Download Lymphokine-Activated Killer Cells Lyse Listeria

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Lymphokine-Activated Killer Cells Lyse Listeria-Infected Hepatocytes and
Produce Elevated Quantities of Interferon-y
Stephen H. Gregory, Xiaosui Jiang, and Edward J. Wing
Department of Medicine, University of Pittsburgh Medical Center,
Pittsburgh, Pennsylvania
The bulk of Listeria monocytogenes injected intravenouslyinto mice is taken up in the liver, where
hepatocytes serve as the principal site of intracellular replication. NK cells have been implicated in
host defenses to a variety of intracellular pathogens. To explore the role of NK cells in resistance
to listerial infections of the liver, lymphokine-activated natural killer (LAK) cells were cocultured
with Listeria-infected hepatocytes. The aspartate aminotransferase activity in the medium (evidence
of cytotoxicity and hepatocyte damage) was elevated significantlyin these cultures. Conversely, the
viability of intracellular Listeria organisms was reduced. Increased quantities of interferon-y (IFN1') were also detected. IFN-1' production by LAK cells was modulated by interleukin (lL)-2 and IL12.These findings suggest that the response of LAK cellsto infected hepatocytes may playa critical
role in host defenses to Listeria organisms taken up in the liver.
Listeria monocytogenes is a gram-positive bacterium capable
of replicating intracellularly. Listeriosis in mice is an experimental model used widely to examine the factors that affect
host resistance to intracellular pathogens [1]. NK cells have
been implicated in nonspecific host defenses to a broad range
of pathogens. In the case of Listeria organisms, the number of
NK cells is increased significantly in the livers of mice infected
intravenously (iv) [2]. The role of NK cells in host resistance
is suggested by the fact that T cell-deficient (but NK-sufficient) mice, such as scid [3] and athymic nude [4], effectively
control the early phase of listerial infections. Furthermore, scid
mice depleted of NK cells before iv infection exhibit a 6-fold
increase in the number of Listeria organisms recovered in the
spleen on day 3 after infection [5]. Similarly, NK cell-depleted
normal mice infected subcutaneously in the footpad exhibited
marked increases in Listeria organisms subsequently recovered
in the footpad and draining lymph nodes [6]. It has been suggested that a major function of NK cells in host defenses to
intracellular pathogens may be to lyse infected host cells that
otherwise serve as a protected environment for the growth of
such organisms [7-10]. In addition, NK cells may synthesize
and secrete soluble factors, such as interferon-y (lFN-y), that
promote the resistance or antimicrobial activity of host cells
[6, 10-12].
Most pathogens that enter the bloodstream are cleared by the
liver [13-15]. In the case of Listeria, > 90% of the organisms
Received 24 October 1995; revised 17 June 1996.
Presented in part: 9th International Congress of Immunology, San Francisco,
July 1995 (abstract 2010).
This project was approved by the University of Pittsburgh Institutional Animal Care and Use Committee (assurance number A3I87-01).
Grant support: NIH (DK-44367).
Reprints or correspondence: Dr. Stephen H. Gregory, Dept. of Medicine,
Montefiore University Hospital, 200 Lothrop St., Pittsburgh, PA 15213-2582.
The Journal of Infectious Diseases 1996; 174:1073-9
© 1996 by The University of Chicago. All rights reserved.
0022-1899/96/7405-0024$0 1.00
recovered in the liver at 2 h after iv infection are associated
with the parenchymal cells (hepatocytes) [16]. Hepatocytes
constitute the principal site of listerial replication in the livers
of nonimmune mice [16, 17]. Listeria organisms injected into
immune animals, on the other hand, are taken up in the liver
and rapidly eliminated [16]. Using a population oflymphokineactivated natural killer (LAK) cells, we undertook a series of
in vitro experiments to explore the interaction ofNK cells and
hepatocytes.
Materials and Methods
Bacteria. L. monocytogenes (EGD strain) was cultured and
maintained as described [18]. Virulence of the organism was sustained by routine passage in mice. Listeria organisms harvested
from cultures growing exponentially were used in the experiments
described.
Animals. Female C57BL/6J mice purchased from Jackson
Laboratories (Bar Harbor, ME) were housed and cared for in accordance with guidelines set forth by the Institutional Animal Care
and Use Committee, University of Pittsburgh. Hepatocytes and
LAK cells were derived from animals that were between 2 and 4
months of age.
Hepatocytes. Purified parenchymal cells (>96% viable hepatocytes) were obtained after perfusion of the liver with collagenase
using the two-step method we reported previously [16, 19]. Hepatocytes were cultured in HEPES-buffered RPMI 1640 medium
(BioWhittaker, Walkersville, MD) supplemented with I mM sodium pyruvate, 10- 7 M recombinant human insulin (Humulin R;
Eli Lilly, Indianapolis), and 10% heat-inactivated fetal bovine serum (Sterile Systems, Logan, UT). Freshly isolated hepatocytes
were analyzed by flow cytometry and found to be 50%-60% major
histocompatibility complex (MHC) class I-positive and 0% MHC
class II-positive; hepatocytes cultured for 12 h under nonadherent
conditions were 50%-60% MHC class I-positive and 20%-30%
MHC class II-positive.
LAK cells. LAK cells were derived from nylon wool-monadherent, erythrocyte-depleted mouse splenocytes by culture in the
presence of 1000 U/mL recombinant human interleukin (lL)-2
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Gregory et al.
(Hoffmann-La Roche, Nutley, NJ) according to the methods described by Gunji et at. [20]. Cell cytometric analysis ofthe resultant
population indicated that the majority of cells expressed surface
markers characteristic of murine NK cells, that is, ~99% Thy1.2+ and ~88% NK-l.l + [20]. For experimental use, the cells were
suspended in HEPES-buffered RPMI 1640 medium supplemented
with 1 mM sodium pyruvate, 1 mM glutamine, 5 X 10- 5 M 2mercaptoethanol, 10% fetal bovine serum, 5 ;..tg/mL gentamicin,
and the following cytokines unless noted otherwise: 30 U/mL IL2, 100 U/mL recombinant human tumor necrosis factor (TNF)-a
(Genentech, South San Francisco, CA), and 50 pg/mL recombinant
murine IL-12 (provided by Maurice Gately, Hoffmann-La Roche).
Cocultures. Hepatocytes were seeded into 96-well tissue culture plates (2 X 104 cells/well) and the cells were incubated overnight. On the following day, the plates were inoculated with Listeria organisms, centrifuged at 200 g for 10 min at room temperature
to facilitate contact between the bacteria and cells, and incubated
for 4 h at 37°C. Gentamicin (5 ;..tg/mL final concentration) was
then added to kill extracellular Listeria organisms, and the cells
were incubated overnight. On the following day, LAK cells were
added to the appropriate wells, and the cells were cocultured for
the time periods indicated in the text.
To determine whether direct contact between cell populations
was required to stimulate IFN--y production by LAK cells, LAK
cells and hepatocytes cocultured in the same well were separated
by membrane inserts (0.45-;..tm Cyclopore membrane; Becton
Dickinson Labware, Lincoln Park, NJ). Hepatocytes (lOs/well) in
24-well tissue culture plates were infected or not infected with 106
Listeria organisms. The next day, 106 LAK cells were added directly to the wells or to inserts placed in the wells. IFN--y in the
culture supernates was assessed after 24 h of incubation.
Cytotoxicity assay. To assess LAK cell-mediated lysis of hepatocytes, the cells were cocultured at various effector-to-target
cell ratios under the conditions described above. Hepatocyte lysis
was estimated from the level of aspartate aminotransferase (AST)
activity in an aliquot of the culture supernate as described by
Feutren et al. [21]. AST levels were quantified by the Clinical
Chemistry Laboratory, University of Pittsburgh Medical Center,
using an automated spectrophotometric assay. Percentage of specific cytotoxicity was calculated as follows: [(experimental AST
- spontaneous AST)/(maximum AST - spontaneous AST)] X
100. Complete lysis of 2 X 104 hepatocytes yielded AST activity
of 250-300 IU/L. Supernates derived from LAK cells cultured
alone had <3 lUll AST activity.
Lysis of infected hepatocytes resulted in the influx of culture
medium and the death of intracellular Listeria organisms exposed
to gentamicin. Viable Listeria organisms that remained were quantified as an alternative approach to assessing cytolysis of infected
hepatocytes. At the end of the incubation period, the culture supernates were aspirated, the cell monolayers were lysed by treatment
with 0.05% Triton X-IOO in trypticase soy broth, and the number
of viable intracellular Listeria organisms released was estimated
using a modification of the MTT assay described by Peck [22].
Briefly, 1 mg/mL (final concentration) MTT (Sigma, St. Louis)
was added to the cell lysates, and the plates were incubated for 4
h at 37°C. The formazan product ofMTT metabolism was solubilized by the addition of an equal volume of 10% SDS in 0.01 N
HCI, and the plates were incubated for an additional 18 h at 37°C.
The plates were read on a microplate reader using a 570-nm test
JlO 1996; 174 (November)
20
3
Listeria
• 1 x 104
v 1 x 105
T 1 x 10
10
1:1
10:1
3:1
Effector:Target cell ratio
Figure 1. Listeria-infected hepatocytes cocultured with LAK cells
in presence of gentamicin exhibit marked reduction in viable intracellular bacteria. LAK cells and hepatocytes infected 24 h previously at
concentrations shown were cocultured for 5 h in presence of 5 pg/
mL gentamicin. Data are mean ± SD absorbanceobtained fromtriplicate wells in single experiment;2 additional experimentsgave comparable results.In all cases, absorbanceobtained for infectedhepatocytes
cocultured with LAK cells was significantly less than that obtained
for infected hepatocytes cultured alone; P < .05 (one-way analysis
of variance).
filter and a 630-nm reference filter; wells containing hepatocytes
with or without LAK cells and no Listeria organisms served as
the blank. Alternatively, surviving intracellular Listeria organisms
were estimated from the colonies that grew on trypticase soy agar
plates inoculated with aliquots of diluted cell lysate.
IFN-y detection by ELISA. IFN-y in the culture supemates
was quantified by ELISA as described [23].
Statistical analysis. Results were analyzed using the SigmaStat
statistics program (Jandel Scientific, San Rafael, CA). Multiple
treatment groups were compared by analysis of variance followed
by a Student-Newman-Keuls test. Differences at the P < .05 level
were considered significant.
Results
Infected hepatocytes cocultured with LAK cells exhibit a
marked reduction in viable, intracellular Listeria organisms.
The addition of LAK cells to cultures of infected hepatocytes
resulted in a marked reduction in the viability of intracellular
Listeria organisms assessed in terms of MTT metabolism (figure I). This reduction was significant at an effector-to-target
JID 1996;174 (November)
LAK Cells Lyse Listeria-Infected Hepatocytes
cell ratio of ~ 1:1 and was apparent in hepatocyte cultures
inoculated with 103 , 104 , or 105 Listeria organisms. Similar
results were obtained when the number of surviving intracellular Listeria organisms was estimated from the colonies that
grew on trypticase soy agar plates inoculated with an aliquot
of cell lysate (figure 2). It is pertinent to note that in the absence
of LAK cells, the maximum number of intracellular bacteria
was determined in cultures infected with 104 Listeria organisms/well. Between 90% and 95% of hepatocytes contained
intracellular Listeria organisms at the time ofLAK cell addition
as judged by Gram's staining. Infection with a larger dose,
105 organisms/well, resulted in an overall loss in hepatocyte
viability and the death of bacteria exposed to gentamicin contained in the medium.
LAK cells lyse Listeria-infected hepatocytes preferentially.
The viability of intracellular bacteria correlated inversely with
the cytotoxic activity expressed by LAK cells cocultured with
Listeria-infected hepatocytes (figure 3). As the percentage of
cytotoxicity increased with an increasing ratio of LAK cells to
infected hepatocytes, MTT metabolism by intracellular Listeria
organisms decreased concomitantly. Moreover, LAK cells
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Figure 3. LAK cells lyse Listeria-infected hepatocytes preferentially. Uninfected hepatocytes (dashed rule) or hepatocytes infected
with 104 Listeria/well (solid rule) were cocultured with LAK cells for
5 h. Data are mean ± SE derived from 6 wells in single experiment;
comparable results were obtained in 5 similar experiments. LAK cells
cocultured with Listeria-infected hepatocytes exhibited significantly
more cytolytic activity (e) than did cells cocultured with uninfected
hepatocytes; P = .042 (two-way analysis of variance). Infected hepatocytes cocultured with LAK cells (effector-to-target cell ratio ~ I)
contained significantly fewer viable intracellular Listeria organisms
(T) than did infected hepatocytes cultured alone; P < .05 (one-way
analysis of variance).
1:1
3:1
10:1
Effector:Target cell ratio
Figure 2. Viable intracellular Listeria organisms are reduced in
cocultures that contain LAK cells and gentamicin (colony-forming
unit assay). LAK cells and Listeria-infected hepatocytes were cocultured 8 h. Data are mean ± SE cfu obtained in single experiment; 2
additional experiments yielded similar results. Values obtained for
infected hepatocytes cocultured with LAK cells at effector-to-target
cell ratios ~ 3:I were significantly less than those obtained for infected
hepatocytes cultured alone; P < .05 (one-way analysis of variance).
lysed Listeria-infected hepatocytes preferentially. The percentage of cytotoxicity expressed by LAK cells was elevated in
cocultures that contained infected, relative to those that contained uninfected, hepatocytes. This was particularly evident
at lower effector-to-target cell ratios, that is, 3: I, at which
nonspecific lysis of uninfected hepatocytes was limited.
LAK cells cocultured with Listeria-infected hepatocytes produce elevated levels ofIFN-y. In addition to exhibiting cytolytic activity, LAK cells cocultured with Listeria-infected hepatocytes produced IFN-y. The amount of IFN-y produced was
proportional to the number of LAK cells present. IFN-y was
not detectable in supemates obtained from cultures of hepatocytes incubated in the absence of LAK cells (figure 4). Quantities of 2 X 105 LAK cells produced 5- to l O-fold more IFNy than did 2 X 104 LAK cells cocultured with hepatocytes
under comparable conditions. In all cases, LAK cells cocultured
with Listeria-infected hepatocytes secreted 2-4 times more
IFN-y than did an equivalent number of LAK cells cocultured
with uninfected hepatocytes.
IL-2 and IL-12 act synergistically to stimulate IFN-y production by LAK cells cocultured with infected hepatocytes.
JID 1996; 174 (November)
Gregory et al.
1076
increase in IFN-y production. LAK cells incubated for 24 h
in the presence or absence of 103 _10 5 bacteria produced comparable levels ofIFN-y (table I). In contrast, LAK cells cocultured with hepatocytes infected with the same number of Listeria organisms produced significantly more IFN-y (2- to 4-fold)
than did LAK cells incubated with uninfected hepatocytes.
Hepatocytes synthesize and secrete a variety of soluble factors, such as IL-I, IL-6, macrophage colony-stimulating factor,
and granulocyte-macrophage colony-stimulating factor, that
possess immunomodulating activity [28, 29]. Soluble factors
produced by hepatocytes did not contribute to the elevated
levels ofIFN-y produced by LAK cells in our coculture system.
Medium conditioned by the culture of uninfected or Listeriainfected hepatocytes had no effect on the subsequent production
of IFN-y by LAK cells cultured alone (table 2).
While there was no evidence to indicate that soluble factors
secreted by hepatocytes affected IFN-y production by LAK
Listeria/well
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Figure 4. IFN-y production by LAK cells cocultured with hepatocytes is modulated by LAK cell concentration and listerial infection.
Hepatocytes (2 X 104/well) infected at concentrations indicated and
increasing number of LAK cells were cocultured for 24 h. Values
are mean IFN-y concentrations in supemates obtained from triplicate
wells. Three experiments yielded comparable results.
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The effects ofIL-2, IL-12, and TNF-a on the biologic activities
expressed by NK and LAK cells are well-documented [5,20,
24-27]. IFN-y production by LAK cells cocultured with Listeria-infected hepatocytes was increased significantly by the
presence ofIL-2 and IL-12. LAK cells incubated in the absence
of exogenous cytokines produced a negligible quantity of IFNy (figure 5). While the addition of either IL-2 or IL-12 alone
caused a detectable increase, optimal IFN-y production was
attained only in cocultures that contained both IL-2 and IL-12.
The effects of IL-2 and IL-12 were synergistic; the concentration of IFN-y in supemates derived from cultures treated with
both cytokines was significantly greater than that expected if
IL-2 and IL-12 had exerted additive effects on IFN-y production. In contrast to IL-2 or IL-12, the presence ofTNF-a failed
to stimulate IFN-y production by LAK cells. Moreover, in
comparable experiments (not shown), TNF-a had a slight inhibitory effect on the production of IFN-y by LAK cells cultured in the presence of either IL-2 or IL-12 alone.
Contact with Listeria-infected hepatocytes stimulates IFNy production by LAK cells. Experiments were undertaken to
examine the mechanisms that effected the elevated production
ofIFN-y by LAK cells cocultured with Listeria-infected hepatocytes. Listeria organisms alone could not account for the
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Figure 5. IFN-y production by LAK cells cocultured with Listeriainfected hepatocytes is modulated by interleukin (IL)-2 and -12. LAK
cells (6 X 104/well) and hepatocytes infected 18 h previously with
104 Listeria organisms were cocultured for 24 h in presence or absence
of 30 U/mL IL-2, 50 pg/mL IL-12, and 100 UlmL tumor necrosis
factor (TNF)-a. Values are mean ± SE IFN-y concentrations in
supemates obtained from 8 comparable wells. Two additional experiments yielded similar results.
LAK Cells Lyse Listeria-Infected Hepatocytes
JID ] 996; ]74 (November)
Table 1. Hepatocytes stimulate IFN-y production by LAK cells.
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Table 3. Direct contactwith hepatocytes stimulates IFN-y production by LAK cells.
IFN-y (pg/mL)
Location of cells
Inoculum/well
2 X 104 hepatocytes
Without hepatocytes
Well
Control
103
104
105
2]
51
86
79
6 ± 5
6±3
4 ± 2
15 ± 9
HC + LAK
L-HC + LAK
HC
L-HC
± 12*
± 10*
± 11*
NOTE. Flat-bottom microtiter wells with or without hepatocytes were inoculated with Listeria organisms at concentration indicated. After 4 h of incubation, gentamicin was added and plates were incubated overnight. Next day, 6
X 104 LAK cells were added to each well; supernates were collected from 8
identical wells 24 h later. Values are mean ± SO TFN-y concentrations obtained
in single experiment representative of 5 similar experiments.
* Significantly greater than LAK cells cocultured with uninfected hepatocytes or cultured alone in wells inoculated with comparable concentration of
Listeria organisms; P < .05 (one-way analysis of variance).
cells, direct contact with Listeria-infected hepatocytes stimulated IFN-y production markedly. LAK cells cultured in contact with infected hepatocytes produced significantly more IFNy than did LAK cells cultured with uninfected hepatocytes
(table 3). IFN-y production by LAK cells was diminished,
however, when LAK cells and infected hepatocytes cocultured
in the same well were separated by a membrane insert.
Discussion
NK cells have been implicated in host defenses to a broad
range of microbial pathogens, including Legionella pneumophila [7, 11], Mycobacterium avium complex [30], Toxoplasma
gondii [31], Shigella fiexneri [9, 12], Salmonella typhimurium
[12], Leishmania major [32], Cryptococcus neoformans [33],
and L. monocytogenes [3-6, 10, 34]. In the case of Listeria
organisms, NK cells exhibit a transient 3- to 4-fold increase in
the livers of mice by day 1 after infection iv with a sublethal
Table 2. Soluble factors secreted by hepatocytes do not influence
the production of IFN-y by LAK cells.
IFN-y (pg/mL)
Conditioned medium
Control
Hepatocytes
Listeria-infected hepatocytes
1:10
1:4
31 ± 3
36 ± 4
33 ± 6
36 ± 2
27 ± 3
31 ± 2
Insert
IFN-y (pg/mL)
±5
NOTE. LAK cells were suspended in medium supplemented with 1:10 or
1:4 dilutions of medium conditioned by 48-h culture of uninfected hepatocytes
or hepatocytes infected with 104 Listeria organisms. Cells were seeded into
flat-bottom microtiter plates at 6 X 104 LAK cells/well and cells were cultured
for 24 h. Culture supemates were collected, and TFN-y production was quantified by ELISA. Values are mean ± SO IFN-y concentrations obtained from
quadruplicate wells in single experiment; 3 experiments provided similar results.
LAK
LAK
142
420
221
265
±
±
±
±
55
109*
88
68
NOTE. LAK cells and uninfected (HC) or Listeria-infected (L-HC) hepatocytes in 24-well tissue culture plates were incubated for 24 h together or
separated by inserts. Data are mean ± SO concentrations of IFN-y in supernates collected from 6 wells in single experiment representative of 3 experiments.
* Significantly greater than values obtained from cultures of LAK cells incubated with un infected hepatocytes or with infected hepatocytes separated by
inserts; P < .05 (one-way analysis of variance).
dose [2]. High levels of NK activity also occur among the
nonadherent population of peritoneal exudate cells obtained
from mice inoculated intraperitoneally with Listeria organisms
[35]. The critical role of NK cells in host defenses to Listeria
organisms is evidenced by the elevated replication of bacteria
and the increased mortality of mice depleted ofNK cells before
infection [5, 6].
The role of NK cells in host resistance to Listeria organisms
remains to be delineated fully. It has been suggested that a
principal function of NK cells may be the lysis of infected
cells that otherwise serve as a protected environment for the
growth of intracellular pathogens. Indeed, in the experiments
reported here, LAK cells exhibited an elevated capacity to lyse
Listeria-infected, relative to uninfected, hepatocytes. Lysis of
infected hepatocytes correlated with a reduction in viable intracellular Listeria organisms that remained at the end of the
incubation period. These results are consistent with previous
reports describing the elevated cytolytic activity of human NK/
LAK cells incubated with Mycobacterium-infected monocytes
[8], Legionella-infected macrophages [7], or Shigella-infected
HeLa cells [9, 12]. Our findings, however, are inconsistent with
a study demonstrating the failure of LAK cells to lyse either
uninfected or Listeria-infected peritoneal macrophages [36].
The conflicting results obtained in the latter case may reflect
the innate sensitivity of the infected target cells (i.e., hepatocytes vs. macrophages) to LAK cell lysis.
IL-2, IL-12, and TNF-a are essential for optimal host defenses to Listeria organisms. Mice administered recombinant
human IL-2 [37], recombinant human TNF-a [38], or recombinant murine IL-12 [39] near the time of infection exhibited
elevated resistance to L. monocytogenes. Conversely, listeriosis
was exacerbated in mice treated with either anti-mouse TNFa [5] or anti-mouse IL-12 [40]. It is likely that each of these
cytokines has a multiplicity of effects on the response to listerial infection and the immune cell populations involved. In
agreement with Gunji et al. [20], for example, we found that
1078
Gregory et al.
IL-2 was crucial for the survival of LAK cells. LAK cells
cultured overnight in the absence of IL-2 exhibited a marked
reduction in cell viability (data not shown). lL-12 and TNFa, added singly or in combination, failed to reverse completely
this loss in cell viability.
In accordance with the reports of others [25, 40], we also
found that IL-2 and IL-12 were essential for optimal production
of IFN-y by LAK cells. While these cytokines had no consistent effect on the cytolytic activity exhibited by LAK cells in
our coculture system (data not shown), IFN-y production by
LAK cells was elevated significantly by a combination of IL2 and IL-12. Undoubtedly, the increased production of IFN-y
observed in cultures that contained IL-2 and IL-12 was due in
part to the enhanced viability of LAK cells incubated in the
presence of exogenous cytokines (i.e., IL-2). Maximum IFNy production by LAK cells cocultured with Listeria-infected
hepatocytes was also dependent on contact between the two cell
populations and was not affected by soluble factors secreted by
hepatocytes.
IFN-y is critical for host defenses to Listeria monocytogenes.
This is demonstrated by the fact that the proliferation of Listeria
organisms is increased significantly in IFN-y-deficient mice
[41] or mice administered monoclonal anti-IFN-y at the time
of infection [19, 42]. Conversely, listerial replication is decreased markedly in animals administered IFN-y [19, 43,44].
IFN-y mRNA expression and/or IFN-y production assessed
in the bloodstreams and organs of Listeria-infected mice are
elevated significantly by day 1 after infection before the onset
of antigen-specific T cell-mediated immunity [19, 45]. This
latter observation has led others to suggest that NK cells may
be responsible for the production of IFN-y detected early during the course of listerial infection [6]. Indeed, mice depleted
of NK cells exhibited a 90% reduction in IFN-y-secreting
cells assessed on day 1 after infection and a marked increase
in the proliferation of Listeria organisms [6]. While the function of IFN-y in host defenses is not completely understood,
it has been shown to stimulate the antimicrobial activity of
macrophages [46], inhibit the replication of Listeria organisms
within hepatocytes [19], and regulate the emergence of the Th1
subset of CD4 T lymphocytes during an immune response to
infection [47, 48].
The parenchymal cells constitute the principal site of listerial
replication in the liver. More than 90% of Listeria organisms
recovered in the livers of mice at ~ 2 h after infection are
associated with the hepatocyte population [16]. Our results
suggest that the interaction between NK cells and infected
hepatocytes may be a significant factor in host resistance to
Listeria organisms expressed within the liver early during the
course of infection. In addition to lysing these infected cells,
infiltrating NK cells may produce elevated concentrations of
IFN-y that are essential for the expression of nonspecific host
resistance and the development a Thl cell-mediated immune
response to infection. The mechanism(s) that enable LAK cells
to distinguish between uninfected and Listeria-infected hepato-
JID 1996; 174 (November)
cytes remains to be determined. Lectin-like receptors, Ly-49
and NK-l.l, have been implicated in the non-MHC-restricted
lysis of target cells by mouse LAK cells [49]. While the ligands
recognized by these receptors have not been delineated, an
inverse correlation exists between the amount of sialic acid
expressed on the target cell surface and NK cell reactivity [50,
51]. Recently, Villanueva et al. [52] reported that the sialylation
of glycoproteins on the surface of two macrophage cell lines
was reduced significantly after listerial infection. Thus, the
decreased sialylation of surface glycoproteins may account for
the elevated reactivity of LAK cells cocultured with Listeriainfected hepatocytes.
Acknowledgment
We acknowledge the excellent technical assistance of Athanasia
J. Sagnimeni.
References
I. Kaufmann SHE. Immunity to intracellular bacteria. Annu Rev Immunol
1993; II: 129-63.
2. Goossens PL, Jouin H, Mi10n G. Dynamics of lymphocytes and inflammatory cells recruited in liver during murine listeriosis: a cytofluorimetric
study. J Immunol 1991; 147:3514-20.
3. Bancroft GJ. Bosma MJ. Bosma GC, Unanue ER. Regulation of macrophage Ia expression in mice with severe combined immunodeficiency:
induction ofIa expression by a T cell-independent mechanism. J Imrnunol 1986; 137:4-9.
4. Newborg MF, North RJ. On the mechanism of T cell-independent antiListeria resistance in nude mice. J Immuno1 1980; 124:571-6.
5. Bancroft GJ. Sheehan KCF, Schreiber RD, Unanue ER. Tumor necrosis
factor is involved in the T cell-independent pathway of macrophage
activation in scid mice. J Immunol1989; 143:127-30.
6. Dunn PL, North RJ. Early gamma interferon production by natural killer
cells is important in defense against murine listeriosis. Infect Immun
1991; 59:2892-900.
7. Blanchard DK, Stewart WE II, Klein TW, Friedman H, Djeu JY. Cytolytic
activity of human peripheral blood leukocytes against Legionella pneumophila- infected monocytes: characterization of the effector cell and
augmentation by interleukin 2. J Immuno1 1987; 139:551-6.
8. Katz P, Yeager H Jr, Whalen G, et al. Natural killer cell-mediated lysis
of Mycobacterium avium complex-infected monocytes. J Clin Immunol
1990; 10:71-7.
9. Klimpel GR, Niesel DW, Klimpe1 KD. Natural cytotoxic effector cell
activity against Shigellaflexneri-infected HeLa cells. J Immunol 1986;
136:1081-6.
10. Guo Y, Niesel DW, Ziegler HK, Klimpel GR. Listeria monocytogenes
activation of human peripheral blood lymphocytes: induction of nonmajor histocompatibility complex-restricted cytotoxic activity and cytokine production. Infect Immun 1992;60:1813-9.
11. Blanchard DK, Friedman H, Stewart WE II, Klein TW, Djeu JY. Role of
gamma interferon in induction of natural killer activity by Legionella
pneumophila in vitro and in an experimental murine infection model.
Infect Immun 1988;56:1187-93.
12. Klimpe1 GR, Niesel DW, Asuncion M, Klimpel KD. Natural kiIler cell
activation and interferon production by peripheral blood lymphocytes
after exposure to bacteria. Infect Immun 1988;56:1436-41.
13. Mackaness GB. CeIlular resistance to infection. J Exp Med 1962; 116:
381 -406.
JID 1996; 174 (November)
LAK Cells Lyse Listeria-Infected Hepatocytes
14. Benacerraf B, Sebestyen M, Schlossman S. A quantitative study of the
kinetics ofblood clearance ofp 32-labelled Escherichia coli and staphylococci by the reticuloendothelial system. J Exp Med 1959; 110:27 -48.
15. Mims CA. The pathogenesis of infectious disease. 3rd ed. London: Academic Press, 1987:105-9.
16. Gregory SH, Barczynski LK, Wing EJ. Effector function of hepatocytes
and Kupffer cells in the resolution of systemic bacterial infections . .I
Leukoc BioI 1992; 51:421-4.
17. Rosen H, Gordon S, North R.I. Exacerbation of murine listeriosis by a
monoclonal antibody specific for the type 3 complement receptor of
myelomonocytic cells. Absence of monocytes at infective foci allows
Listeria to multiply in nonphagocytic cells . .I Exp Med 1989; 170:2737.
18. Wing EJ, Waheed A, Shadduck RK. Changes in serum colony stimulating
factor (CSF) and monocytic progenitor cells during Listeria monocytogenes infection in mice. Infect Immun 1984;45:180-4.
19. Gregory SH, Wing EJ. IFN-gamma inhibits the replication of Listeria
monocytogenes in hepatocytes. J ImmunoI1993;151:1401-9.
20. Gunji Y, Vujanovic NL, Hiserodt JC, Herberman RE, Gorelik E. Generation and characterization of purified adherent Iymphokine-activated
killer cells in mice. J Immunol 1988; 142:1748-54.
21. Feutren G, Lacour B, Bach JF. Immune lysis of hepatocytes in culture:
accurate detection by aspartate aminotransferase release measurement.
.I Immunol Methods 1984;75:85-94.
22. Peck R. A one plate assay for macrophage bactericidal activity. J Immunol
Methods 1985; 82: 131-40.
23. Jiang X, Gregory SH, Wing EJ. Hepatocytes can serve as accessory cells
in the response of immune T lymphocytes to heat-killed Listeria monocytogenes. Infect Tmmun 1995; 63 :926- 33.
24. Wherry JC, Schreiber RD, Unanue ER. Regulation of gamma interferon
production by natural killer cells in scid mice: roles of tumor necrosis
factor and bacterial stimuli. Infect Immun 1991; 59: 1709-15.
25. Tripp CS, Wolf SF, Unanue ER. Interleukin 12 and tumor necrosis factor
Q' are costimulators of interferon gamma production by natural killer
cells in severe combined immunodeficiency mice with listeriosis, and
interleukin 10 is a physiologic antagonist. Proc Nat! Acad Sci USA
1993; 90:3725-9.
26. Gately MK, Warrier RR, Honasoge S, et al. Administration ofrecombinant
TL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo. Tnt Immunol 1994;6:157-67.
27. Chan SH, Perussia B, Gupta JW, et al. Induction of interferon-gamma
production by natural killer cell stimulatory factor: characterization of
the responding cells and synergy with other inducers. J Exp Med 1991;
173:869- 79.
28. Tsukui T, Kikuchi K, Mabuchi A, et al. Production of macrophage colonystimulating factor by adult murine parenchymal liver cells (hepatocytes).
J Leukoc Bioi 1992; 52:383-9.
29. Sakamoto T, Mabuchi A, Kuriya S, et al. Production of granulocytemacrophage colony-stimulating factor by adult murine parenchymal
liver cells (hepatocytes). Reg Immunol 1991;3:260-7.
30. Harshan KV, Gangadharam PRJ. Depletion of natural killer cell activity
leads to enhanced multiplication of Mycobacterium avium complex in
mice. Infect Immun 1991;59:2818-21.
31. Hunter CA, Abrams JS, Beaman MH, Remington JS. Cytokine mRNA in
the central nervous system of SCID mice infected with Toxoplasma
gondii: importance of T-cell- independent regulation of resistance to T.
gondii. Infect Immun 1993;61:4038-44.
32. Laskay T, RollinghoffT, Solbach W. Natural killer cells participate in the
early defense against Leishmania major infection in mice. Eur J Immunol 1993;23:2237 -41.
1079
33. Salkowski CA, Balish E. Role of natural killer cells in resistance to systemic cryptococcosis. J Leukoc Bioi 1991;50:151-9.
34. Emmerling P, Finger H, Hof H. Cell-mediated resistance to infection with
Listeria monocytogenes in nude mice. Infect Immun 1977; 15:382-95.
35. Holmberg LA, Ault KA. Characterization of Listeria mono[ytogenesinduced murine natural killer cells. Immunol Res 1986;5:50-60.
36. Zychlinsky A, Karim M, Nonacs R, Young DE. A homogeneous population of lymphokine-activated killer (LAK) cells is incapable of killing
virus-, bacteria-, or parasite-infected macrophages. Cell Immunoll990;
125:261-7.
37. Haak-Frendscho M, Young KM, Czuprynski C.I. Treatment of mice with
human recombinant interleukin-Z augments resistance to the facultative
intracellular pathogen Listeria monocytogenes. Infect Immun 1989; 57:
3014-21.
38. Kato K, Nakane A, Minagawa T, et al. Human tumor necrosis factor
increases the resistance against Listeria infection in mice. Med MicrobioI Immunol 1989; 178:337-46.
39. Wagner RD, Steinberg H, Brown JF, Czuprynski CJ. Recombinant interleukin-12 enhances resistance to Listeria monocytogenes infection.
Microb Pathog 1994; 17:175-86.
40. Tripp CS, Gately MK, Hakimi J, Ling P, Unanue ER. Neutralization of
IL-12 decreases resistance to Listeria in SCID and C.B-17 mice. J
Immunol 1994; 152: 1883-7.
41. Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA .
Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science 1993;259: 1739-42.
42. Buchmeier NA, Schreiber RD. Requirement of endogenous interferongamma production for resolution of Listeria monocytogenes infection.
Proc Natl Acad Sci USA 1985; 82:7404-8.
43. Kiderlen AF, Kaufmann SHE, Lohmann-Matthes ML. Protection of mice
against the intracellular bacterium Listeria monocytogenes by recombinant immune interferon. Eur J lmmunol 1984; 14:964- 7.
44. Langermans JAM, van der Hulst MEB, Nibbering PH, van der Meide PH,
van Furth R. Intravenous injection of interferon-gamma inhibits the
proliferation of Listeria monocytogenes in the liver but not the spleen
and peritoneal cavity. Immunology 1992;77:354-61.
45. Ehlers S, Mielke MEA, Blankenstein T, Hahn H. Kinetic analysis of
cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity. J Immunoll992; 149:3016-22.
46. Peck R. Gamma interferon induces monocyte killing of Listeria monocytogenes by an oxygen-dependent pathway; alpha- or beta-interferons by
oxygen-independent pathways. J Leukoc Biol 1989;46:434-40.
47. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM.
Development ofTH I CD4+ T cells through IL-12 produced by Listeriainduced macrophages. Science 1993;260:547-9.
48. Hsieh CS, Macatonia SE, O'Garra A, Murphy KM. Pathogen-induced Th l
phenotype development in CD4 + alpha beta-TCR transgenic T cells is
macrophage dependent. Int Immunol 1993; 5:371-82.
49. Yokoyama WM, Seaman WE. The Ly-49 and NKR-PI gene families
encoding lectin-like receptors on natural killer cells: the NK gene complex. Annu Rev Immunol 1993; 11:613-35.
50. Van Rinsum J, Srnets LA, Van Rooy H, Van den Eijnden DH. Specific
inhibition of human natural killer cell-mediated cytotoxicity by sialic
acid and sialo-oligosaccharides. Int J Cancer 1986;38:915-22.
51. Nabi ZF, Zucker-Franklin D, Baten A. Phorbol ester-induced loss ofmembrane sialic acid: implications for tumor cytolysis by natural killer cells.
J Leukoc Bioi 1989;45:183-9.
52. Villanueva MS, Beckers CJM, Pamer EG. Infection with Listeria monocytogenes impairs sialic acid addition to host cell glycoproteins. J Exp
Med 1994; 180:2137-45.