, heart, and brain during the first 24 h were similar in the wild-type and knockout mice. Thus, the differences in outgrowth later in the infection are expected to involve mechanisms unrelated to effects in the initial stages of infection.
TNF is able to stimulate the anticandidal properties of neutrophils (20), and this is associated with an increased production of oxygen radicals (21). These in vitro data, together with the severe course of disseminated candidiasis in neutropenic mice (17), suggest that neutrophils are the main effector cells responsible for the defense against C. albicans infection (15). Indeed, when both wild-type and TNF-/-LT-/- mice were rendered neutropenic by cyclophosphamide, the mortality was high in both groups, and no difference in the colony count of C. albicans in the organs of the two mouse strains was observed. This observation suggests that the presence of neutrophils is crucial for the protective effects of endogenous TNF against Candida. It should, however, be noted that cyclophosphamide treatment also influences, although to a much lesser extent, the lymphocyte population (25, 27), and an influence of this effect on anticandidal defense cannot be excluded.
The lack of functional TNF and LT could affect neutrophil function at several levels. The first step in the action of neutrophils during an infection is attachment to the activated endothelial cells and migration into the infected tissues. In the present study, the histology of the kidneys in the TNF-/-LT-/- mice shows a significant delay in the neutrophil infiltration at the site of infection. Consequently, the neutrophil-mediated inhibition of hyphal formation that is apparent in control mice and that is important for the defense against Candida (37) is severely impaired in the TNF-/-LT-/- mice, leading to extensive growth of C. albicans. Impaired recruitment of neutrophils in TNF-/-LT-/- mice is also present after an i.p. infection with Candida. Similar defects in recruitment of neutrophils have been observed during a Micropolyspora faeni infection in TNF-Rp55-/- mice (38).
The mechanisms responsible for the impaired recruitment of neutrophils
in the TNF-/-LT-/- mice
could involve defective expression of adhesion molecules on the
leukocytes and endothelial cells of these mice. Attachment of
neutrophils to the endothelial cells is mediated through expression of
adhesion molecules such as E-selectin and ICAM-1 on the endothelium and
the corresponding ligands on the neutrophils (39, 40).
TNF is an important stimulus of their expression (41), and
mice deficient in TNFRp55-/- fail to express
normal amounts of adhesion molecules (42). As a result,
homing of lymphocytes in the lymphoid organs of these mice is
defective, with disturbed lymphoid organ architecture
(42). In addition, reduced expression of the adhesion
molecules due to the lack of TNF and LT may also impair recruitment of
neutrophils and have deleterious effects during systemic candidiasis in
the TNF-/-LT-/- mice.
Impaired expression of adhesion molecules such as the
2 integrins may have an additional effect,
because it has been shown that leukocytes utilize CD11b/CD18 for
recognition and binding of C. albicans (43).
The importance of proper expression of these molecules for the
Candida infection is underlined by a recent study showing
that ICAM-1 knockout mice are more susceptible to C.
albicans infection (44). In addition, TNF is an
important stimulus of chemokine production (3), and
macrophages from
TNF-/-LT-/- mice
produced significantly less MIP-1 compared with macrophages from
control TNF+/+LT+/+ mice.
This effect may also have contributed to the reduced and delayed PMN
recruitment in
TNF-/-LT-/-
animals.
Another possible target for the effects of TNF on neutrophils is represented by the candidacidal mechanisms. Phagocytosis of C. albicans by PMN of TNF-/-LT-/- mice was impaired compared with phagocytosis by PMN of control animals, and the requirement of TNF for phagocytosis was confirmed by restoration of phagocytic activity of TNF-/-LT-/- neutrophils after addition of rmTNF. In addition, previous in vitro studies have also shown that TNF is able to enhance the capacity of neutrophils to kill C. albicans, and an impaired killing capacity of the neutrophils of TNF-/-LT-/- mice could have been expected. However, the neutrophils of knockout mice were as potent as their wild-type counterparts to kill Candida, and this was accompanied by a similar production of superoxide. These results are consistent with previous studies showing normal oxygen radical production by neutrophils of TNF-/- mice (36) and normal killing of Leishmania major by macrophages of TNFRp55-/- mice (45). Another possible killing mechanism influenced by TNF could have been the nitric oxide production, but previous studies have shown normal nitric oxide production by cells of TNF-/- (36), TNFRp55-/-, and TNFRp75-/- mice (35).
The data presented in this study suggest that neutrophils are the main
cells mediating the beneficial effects of endogenous TNF during
systemic candidiasis. However, the possible involvement of other cell
types cannot be ruled out. Several studies in the literature have
underlined the involvement of a Th1/Th2 imbalance in the increased
susceptibility to C. albicans infection (for review, see
Ref. 46). Indeed, an impaired Th1 response in
TNF-/- mice has been suggested recently
(47), and this may have contributed to the increased
susceptibility of these mice to C. albicans infection. The
protective effect of a Th1 response is, however, probably mediated
through neutrophils, because IFN- activates polymorphonuclear
neutrophils for killing of Candida (20), and
its beneficial effects during candidiasis are absent in neutropenic
mice (18). The monocytes and macrophages are probably less
involved, as murine macrophages are only able to kill C.
albicans in vitro to a minor degree, and monocyte-depleted mice do
not show an increased susceptibility to candidiasis
(17).
In addition to TNF and LT, the production capacity of other cytokines
such as IL-1, IL-1, and IL-6 by macrophages of
TNF-/-LT-/- mice was
also significantly impaired compared with normal mice. This effect
could be due to either a defect in the ability of
TNF-/-LT-/- macrophages
to produce these cytokines, or an indirect involvement of endogenous
TNF or LT in the production of other cytokines. Indeed, an important
proportion of the LPS-mediated synthesis of IL-1, IL-6, and MIP-1
has been shown to be mediated through intermediary production of
endogenous TNF-like molecules (48, 49). The normal
production of IL-1 and IL-6 by TNF-/- mice in
which the gene for LT was intact (36, 50) suggests an
important role of LT deficiency for the decreased production of these
cytokines in the
TNF-/-LT-/- mice. IL-1
and IL-6 can also contribute to the defense against C.
albicans (51, 52), and the relative deficiency in the
production of these cytokines could have also contributed to the
increased susceptibility of
TNF-/-LT-/- to systemic
candidiasis. In vivo, the higher circulating concentrations of IL-6 in
the TNF-/-LT-/- mice
are probably due to the much higher fungal load in the deficient
animals, resulting in more cellular stimulation than in the wild-type
counterparts.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: LT, lymphotoxin-; MIP-1, macrophage-inflammatory protein-1; PMN, polymorphonuclear neutrophils.
Received for publication July 14, 1998. Accepted for publication May 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  B. Balestrieri, A. Maekawa, W. Xing, M. H. Gelb, H. R. Katz, and J. P. Arm Group V Secretory Phospholipase A2 Modulates Phagosome Maturation and Regulates the Innate Immune Response against Candida albicans J. Immunol., April 15, 2009; 182(8): 4891 - 4898. [Abstract] [Full Text] [PDF]
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 B. J. Spellberg, M. Collins, V. Avanesian, M. Gomez, J. E. Edwards Jr., C. Cogle, D. Applebaum, Y. Fu, and A. S. Ibrahim Optimization of a myeloid cell transfusion strategy for infected neutropenic hosts J. Leukoc. Biol., March 1, 2007; 81(3): 632 - 641. [Abstract] [Full Text] [PDF]
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M. Moller-Kristensen, W. K. E. Ip, L. Shi, L. D. Gowda, M. R. Hamblin, S. Thiel, J. Chr. Jensenius, R. A. B. Ezekowitz, and K. Takahashi Deficiency of Mannose-Binding Lectin Greatly Increases Susceptibility to Postburn Infection with Pseudomonas aeruginosa J. Immunol., February 1, 2006; 176(3): 1769 - 1775. [Abstract] [Full Text] [PDF]
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T. W. Spahn, H.-P. Eugster, A. Fontana, W. Domschke, and T. Kucharzik Role of Lymphotoxin in Experimental Models of Infectious Diseases: Potential Benefits and Risks of a Therapeutic Inhibition of the Lymphotoxin-{beta} Receptor Pathway Infect. Immun., November 1, 2005; 73(11): 7077 - 7088. [Full Text] [PDF]
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H. S. Kim, E. H. Choi, J. Khan, E. Roilides, A. Francesconi, M. Kasai, T. Sein, R. L. Schaufele, K. Sakurai, C. G. Son, et al. Expression of Genes Encoding Innate Host Defense Molecules in Normal Human Monocytes in Response to Candida albicans Infect. Immun., June 1, 2005; 73(6): 3714 - 3724. [Abstract] [Full Text] [PDF]
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A. C. Herring, N. R. Falkowski, G.-H. Chen, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Transient Neutralization of Tumor Necrosis Factor Alpha Can Produce a Chronic Fungal Infection in an Immunocompetent Host: Potential Role of Immature Dendritic Cells Infect. Immun., January 1, 2005; 73(1): 39 - 49. [Abstract] [Full Text] [PDF]
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P. B. Mann, K. D. Elder, M. J. Kennett, and E. T. Harvill Toll-Like Receptor 4-Dependent Early Elicited Tumor Necrosis Factor Alpha Expression Is Critical for Innate Host Defense against Bordetella bronchiseptica Infect. Immun., November 1, 2004; 72(11): 6650 - 6658. [Abstract] [Full Text] [PDF]
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M. G. Netea, R. Sutmuller, C. Hermann, C. A. A. Van der Graaf, J. W. M. Van der Meer, J. H. van Krieken, T. Hartung, G. Adema, and B. J. Kullberg Toll-Like Receptor 2 Suppresses Immunity against Candida albicans through Induction of IL-10 and Regulatory T Cells J. Immunol., March 15, 2004; 172(6): 3712 - 3718. [Abstract] [Full Text] [PDF]
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 M. C. Rodriguez-Galan, C. Sotomayor, M. E. Costamagna, A. M. Cabanillas, B. S. Renteria, A. M. Masini-Repiso, and S. Correa Immunocompetence of macrophages in rats exposed to Candida albicans infection and stress Am J Physiol Cell Physiol, January 1, 2003; 284(1): C111 - C118. [Abstract] [Full Text] [PDF]
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L. Wang, H. Das, A. Kamath, L. Li, and J. F. Bukowski Human V{gamma}2V{delta}2 T Cells Augment Migration-Inhibitory Factor Secretion and Counteract the Inhibitory Effect of Glucocorticoids on IL-1{beta} and TNF-{alpha} Production J. Immunol., May 15, 2002; 168(10): 4889 - 4896. [Abstract] [Full Text] [PDF]
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S. Magez, B. Stijlemans, G. Caljon, H.-P. Eugster, and P. De Baetselier Control of Experimental Trypanosoma brucei Infections Occurs Independently of Lymphotoxin-{alpha} Induction Infect. Immun., March 1, 2002; 70(3): 1342 - 1351. [Abstract] [Full Text] [PDF]
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C. Steele and P. L. Fidel Jr. Cytokine and Chemokine Production by Human Oral and Vaginal Epithelial Cells in Response to Candida albicans Infect. Immun., February 1, 2002; 70(2): 577 - 583. [Abstract] [Full Text] [PDF]
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J. H. Choi, H. M. Ko, J.-W. Kim, H.-K. Lee, S. S. Han, S.-B. Chun, and S. Y. Im Platelet-Activating Factor-Induced Early Activation of NF-{{kappa}}B Plays a Crucial Role for Organ Clearance of Candida albicans J. Immunol., April 15, 2001; 166(8): 5139 - 5144. [Abstract] [Full Text] [PDF]
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A. N. Shakhov, I. G. Lyakhov, A. V. Tumanov, A. V. Rubtsov, L. N. Drutskaya, M. W. Marino, and S. A. Nedospasov Gene profiling approach in the analysis of lymphotoxin and TNF deficiencies J. Leukoc. Biol., July 1, 2000; 68(1): 151 - 157. [Abstract] [Full Text]
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 A. Vazquez-Torres, G. Fantuzzi, C. K. Edwards III, C. A. Dinarello, and F. C. Fang Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages PNAS, February 27, 2001; 98(5): 2561 - 2565. [Abstract] [Full Text] [PDF]
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), heterozygous
TNF+/-LT+/- mice (
), and homozygous
TNF-/-LT-/- mice (
) were infected i.v.
with 5 x 104 CFU Candida per
mouse. Both homozygous and heterozygous deficient mice
had an increased mortality compared with wild-type controls
(p < 0.01, log rank test).
