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Department of Immunology and Microbiology, Wayne State University, Detroit MI 48201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-10 production in vitro,
whereas levels of the 
-chemokine monocyte chemoattractant protein-1
were significantly elevated. Flow cytometry studies suggest that the NK
cells may play an important role in regulating both normal and
autoimmune T cell responses by exerting a direct effect on activated,
autoantigen-specific T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells of the innate immune system (i.e., NK cells), for example, can
affect the differentiation of naive T cells (reviewed in Ref.
1). IFN- is secreted by NK cells, and IFN-, in turn,
drives CD4+ T cells to differentiate into Th1
cells. NK cells can also influence B cell function and Ab secretion
(2). Conversely, production of IL-13 by NK cells can
influence macrophage function and Th2 differentiation (3).
Clearly, NK cells play a crucial role in both innate and adaptive
immune responses.
Recent data have suggested that NK cells may play an important role in
experimental autoimmune encephalomyelitis
(EAE),3 a rodent model
for the human demyelinating disease multiple sclerosis (4, 5). While these studies showed that NK cells might play an
important role in recovery from EAE, they did not investigate direct NK
cell-T cell interactions. We chose to study NK cell regulation of T
cell responses in the DA rat strain, which is highly susceptible to
various autoimmune diseases, including EAE and collagen-induced
arthritis (6, 7, 8). However, DA rats do not develop
spontaneous autoimmune disease, suggesting that autoreactivity is
strictly regulated. The goal of the present study was to investigate
the ability of NK cells to regulate encephalitogenic T cell responses
in vitro. We demonstrate that rat bone marrow-derived NK cells (BMNK)
inhibit proliferation, and IFN- and IL-10 production by
encephalitogenic Th1 cells at low effector to target ratios. BMNK
inhibition of T cell responses was associated with a dramatic increase
in levels of the -chemokine monocyte chemoattractant protein
(MCP)-1. These results have important implications with respect to how
NK cells might regulate adaptive immune responses, particularly
autoimmune diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DA rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN), maintained in our American Association for the Accreditation of Laboratory Animal Care accredited facility, and used at 10–20 wk of age. Naive rats were used as a source of bone marrow (BM) cells. For studies of myelin basic protein (MBP)-primed T cells, rats were immunized s.c. with 25 µg of guinea pig MBP or MBP peptide 63–81 emulsified in IFA supplemented with 200 µg Mycobacterium butyricum (Difco, Detroit, MI) as described previously (9).
Preparation of cell suspensions
BM was obtained from naive rats by flushing the cavities of the
femurs and tibias with cold RPMI 1640 supplemented with 5% FCS (Life
Technologies, Grand Island, NY) using an 18-gauge needle. A single cell
suspension was prepared by gentle aspiration of the marrow with needles
of increasingly smaller gauge. BM cells were treated with
Tris-NH4Cl (pH 7.2) to remove erythrocytes, then
depleted of macrophages by adherence on plastic
(250-cm2 tissue culture flasks; Falcon, Lincoln
Park, NJ) for 
2–3 h at 37°C. Nonadherent BM cells were poured
through nylon filters and collected in 50-ml conical centrifuge tubes,
washed with complete medium, then layered onto Percoll (Pharmacia,
Uppsala, Sweden) density gradients to enrich for NK cells.
Responder T cells were obtained by preparing single cell suspensions of spleen or draining inguinal lymph nodes, depleted of macrophages by a 1-h adherence on plastic, followed by enrichment on T cell columns (Cytovax Biotechnologies, Edmonton, Alberta, Canada). T cells were cultured with irradiated syngeneic thymocytes (2000 rads) as APC at a 1:1 ratio (9).
Percoll fractionation
Nonadherent BM cells were layered onto Percoll density gradients consisting of 75, 65, 55, and 45% Percoll, and HBSS in 15-ml conical centrifuge tubes (Falcon; Becton Dickinson, Franklin Lakes, NJ) (10). Cells were centrifuged at 2000 rpm for 15 min. Cells in the 45 and 55% Percoll fraction were pooled as Fraction 1, and the 65% Percoll fraction was designated Fraction 2. The Fraction 1 and 2 cells were washed twice with cold HBSS and were resuspended in RPMI 1640 medium containing 5% FBS. Effector cells (BM cells) and target cells (T cells) were combined in different proportions (E:T) for use in proliferation assays.
Proliferation assay
T cell proliferation assays were performed as previously described (9). Briefly, lymph node cells were depleted of adherent cells, enriched for T cells on Cytovax columns, and plated in microtiter wells. Each well received 5 x 105 T cells and 5 x 105 syngeneic, irradiated thymocytes (2000 rads) as APC. Cells were cultured in RPMI 1640 containing 5% FCS. The cells were stimulated either with 5 µM MBP, which was found optimal in previous studies, or with Con A at a concentration of 2.5 µg/ml. The cultures were incubated for either 72 h (Con A), or 96 h (MBP), and pulsed with 0.5 µCi of [3H]thymidine for the last 18 h. The plates were harvested using a Tomtec Harvester 96 and counted in a 1450 Microbeta Plus liquid scintillation counter (Wallac, Gaithersburg, MD). Results are presented as cpm ± SD.
Cytokine analysis
We focused on one proinflammatory Th1 cytokine that is also
secreted by NK cells (IFN-), two Th2 cytokines (IL-4 and IL-10), and
one chemokine (MCP-1). The latter was selected because it has been
reported that in vivo neutralization of MCP-1 reverses the protective
effect of oral tolerization on EAE (11). Culture
supernatants from MBP or Con A-stimulated T cells were evaluated for
IFN-, IL-4, IL-10, and MCP-1, using rat-specific commercial ELISA
kits (Biosource International, Camarillo, CA; Life Technologies)
according to the manufacturers’ instructions. Recombinant rat MCP-1,
and anti-rat/mouse cross-reactive MCP-1 mAb (clone 2H5) were from
Biosource International and PharMingen (San Diego, CA),
respectively.
Flow cytometry
Cells were prepared for flow cytometry by first incubating
1 x 106 Percoll-isolated BM cells with PBS
containing 0.02% sodium azide, 1% BSA (PBS-A-BSA) and 1% normal rat
serum for 30 min as a blocking step, as previously described
(12). The cells were subsequently washed in PBS-A-BSA and
incubated with the appropriate chromogen-labeled Ab for 20 min. Abs
used were: FITC- or PE-labeled R73 (anti-rat 
TCR),
PE-labeled G4.18 (anti-CD3), FITC-labeled anti-rat NKR-P1 (NK
cells), FITC-labeled anti-rat B220 (B cells), and FITC anti-rat
DNP as an isotype control (all purchased from PharMingen). Cells were
analyzed in a FACScan flow cytometer using PC LYSIS software (Becton
Dickinson, Mountain View, CA).
Cytotoxicity assay
Cytotoxicity assays were performed using the flow cytometric method of Slezak and Horan (13). Briefly, 2 x 106 column-enriched T cells were combined with 1 x 106 syngeneic thymocytes, and the cells were labeled with the lipophilic green dye PKH26 (Sigma, St. Louis, MO). We did not irradiate the thymocyte APCs because irradiated thymocytes might not have survived for the 72-h culture period, and would therefore have taken up the propidium iodide (PI) used to delineate the nonviable cell population. Uniform labeling of cells was confirmed by visualization using a fluorescent microscope. Labeled target cells were then incubated with 2.5 x 105 nonlabeled Percoll-enriched BMNK cells (1:8 E:T) and 2.5 µg/ml Con A in 24-well plates for a total of 72 h. Cells were then collected, stained with PI, and analyzed by flow cytometry. Controls consisted of Con A- or MBP 63–81-stimulated T cells alone (no BM), as well as a BM fraction lacking NK cells. Cytotoxicity was determined by calculating the percentage of cells positive for both PI (red) and PKH (green).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the regulatory effects of BMNK cells on T cells, it was important to utilize an appropriate source of NK cells. BM has traditionally been a source of NK cells. Percoll density gradients were therefore utilized to separate and enrich for these cells, which are characterized as large granular cells. Plastic nonadherent BM cells from naive rats were applied to gradients consisting of 75, 65, 55, and 45% Percoll, overlayered with HBSS. After centrifugation, cells from the 45–55% interfaces (designated as Fraction 1) as well as the 65% interface (Fraction 2) were harvested, washed, and evaluated for suppressive activity.
In two initial experiments, we evaluated Fraction 1 and Fraction 2 BM
cells obtained from Lewis (LEW) and DA rats. As shown in Fig. 1
, the Fraction 1 BM cells suppressed Con
A-induced proliferative responses, whereas the Fraction 2 BM cells were
not suppressive. Since DA Fraction 1 cells appeared to exert a more
profound inhibitory effect on DA T cells relative to that achieved with
LEW Fraction 1 cells on the LEW T cell proliferative response, we
decided to focus on the DA strain for this investigation.
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and
3). As shown in Fig. 2, two populations
of cells can be discerned for each BM fraction (Fraction 1 and Fraction
2), and these are gated R1-R4. R1 and R3 represent small cells, and
include a majority of B cells (B220+ cells, Fig. 3B), and a very small percentage of
TCR+ T cells (TCR+ cells,
Fig. 3C). R2 and R4 represent large granular cells. In four
independent experiments, an average of 27 ± 0.5% of the cells in
Fraction 1, region R2 express NKR-P1 (the NK cell marker), whereas only
5% of the R4 cells in Fraction 2 are NKR-P1+
(Fig. 3D). Thus, the NK cells are enriched in BM Fraction 1,
and more specifically, in region R2 (Fig. 3D). Employing
two-color flow cytometric analyses with FITC-anti NKR-P1 and
PE-anti- TCR, 1% of the NKR-P1+
cells in Fraction 1 expressed TCR (Fig. 4). Similar results were obtained when
the BM Fraction 1 cells were stained with FITC-anti NKR-P1 and
PE-anti-CD3 mAb (data not shown). Therefore, it is unlikely that
NK-T cells play a major role in suppression of the T cell proliferative
responses (Fig. 1
, and data presented below). Moreover, we failed to
detect IL-4 in the Fraction 1-containing supernatants, and NK-T cells
reportedly produce IL-4 upon stimulation (14).
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To determine how NK cells might regulate T cell responses, BMNK
cells were added at various ratios to T cells, and proliferative
responses were evaluated. In the first group of experiments, splenic T
cells were stimulated in vitro with Con A and syngeneic APCs in the
presence of either Fraction 1 or Fraction 2 cells. BM cells were added
at E:T ranging from 1:4 to 1:128. As shown in Fig. 5, only Fraction 1 cells
(BMNK+) were capable of inhibiting T cell
proliferation to Con A. This inhibition was observed even at an E:T of
1:32; however, there was no significant inhibition of T cell
proliferation by Fraction 2 cells, which do not contain BMNK cells
(Fig. 5).
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, only BMNK+
(Fraction 1) cells were capable of inhibiting T cell proliferation to
MBP, and significant inhibition was observed at an E:T of 1:64. No
inhibition was observed with Fraction 2 cells. Similar results were
obtained in seven independent proliferation experiments (four with Con
A, and three with MBP).
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To correlate BMNK inhibition of Con A- and MBP-induced T cell
proliferation with cytokine secretion, culture supernatants were
analyzed for IFN-, IL-4, and IL-10 production by ELISA. IL-4 was not
detected in any of the supernatants (data not shown). IFN- and IL-10
production, Th1 and Th2 cytokines, respectively, was significantly
reduced in response to either Con A or MBP in the presence of
either unfractionated BM cells or Fraction 1 cells (Table I). However, no decrease in IFN- or
IL-10 production was observed in cultures containing Fraction 2 cells
(Table I). Note that IFN- is expressed in ng/ml, and IL-10 and MCP-1
(see below) are expressed in pg/ml (Table I).
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The decrease in IFN- production in cultures with BMNK cells was
not surprising since proliferation was also inhibited. To extend our
analysis of cytokine production, we also evaluated production of the
-chemokine MCP-1 in the culture supernatants from Con A- and
MBP-activated T cells. This chemokine has been implicated in the
regulation of EAE (11). We found that MCP-1 levels were
dramatically elevated in cultures that contained the Fraction 1 BMNK
cells. The results of one experiment are presented in Fig. 7. Table I summarizes the results of
three experiments, and includes data from Fig. 7. A fourth
experiment gave similar results (data not shown). The supernatants that
contained high levels of MCP-1 (152–411 pg/ml) were from T cell
cultures that exhibited suppressed proliferative responses (compare
Table I with Figs. 5 and 6). MCP-1 levels were low in wells not
containing BMNK cells. MCP-1 production by BMNK cells in the absence of
stimulation was also low. (Fig. 7 and Table I). Furthermore,
anti-MCP-1 mAb 2H5 competitively inhibited in ELISA experiments,
supporting the conclusion that MCP-1 was actually present in the
cultures that contained the activated T cells and BMNK cells
(Fig. 8).
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, MCP-1 dramatically suppressed Ag- and
mitogen-stimulated proliferative responses.
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Because BMNK cells were potent inhibitors of T cell proliferation, it was important to determine whether the NK cells had a direct effect on the T cells. This was investigated using a flow cytometric assay in which T cells were labeled with the lipophilic green dye PKH26, then stimulated with Con A in the presence or absence of nonlabeled BMNK cells (E:T, 1:8) (13). After 72 h of culture, cells were harvested and stained with PI. Dead target T cells are double-positive for both PKH26 (green) and PI (red), while live T cell targets stain only with PKH26. Dead BM effector cells take up only PI.
Fig. 10 depicts the results of one
experiment. PKH26-labeled cells showed a uniform mean fluorescence
intensity of 103 at the start of culture;
virtually all of the cells were labeled. This is presented in Fig. 10A, in which the PKH26-labeled histogram (checkered
pattern, M1) has been superimposed on the histogram of the isotype
control (crosshatched pattern). After incubation, the control Con
A-stimulated T cells (Fig. 10B), and stimulated T cells
cultured with Fraction 2 (BMNK-) cells (Fig. 10D), revealed two major stained regions. The cells in the
region gated R1 (lower right quadrant) are PKH26+
PI- viable target T cells, and the cells in
region R2 (upper right quadrant) are double-stained
PKH26+ PI+ (dead) T cells.
The PKH26- PI+ population
includes dead BM cells (upper left quadrant). In cultures containing
Con A-activated T cells and Fraction 1 (BMNK+)
cells, there was a significant reduction in viable cells in gated
region R1 and a corresponding increase in PKH26+
PI+ cells in region R2 (Fig. 10C).
This suggests that BMNK cells inhibit T cell proliferation, leading to
cell death (Fig. 10C). There was significantly less
cytotoxicity when T cells were cultured in the presence of BMNK
Fraction 1 or Fraction 2 cells without Con A (Fig. 10, E and
F).
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show the PI staining pattern of
T cells from DA rats cultured with Con A and APCs alone, or with
Fraction 2 or Fraction 1 BM cells, respectively (E:T, 1:8). After
72 h, 43 and 52% of the cells cultured without BM or with BM
Fraction 2 cells were viable (did not incorporate PI). In contrast,
only 18% of the T cells cocultured with Fraction 1 BM cells were
viable. As shown in the lower panels of Fig. 11, there were two major
cell populations in each culture. When we gated on live target T cells
(Fig. 11, R1), the pattern of staining of control Con A-stimulated T
cells was similar to that seen when Fraction 2 cells were cultured with
T cells (left and center panels, gated
area R1). In contrast, few viable target T cells remain in gated area
R1 of cultures containing Fraction 1 (BMNK+)
cells (right panel). Furthermore, PI-positive cells (gated
area R2) increased from 15–19% in the control and Fraction
2-containing cultures (Fig. 11, left and center
panels, respectively, gated area R2) to 37% in the T cell
culture incubated with Fraction 1 cells (right panel, gated
area R2). This suggests that suppressed T cell proliferation is a
reflection, in part, of killing of the target T cells by the NK cells
in Fraction 1. Regions R1 and R2 account for 61–65% of the stained
cells, and most of the remaining cells are distributed in the region
between R1 and R2 (Fig. 11). Similar results were obtained in a third
cytotoxicity experiment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we investigated NK cell regulation of T cells. We used
a more traditional source of NK cells (BM rather than splenic
NK cells). BMNK cells were potent inhibitors of T cell proliferation to
both Con A and MBP, a CNS autoantigen. Inhibition was evident at low
E:T ratios (E:T < 1:16) and was accompanied by a corresponding
decrease in levels of IFN- and IL-10. Interestingly, we found high
levels of MCP-1 in cultures containing BMNK cells. We consider it
likely that one source of the MCP-1 is the BMNK cells because maximal
levels were present in the cultures containing Fraction 1 BMNK
cells (Table I). However, we cannot exclude the possibility that T
cells or APCs also secrete MCP-1, or that the BMNK cells indirectly
induce T cells or APCs to produce this chemokine. Recombinant MCP-1
appears to inhibit T cell proliferative responses (Fig. 9), which is
consistent with a previous report that this chemokine can have
protective effects on the development of EAE in mice orally tolerized
with encephalitogenic peptide (11). It has also been
reported that MCP-1 acts as a lymphocyte chemoattractant for memory T
cells (19). Therefore, one might speculate that the MCP-1
is produced by the NK cells, which, in turn, attract the activated T
cells.
Moreover, as shown in Figs. 10 and 11, inhibition of T cell
proliferation by BMNK cells is associated with a significant increase
in the number of PI-positive (i.e., dead) T cells when Con A-activated
T cells are cultured with BMNK cells for 72 h. This suggests
that one mechanism by which the BMNK cells inhibit proliferation may
involve cytotoxic activity directed against the autoreactive T cells.
The possibility that perforin- and/or Fas/FasL-mediated
cytotoxicity is involved is currently under investigation. However, the
mean fluorescence intensity of the cells that did not stain with PI
was not decreased, as would be expected if the cells had
proliferated (Figs. 10 and 11). This might suggest that
cytokines also play a role in BMNK-mediated inhibition of T
cell responses by suppressing proliferation. We previously found that
regulatory T cells produce TGF-, which inhibits autoreactive T cell
responses in EAE (20). This issue is also under
investigation.
There is evidence that NK cells play an important role in autoimmune disease. In the report by Matsumoto et al. (5), an increase in peripheral blood NK cells in Lewis rats was observed during both clinical EAE and recovery, but NK cells were present in the spinal cord only during clinical disease. Despite the increase in peripheral blood NK cells, there was no corresponding change in the percentage of splenic NK cells. Thus, it is possible that BMNK cells are recruited to the CNS via the bloodstream, participate in recovery from EAE, and exit from the CNS after the disease-inducing effector T cells have been lysed. Furthermore, depletion of NK cells in both rats and mice resulted in disease exacerbation and relapse (4, 5). These results suggest that NK cells play a role in recovery from EAE, and perhaps multiple sclerosis (21), by inhibiting the induction of new T cell responses within the CNS, e.g., via epitope spreading (22, 23). Mice depleted of NK cells developed a more severe form of colitis, which is correlated with the expansion and increase in the number of donor T cells that mediate colitis (16). Studies are in progress to elucidate the mechanism by which BMNK cells inhibit T cell responses and to ascertain the role chemokines play in the activation and function of NK cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert H. Swanborg, Department of Immunology and Microbiology, Wayne State University School of Medicine, 540 East Canfield, Room 7263, Detroit, MI 48201. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; BM, bone marrow; BMNK, BM-derived NK cell; MCP, monocyte chemoattractant protein; PI, propidium iodide.
Received for publication January 9, 1999. Accepted for publication May 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Immunol. 162:51.
T cells in acute autoimmune encephalomyelitis. Eur. J. Immunol. 28:1681.[Medline]
8.2+ T cells and encephalitogenicity in the Lewis rat. J. Immunol. 162:829.. J. Immunol. 146:1163.[Abstract]
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