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IMMUNOPHARMACOLOGY A N D IMMUNOTOXICOLOGY, 13(3), 237-250 (1991)
EFFECTS OF BIOLOGICAL RESPONSE MODIFIERS ON LUNG NATURAL KILLER ACTIVITY
Wallace Lauzon and Irma Lemaire. Laboratory of Immunopharmacology, Department of Pharmacology Faculty of Medicine, University of Ottawa, Ottawa, Ontario Canada K1H 8M5
ABSTRACT
Natural killer (NK) activity plays an important role in host defense against tumors, especially once augmented by immunomodulators. It is likely that the modulation of NK cells is a reflection of the environment in which they reside. The current study was undertaken to characterize the response profile of lung interstitial lymphocyte natural killer (LLNK) activity to various biological response modifiers (BRM) in vitro after short term incubation (18h). The presented data show that treatment of lung lymphocytes with human recombinant interleukin 2 (rlL-2), purified rat interferon alpha/beta (IFN-a/B), or murine recombinant tumor necrosis factor alpha (rTNF-a) resulted in a dose-dependent increase in LLNK activity. The maximum stimulation was similar for rlL-2 and IFN-a/B, although a much higher concentration of IFN-a/O was required to reach this level of stimulation. The maximum response to rTNF-a treatment was about half that seen with rll-2 or IFN-a/B and it, too, required a high concentration. By contrast, rat recombinant interferon gamma (rlFN-y) or murine recombinant interleukin 1 (rlL-1) failed to alter LLNK activity significantly when used alone. Furthermore, doses of IFN-a/B and rTNF-a! that had little enhancing effect were able to synergize with a suboptimal dose of rlL-2, whereas rlL-1 and rlFN-y failed to do so. These data demonstrate the response of lung NK activity to BRM treatment, which is important for the responsible and effective use of BRM. However the spectrum of lung NK cell response to BRM is smaller than that previously reported for NK cells from other anatomic compartments. 237 Copyright 0 1991 by Marcel Dekker, Inc.
238
LAUZON AND LEMAIRE
INTRODUCTION Natural killer (NK)’ cells appear to play an important early role in Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/07/15 For personal use only.
immune surveillance against tumors and control of metastasis (1-4). Thus, understanding the regulation of NK activity may provide a powerful tool for cancer therapy. Much effort has been spent on studying the modulation of peripheral blood and spleen NK activity. However, significant levels of NK activtty have been reported for lymphocytes of the lung, liver, lymph nodes and the gut (5-9), although, the local regulation in these organs has largely been neglected. There is growing evidence that the modulation of NK activity may be compartmentalized, in that, target organs can show enhanced NK activity in the absence of a systemic increase in the peripheral blood NK activity (10,ll). Thus, it is essential that the modulation of NK activtty in the target organ be understood, in a rational approach to the design of more effective treatment methods. The lung is an organ of particular significance with respect to cancer because of its exposure to airborne carcinogens and the potential for metastasis via the vasculature.
Therefore, lung NK activity and its
modulation, in particular, merit further study. We have previously shown that rat lung contains NK cells which display high intrinsic activity (9). In addition, we have demonstrated that lung NK activity differs from spleen and peripheral blood NK activity in its responsiveness to certain biological response modifiers (BRM)’ (9). The purpose of the present study was to characterize further, the in vitro potential for augmentation of natural killer activity from lung interstitial lymphocytes. Accordingly, we have treated lung lymphocytes (LL)’ with various BRM, which have been shown to have a potentiating effect on peripheral blood or spleen NK activity. These BRM include interleukin-1 (IL-l)’, interleukin-2 (lL-2)’, interferon alpha/beta (IFNa / @ ) ’interferon-gamma , (IFN-y)’ and tumor necrosis factor alpha (TNF-a)’
(9,ll-15).
NATURAL KILLER ACTIVITY
239
MATERIALS AND METHODS
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Animals Male Wistar rats weighing 250 to 3009 were purchased from Charles River Canada, Inc. (St-Constant, Quebec). These animals were derived from a pathogen-free colony, shipped behind filter barriers and housed in isolated temperature controlled quarters in an animal isolator unit (Upjohn Scientific). Cell Preparation Isolated rat lung cells were prepared as previously described (16). Briefly, the lungs were perfused with Krebs buffer containing 2.5% bovine serum albumin and 5 mM glucose (37OC, pH 7.4) via the pulmonary artery to remove all blood elements, immediately after sacrifice. The lung and trachea were removed from the animal and perfused for 10 min with Ca", Mg' free Krebs buffer containing 1 mg/mL protease type VII (Sigma) and +
1 mM EDTA (150 mL, 37'C, pH 7.4). The lung tissue was minced and
dispersed via repeated passage through a 60 cc syringe and filtered through Nytex (110 um).
Mononuclear cells (MNC)' were isolated by
fractionation on a Ficoll-Hypaque (Pharmacia) gradient (specific density, 1.077). MNC were depleted of macrophage by passage through a nylon wool fiber column (Wako Chemicals, Dallas, Texas). The resulting cell preparation was found to contain approximately, 2% macrophage by light microscopy and Wright-Giemsa staining. Biological Response Modifiers Human recombinant interleukin 2 (rlL-2) with specific activity of 5 x 10' U/mg of protein was kindly provided by Cetus Corp., Emeryville, CA.
or purchased from Gibco/BRL, Burlington, Ont. (specific activity of 3.1 x lo6 U/mg of protein). Both preparations were used in the experiments reported here, with equal effectiveness. Murine recombinant interleukin 1 (rlL-1) was purchased from Genzyme, Boston, MA, with a specific activity of 1 x 10' U/mg of protein. Rat recombinant interferon gamma (rlFN-y) with specific activity of 4 x 10' U/mg of protein was purchased from Amgen, Thousand
2 40
LAUZON AND LEMAIRE
Oaks, CA. Purified rat interferon alpha/beta (IFN-a/O) with specific activity of 3.5 x
lo6 U/mg of protein was purchased from Lee Biomolecular, San
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Diego, CA. Murine recombinant tumor necrosis factor alpha (rTNF-a) with a specific activity of 4 x lo7 U/mg of protein was purchased from Genzyme, Boston, MA. lncubatlon with BRM Nylon wool non-adherent lung lymphocytes (NNLL)' were placed into O5 cells/well) in 96-well microtiter plates at 2-fold dilutions (2.0-0.25~1
triplicate in 0.1 mL of culture media. The desired concentration of BRM(s) was then added to the wells in 0.1 mL of media. The plates were incubated for 18h at 37%. The plates were centrifuged and 0.1 mL of the supernatant was discarded from each well. Cytotoxlcfty Assay Cytolytic activity was tested in a typical 5'Cr release assay. Yac-1 target cells were labelled for 90 min with "Cr (New England Nuclear, 0.1 mCi).
Labelled target cells were distributed into microtiter wells at a
concentration of
lo4 cells/well in 0.1 mL of media to give effector:
target
ratios of 2O:l to 2.51. Other wells contained BRM(s) with target cells and media with target cells. The plates were incubated for 4h and 0.1 mL of the supernatant was removed and 51Crcontent was measured in a gammacounter (Raytest). Cytotoxicity was expressed for each ratio as the percent specific 5'Cr release using the following formula: % specific ''Cr release = ER - SR x 100 TR - SR
where ER represents cpm in the presence of lymphocytes; SR,cpm due to spontaneous release and TR, cpm due to the total incorporated 5'Cr in
lo4target cells.
The lytic activity of the lymphocytes was expressed as lytic
units derived by a computer program generously provided by Dr. H. Pross (Queen's University) (17). One lytic unit was defined as the number of effector cells required to cause 20% lysis of lostarget cells. The data were
241
NATURAL KILLER ACTIVITY
expressed as percent stimulation, which was calculated as follows: S.I.
=
U, from experimental x 100
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LU,
from control
Statistical Analysis Results were expressed as mean values
2
SEM.
Statistical
significance was determined using unpaired Student’s T test (p
-
0
z n
E
m
.r(
c , b p
m -
"
0
25 50 75 100 125 250 500 1000 Concentration: IFN-a/B (U/mLl
FIG. 2 Effects of rll-2 on IFN-a/R enhancement of lung NK activity. Lung lymphocytes were incubated with various concentrations of IFN-a/B alone ( A ) or in the presence of 10 U/mL rlL-2 ( A ) for 18h. The data were calculated and expressed in the same manner as figure 1. The values represent the mean 5 SEM of at least 5 experiments.
243
NATURAL KILLER ACTIVITY
o without IL-2
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0
with 10 U/mL IL-2
Concentrat ion:
TNF-a (U/mL)
FIG. 3 Effects of rlL-2 on rTNF-a enhancement of lung NK activity. Lung lymphocytes were incubated with indicated concentrations of rTNF-a alone ( o ) or with 10 U/mL rlL-2 ( 0 ) for 18h. The data were calculated and expressed in the same manner as figure 1. The values represent the mean 2 SEM of at least 3 experiments.
a/O and 10 U/mL rlL-2. Higher concentrations of IFN-a/O in conjunction
with 10 U/mL rlL-2 were ineffective in further boosting the LLNK activity. A similar pattern was found for rTNF-a, as shown in Fig. 3. In this case, the synergism is even more pronounced at concentrations of rTNF-a that were ineffective in enhancing LLNK activity in the absence of rlL-2. However, the presence of rlL-2 became less a factor at concentrations where rTNF-a was shown to be an effective modulator of LLNK activity. Neither rlL-1 (Fig. 4) or rlFN-y (Fig. 5) significantly affected the enhancement of lung NK activity by low concentrations of rlL-2. DISCUSSION
Several lymphocyte and monocyte derived cytokines have been shown to modulate peripheral blood or spleen natural killer activity (9,12,13,18-24). We have previously shown that, in the rat, the in vitro
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2 44
LAUZON AND LEMAIRE
FIG. 4 Effects of rlL-1 on lung NK activity with and without rlL-2. Lung lymphocytes were treated with the indicated concentrations of rlL-1 ( 0 )or with rlL-1 and 10 U/mL rlL-2 ( ). The data were calculated and expressed as in figure 1. The values represent the mean 2 SEM of at least 3 experiments.
+
50 45 -
without I L - 2 + with 10 U/mL IL-2 0
40 35 30 25 20 15 10 -
10 U l ________ l L IL-2 5'-----
0
ax
1
FIG. 5 Effects of rlFN-y on lung NK activity alone and in concert with rlL-2. Lung lymphocytes were treated with the indicated concentrations of rlFN-y ( 0 ) or with rlFN-y and 10 U/mL rlL-2 ( ). The data were calculated and expressed as in figure 1. The values represent the mean 2 SEM of at least 4 experiments.
+
245
NATURAL KILLER A C T I V I T Y
response to BRM varied both with the BRM used and the effector cell origin (9). In the current study, we determined the response of lung NK cells to Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/07/15 For personal use only.
short-term pre-incubation with rlL-1, rlL-2, IFN-a/B, rlFN-y and rTNF-a alone, as well as, in combination with a suboptimal dose of rlL-2. The most efficient enhancer of lung NK activity was rlL-2.
Relatively low
concentrations of rlL-2 produced significant enhancement and the maximum was quickly reached.
In contrast, IFN-a/B required a much higher
concentration for maximum effect, although the maximum stimulation was similar to that found for rlL-2. When the lung NK cells were treated with a combination of IFN-a/B of various concentrations and 10 U/mL of rlL-2, there was a synergistic effect on NK activity. This effect was only observed at low concentrations of IFN-a/B, concentrations that have little enhancing activity on their own. The stimulation reaches a maximum at 50 U/mL of IFN-a/B and, interestingly, this maximum is similar to the maximum stimulation observed for IFN-a/B or rlL-2 alone. This level of stimulation may represent a physical limit to the stimulation of our lung NK population. However, it is unlikely that it is a limitation imposed by the assay system because we have previously shown greater stimulation with spleen lymphocytes (9). Perhaps lung NK cells, which possess superior basal activity to spleen or PBL natural killer cells (9),are limited in their ability to respond to external stimulation by some organ-associated process. Alternatively, this limit may be a property of natural killer activity in general and the lower limit observed for lung NK activity is a reflection of prior stimulation either in situ or as a consequence of isolation. However, this last possibility seems unlikely because we have shown that protease treatment does not enhance NK activity in the lung other than to enrich the NK population (9). Additionally, we have found that treatment of spleen cells with protease does not stimulate NK activity (data not shown). Interestingly, we have found that lung NK activity was insensitive to treatment with rlFN-y and that rlFN-y did not augment the effect of rlL-2 at
246
LAUZON A N D LEMAIRE
any concentration. Brunda et.al. (19) have reported little activity of rlFN-y in augmenting murine spleen NK activity and variable effect when combined
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with 11-2. However, Platsoucas et.al. (18) reported that human rlFN-y is a potent enhancer of PBL NK activity.
This may represent a species
difference where murine and rat NK cells are not responsive to rlFN-y while human NK cells are. However, we have found that rat PBL is responsive to rlFN-y treatment (9),indicating that the discrepencies, in the literature, may reflect organ-associated differences.
It is unclear whether this
difference is caused by a lack of functional IFN-y receptors which are known to differ from IFN-a/B receptors (25), or a more fundamental difference in NK cells from different tissues. Recombinant TNF-a stimulated lung NK activity in a dose-dependent manner similar to IFN-a/B but the maximum stimulation by rTNF-a was only about half. Talmadge et.al. (24) has reported an In vivo but no In vltro effect of TNF-a on murine spleen NK activity, which is in direct contrast with this finding. However, Ostensen et al (23) reported that TNF-a alone has a small enhancing effect on human PBL NK activity and augmented low dose 11-2 boosting. This is entirely consistent with our results. When the effect of rTNF-a was determined in the presence of 10 U/mL rlL-2, it was found that a synergistic relationship existed for low doses of rTNF-a and again a maximum stimulation was quickly reached. In this case, the maximum stimulation was lower than the maximum found for IFN-a/B. When an optimal dose of rlL-2 was used, rTNF-a was seen to have no significant effect (unpublished data). The reason for these differences can not easily be attributed to any single factor given the range of animals and tissues used.
However, the degree of similarity between the finding
presented here and those reported by Ostensen et.al. (23) tends to indicate that TFN-a has an immunomodulatory function for NK activity. We found no consistent effect of rlL-1 on lung NK activity. This is different from the finding of Dinarello et al (20) that IL-1 alone is a potent
247
NATURAL KILLER ACTIVITY
activator of PBL NK activity in humans. Dempsey et al (21) using purified IL-1 on human PBL NK, reported an effect only in the presence of T cell
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growth factor (TCGF) or IFN. However, we have found that NK cells do not respond to rlL-1 even in combination with rlL-2. It is possible that the discrepency is a result of some contamination of the native proteins used in the Dempsey report. This seems unlikely because Dinarello et al. (20) have found an enhancement after treatment with recombinant 11-1 and 11-2. We feel that it is more likely a reflection of the difference in effector cell origin. In conclusion, we have characterized the responsiveness of lung interstitial lymphocyte NK activity to a battery of BRM that have been reported to enhance NK activity in other compartments. We have found some discrepencies in response of lung NK activity when compared to the published reports and we suggest that these differences result from organassociated alterations in the NK cell. These data provide insights into the regulation of natural immunity in the lung that should be considered in the development of rational therapeutic approaches. ACKNOWLEDGEMENTS
This work was supported by a grant from the National Cancer Institute of Canada. W. Lauzon is an Ontario Graduate Scholar. Dr. 1. Lemaire is a scholar of MRC (Medical Research Council of Canada). We thank Mrs. Carole blonde for excellent secretarial assistance. FOOTNOTES ‘Abbreviations:
NK, natural killer; BRM, biological response modifiers;
LL, lung lymphocytes;
LGL, large granular lymphocytes;
MNC,
mononuclear cells; NNLL, nylon wool nonadherent lung lymphocytes; rlL1, recombinant interleukin-1; rlL-2, recombinant interleukin-2; rTNF-a, recombinant tumor necrosis factor-a; IFN-a/O, interferon-a/B; rlFN-y, recombinant interferon-y; 51Cr,chromium-51; LU, lytic unit; SI, stimulatory index.
248
LAUZON AND LEMAIRE
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REFERENCES 1.
Woodruff MFA: The cytolytic and regulatory role of natural killer cells in experimental neoplasia. Blochlmlca Blophyslca Acta =:43, 1986.
2.
Barlouari TI Leonhardt J, Wiltrout RH, Herberman RB and Reynolds CW: Direct evidence for the role of LGL in the inhibition of experimental tumor metastases. J. Immunol. 134:2783, 1985.
3.
Nolibe D, Aumaitre E and Thang MN: In vivo augmentation of rat lung natural killer cell activity and inhibition of experimental metastases by double stranded polynucleotides. Cancer Res. 4774, 1985.
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4.
Nolibe D and Poupon MF: Enhancement of pulmonary metastases induced by decreased lung natural killer activity. J. Natl. Cancer Inst. 77:99, 1986.
5.
Reynolds CW, Timonen T and Herberman RB: Natural killer (NK) cell activity in the Rat. I. Isolation and characterization of the effector cells. J. Immunol. 127:282, 1981.
6.
Wiltrout RH, Mathieson BJ, Talmadge JE, Reynolds CW, Zhang S-R, Herberman RB and Ortaldo JR: Augmentation of organ-associated natural killer activity by biological response modifiers: Isolation and characterizationof large granular lymphocytesfrom the liver. J. Exp. M e d . m : 1431, 1984.
7.
Nuntirooj K, Bozelka BE, deShazo RD and Salvaggio JE: Analytical and functional assessment of lymphoid cells within murine lung. Exp. Lung Res. B: 13, 1987.
8.
ltoh H, Abo TI Sugawara S, Kanno A and Kumagai K: Age-related variation in the proportion and activity of murine liver natural killer cells and their cytotoxicity against regenerating hepatocytes. J. Immunol. 141:315, 1988.
9.
Lauzon W, Yang H and Lemaire I: Comparative analysis of natural killer function in lung, spleen and peripheral blood lymphocytes: evidence of differential characteristics. Reglonal Immunol., in press, 1990.
10.
Wiltrout RA, Herberman RB, Zhang SR, Chirigos MA, Ortaldo JR, Green KM and Talmadge JE: Role of organ-associated NK cells in
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N A T U R A L KILLER A C T I V I T Y
decreased formation of experimental metastases in lung and liver. J. Immunol. 4267, 1985.
m:
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11.
Stein-Streilein J, Bennett MI Mann D and Kumar V: Natural killer cells in mouse lung: surface phenotype, target preference and response to local influenza virus infection. J. Immunol. 2699, 1983.
m:
12.
Sayers TJ, Mason AT and Ortaldo JR: Regulation of human natural killer activity by IFN-y: lack of a role in interleukin-2 mediated augmentation. J. Immunol. 2176, 1986.
m:
13.
Voth R, Rossol S,Gallati HI Pracht I, Lauberstein HP, Hess GI Muller WE, Schroder HC, Jochum C, Meyer zum and Buschenfeld KH: In vlvo and In vltro induction of natural killer cells by cloned human tumor necrosis factor. Cancer Immunol, lmmunotherapy2J: 128, 1988.
14.
Endo Y, Matsushima K and Oppenhiem JJ: Mechanism of in vitro antitumor effects of interleukin-1 (IL-1). 1mmunobiol.m: 316, 1986.
15.
Welsh RM: Natural killer cells and interferon. CRC Critical Rev. Immunol. 5: 55, 1986.
16.
Day R, Lemaire S, Nadeau D, Keith I and Lemaire I: Changes in autacoid and neuropeptide contents of lung cells in asbestosinduced pulmonary fibrosis. Am. Rev. Respls. Dis. =: 908, 1987.
17.
Pross HF, Baines MG, Rubin PI Shragge P and Patterson MS: Spontaneoushuman lymphocyte-mediatedcytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity. J. Clin. Immunol. 1:51, 1981.
18.
Platsoucas CD, Fernandes GI Good RA and Gupta A: Augmentation of natural killer cytotoxicity by alpha or gamma natural and recombinant interferons and interferon inducers: effect of monocytes. Int. Archs Allergy appl. Immun. 1, 1986.
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19.
Brunda MJ, Tarnowski D and DavatelisV: Interaction of recombinant interferons with recombinant interleukin-2: differential effects on natural killer cell activity and interleukin-2-activatedkiller cells. Int. J. Cancer X : 787, 1986.
20.
Dinarello CAI Conti P and Mier JW: Effects of human interleukin-1 on natural killer cell activity: is fever a host defense mechanism for tumor killing? Yale J. Blol. Med. 3: 97, 1986.
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21.
Dempsey RA, Dinarello CA, Mier JW, Rosenwasser W, Allegretta MI Brown TE and Parkinson DR.: The differential effects of human leukocytic pyrogen/lymphocyte-activatingfactor, T cell growth factor and interferon on human natural killer activity. J. Immunol. 129: 2504, 1982.
22.
Mannel DN, Kist A, Ho AD, Rath U, Reichardt PI Wiedenmann 6 , Schlick E and Kirchner H: Tumor necrosis factor production and natural killer cell activity in peripheral blood during treatment with recombinant tumor necrosis factor. Br. J. Cancer 6p:585, 1989.
23.
Ostensen ME, Thiele DL and Lipsky PE: Tumor necrosis factoralpha enhances cytolytic activity of human natural killer cells. J. Immunol. B: 4185, 1987.
24.
Talmadge JE, Tribble HR, Pennington RW, Phillips H and Wiltrout RH: lmmunomodulatory and immunotherapeutic properties of recombinant gamma-interferon and recombinant tumor necrosis factor in mice. Cancer Res. 47:2563, 1987.
25.
Branca AA and Baglioni C: Evidence that type I and II interferons have different receptors. Nature 768, 1981.
a:
IMMUNOPHARMACOLOGY A N D IMMUNOTOXICOLOGY, 13(3), 237-250 (1991)
EFFECTS OF BIOLOGICAL RESPONSE MODIFIERS ON LUNG NATURAL KILLER ACTIVITY
Wallace Lauzon and Irma Lemaire. Laboratory of Immunopharmacology, Department of Pharmacology Faculty of Medicine, University of Ottawa, Ottawa, Ontario Canada K1H 8M5
ABSTRACT
Natural killer (NK) activity plays an important role in host defense against tumors, especially once augmented by immunomodulators. It is likely that the modulation of NK cells is a reflection of the environment in which they reside. The current study was undertaken to characterize the response profile of lung interstitial lymphocyte natural killer (LLNK) activity to various biological response modifiers (BRM) in vitro after short term incubation (18h). The presented data show that treatment of lung lymphocytes with human recombinant interleukin 2 (rlL-2), purified rat interferon alpha/beta (IFN-a/B), or murine recombinant tumor necrosis factor alpha (rTNF-a) resulted in a dose-dependent increase in LLNK activity. The maximum stimulation was similar for rlL-2 and IFN-a/B, although a much higher concentration of IFN-a/O was required to reach this level of stimulation. The maximum response to rTNF-a treatment was about half that seen with rll-2 or IFN-a/B and it, too, required a high concentration. By contrast, rat recombinant interferon gamma (rlFN-y) or murine recombinant interleukin 1 (rlL-1) failed to alter LLNK activity significantly when used alone. Furthermore, doses of IFN-a/B and rTNF-a! that had little enhancing effect were able to synergize with a suboptimal dose of rlL-2, whereas rlL-1 and rlFN-y failed to do so. These data demonstrate the response of lung NK activity to BRM treatment, which is important for the responsible and effective use of BRM. However the spectrum of lung NK cell response to BRM is smaller than that previously reported for NK cells from other anatomic compartments. 237 Copyright 0 1991 by Marcel Dekker, Inc.
238
LAUZON AND LEMAIRE
INTRODUCTION Natural killer (NK)’ cells appear to play an important early role in Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/07/15 For personal use only.
immune surveillance against tumors and control of metastasis (1-4). Thus, understanding the regulation of NK activity may provide a powerful tool for cancer therapy. Much effort has been spent on studying the modulation of peripheral blood and spleen NK activity. However, significant levels of NK activtty have been reported for lymphocytes of the lung, liver, lymph nodes and the gut (5-9), although, the local regulation in these organs has largely been neglected. There is growing evidence that the modulation of NK activity may be compartmentalized, in that, target organs can show enhanced NK activity in the absence of a systemic increase in the peripheral blood NK activity (10,ll). Thus, it is essential that the modulation of NK activtty in the target organ be understood, in a rational approach to the design of more effective treatment methods. The lung is an organ of particular significance with respect to cancer because of its exposure to airborne carcinogens and the potential for metastasis via the vasculature.
Therefore, lung NK activity and its
modulation, in particular, merit further study. We have previously shown that rat lung contains NK cells which display high intrinsic activity (9). In addition, we have demonstrated that lung NK activity differs from spleen and peripheral blood NK activity in its responsiveness to certain biological response modifiers (BRM)’ (9). The purpose of the present study was to characterize further, the in vitro potential for augmentation of natural killer activity from lung interstitial lymphocytes. Accordingly, we have treated lung lymphocytes (LL)’ with various BRM, which have been shown to have a potentiating effect on peripheral blood or spleen NK activity. These BRM include interleukin-1 (IL-l)’, interleukin-2 (lL-2)’, interferon alpha/beta (IFNa / @ ) ’interferon-gamma , (IFN-y)’ and tumor necrosis factor alpha (TNF-a)’
(9,ll-15).
NATURAL KILLER ACTIVITY
239
MATERIALS AND METHODS
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/07/15 For personal use only.
Animals Male Wistar rats weighing 250 to 3009 were purchased from Charles River Canada, Inc. (St-Constant, Quebec). These animals were derived from a pathogen-free colony, shipped behind filter barriers and housed in isolated temperature controlled quarters in an animal isolator unit (Upjohn Scientific). Cell Preparation Isolated rat lung cells were prepared as previously described (16). Briefly, the lungs were perfused with Krebs buffer containing 2.5% bovine serum albumin and 5 mM glucose (37OC, pH 7.4) via the pulmonary artery to remove all blood elements, immediately after sacrifice. The lung and trachea were removed from the animal and perfused for 10 min with Ca", Mg' free Krebs buffer containing 1 mg/mL protease type VII (Sigma) and +
1 mM EDTA (150 mL, 37'C, pH 7.4). The lung tissue was minced and
dispersed via repeated passage through a 60 cc syringe and filtered through Nytex (110 um).
Mononuclear cells (MNC)' were isolated by
fractionation on a Ficoll-Hypaque (Pharmacia) gradient (specific density, 1.077). MNC were depleted of macrophage by passage through a nylon wool fiber column (Wako Chemicals, Dallas, Texas). The resulting cell preparation was found to contain approximately, 2% macrophage by light microscopy and Wright-Giemsa staining. Biological Response Modifiers Human recombinant interleukin 2 (rlL-2) with specific activity of 5 x 10' U/mg of protein was kindly provided by Cetus Corp., Emeryville, CA.
or purchased from Gibco/BRL, Burlington, Ont. (specific activity of 3.1 x lo6 U/mg of protein). Both preparations were used in the experiments reported here, with equal effectiveness. Murine recombinant interleukin 1 (rlL-1) was purchased from Genzyme, Boston, MA, with a specific activity of 1 x 10' U/mg of protein. Rat recombinant interferon gamma (rlFN-y) with specific activity of 4 x 10' U/mg of protein was purchased from Amgen, Thousand
2 40
LAUZON AND LEMAIRE
Oaks, CA. Purified rat interferon alpha/beta (IFN-a/O) with specific activity of 3.5 x
lo6 U/mg of protein was purchased from Lee Biomolecular, San
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Diego, CA. Murine recombinant tumor necrosis factor alpha (rTNF-a) with a specific activity of 4 x lo7 U/mg of protein was purchased from Genzyme, Boston, MA. lncubatlon with BRM Nylon wool non-adherent lung lymphocytes (NNLL)' were placed into O5 cells/well) in 96-well microtiter plates at 2-fold dilutions (2.0-0.25~1
triplicate in 0.1 mL of culture media. The desired concentration of BRM(s) was then added to the wells in 0.1 mL of media. The plates were incubated for 18h at 37%. The plates were centrifuged and 0.1 mL of the supernatant was discarded from each well. Cytotoxlcfty Assay Cytolytic activity was tested in a typical 5'Cr release assay. Yac-1 target cells were labelled for 90 min with "Cr (New England Nuclear, 0.1 mCi).
Labelled target cells were distributed into microtiter wells at a
concentration of
lo4 cells/well in 0.1 mL of media to give effector:
target
ratios of 2O:l to 2.51. Other wells contained BRM(s) with target cells and media with target cells. The plates were incubated for 4h and 0.1 mL of the supernatant was removed and 51Crcontent was measured in a gammacounter (Raytest). Cytotoxicity was expressed for each ratio as the percent specific 5'Cr release using the following formula: % specific ''Cr release = ER - SR x 100 TR - SR
where ER represents cpm in the presence of lymphocytes; SR,cpm due to spontaneous release and TR, cpm due to the total incorporated 5'Cr in
lo4target cells.
The lytic activity of the lymphocytes was expressed as lytic
units derived by a computer program generously provided by Dr. H. Pross (Queen's University) (17). One lytic unit was defined as the number of effector cells required to cause 20% lysis of lostarget cells. The data were
241
NATURAL KILLER ACTIVITY
expressed as percent stimulation, which was calculated as follows: S.I.
=
U, from experimental x 100
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LU,
from control
Statistical Analysis Results were expressed as mean values
2
SEM.
Statistical
significance was determined using unpaired Student’s T test (p
-
0
z n
E
m
.r(
c , b p
m -
"
0
25 50 75 100 125 250 500 1000 Concentration: IFN-a/B (U/mLl
FIG. 2 Effects of rll-2 on IFN-a/R enhancement of lung NK activity. Lung lymphocytes were incubated with various concentrations of IFN-a/B alone ( A ) or in the presence of 10 U/mL rlL-2 ( A ) for 18h. The data were calculated and expressed in the same manner as figure 1. The values represent the mean 5 SEM of at least 5 experiments.
243
NATURAL KILLER ACTIVITY
o without IL-2
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0
with 10 U/mL IL-2
Concentrat ion:
TNF-a (U/mL)
FIG. 3 Effects of rlL-2 on rTNF-a enhancement of lung NK activity. Lung lymphocytes were incubated with indicated concentrations of rTNF-a alone ( o ) or with 10 U/mL rlL-2 ( 0 ) for 18h. The data were calculated and expressed in the same manner as figure 1. The values represent the mean 2 SEM of at least 3 experiments.
a/O and 10 U/mL rlL-2. Higher concentrations of IFN-a/O in conjunction
with 10 U/mL rlL-2 were ineffective in further boosting the LLNK activity. A similar pattern was found for rTNF-a, as shown in Fig. 3. In this case, the synergism is even more pronounced at concentrations of rTNF-a that were ineffective in enhancing LLNK activity in the absence of rlL-2. However, the presence of rlL-2 became less a factor at concentrations where rTNF-a was shown to be an effective modulator of LLNK activity. Neither rlL-1 (Fig. 4) or rlFN-y (Fig. 5) significantly affected the enhancement of lung NK activity by low concentrations of rlL-2. DISCUSSION
Several lymphocyte and monocyte derived cytokines have been shown to modulate peripheral blood or spleen natural killer activity (9,12,13,18-24). We have previously shown that, in the rat, the in vitro
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2 44
LAUZON AND LEMAIRE
FIG. 4 Effects of rlL-1 on lung NK activity with and without rlL-2. Lung lymphocytes were treated with the indicated concentrations of rlL-1 ( 0 )or with rlL-1 and 10 U/mL rlL-2 ( ). The data were calculated and expressed as in figure 1. The values represent the mean 2 SEM of at least 3 experiments.
+
50 45 -
without I L - 2 + with 10 U/mL IL-2 0
40 35 30 25 20 15 10 -
10 U l ________ l L IL-2 5'-----
0
ax
1
FIG. 5 Effects of rlFN-y on lung NK activity alone and in concert with rlL-2. Lung lymphocytes were treated with the indicated concentrations of rlFN-y ( 0 ) or with rlFN-y and 10 U/mL rlL-2 ( ). The data were calculated and expressed as in figure 1. The values represent the mean 2 SEM of at least 4 experiments.
+
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NATURAL KILLER A C T I V I T Y
response to BRM varied both with the BRM used and the effector cell origin (9). In the current study, we determined the response of lung NK cells to Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/07/15 For personal use only.
short-term pre-incubation with rlL-1, rlL-2, IFN-a/B, rlFN-y and rTNF-a alone, as well as, in combination with a suboptimal dose of rlL-2. The most efficient enhancer of lung NK activity was rlL-2.
Relatively low
concentrations of rlL-2 produced significant enhancement and the maximum was quickly reached.
In contrast, IFN-a/B required a much higher
concentration for maximum effect, although the maximum stimulation was similar to that found for rlL-2. When the lung NK cells were treated with a combination of IFN-a/B of various concentrations and 10 U/mL of rlL-2, there was a synergistic effect on NK activity. This effect was only observed at low concentrations of IFN-a/B, concentrations that have little enhancing activity on their own. The stimulation reaches a maximum at 50 U/mL of IFN-a/B and, interestingly, this maximum is similar to the maximum stimulation observed for IFN-a/B or rlL-2 alone. This level of stimulation may represent a physical limit to the stimulation of our lung NK population. However, it is unlikely that it is a limitation imposed by the assay system because we have previously shown greater stimulation with spleen lymphocytes (9). Perhaps lung NK cells, which possess superior basal activity to spleen or PBL natural killer cells (9),are limited in their ability to respond to external stimulation by some organ-associated process. Alternatively, this limit may be a property of natural killer activity in general and the lower limit observed for lung NK activity is a reflection of prior stimulation either in situ or as a consequence of isolation. However, this last possibility seems unlikely because we have shown that protease treatment does not enhance NK activity in the lung other than to enrich the NK population (9). Additionally, we have found that treatment of spleen cells with protease does not stimulate NK activity (data not shown). Interestingly, we have found that lung NK activity was insensitive to treatment with rlFN-y and that rlFN-y did not augment the effect of rlL-2 at
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LAUZON A N D LEMAIRE
any concentration. Brunda et.al. (19) have reported little activity of rlFN-y in augmenting murine spleen NK activity and variable effect when combined
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with 11-2. However, Platsoucas et.al. (18) reported that human rlFN-y is a potent enhancer of PBL NK activity.
This may represent a species
difference where murine and rat NK cells are not responsive to rlFN-y while human NK cells are. However, we have found that rat PBL is responsive to rlFN-y treatment (9),indicating that the discrepencies, in the literature, may reflect organ-associated differences.
It is unclear whether this
difference is caused by a lack of functional IFN-y receptors which are known to differ from IFN-a/B receptors (25), or a more fundamental difference in NK cells from different tissues. Recombinant TNF-a stimulated lung NK activity in a dose-dependent manner similar to IFN-a/B but the maximum stimulation by rTNF-a was only about half. Talmadge et.al. (24) has reported an In vivo but no In vltro effect of TNF-a on murine spleen NK activity, which is in direct contrast with this finding. However, Ostensen et al (23) reported that TNF-a alone has a small enhancing effect on human PBL NK activity and augmented low dose 11-2 boosting. This is entirely consistent with our results. When the effect of rTNF-a was determined in the presence of 10 U/mL rlL-2, it was found that a synergistic relationship existed for low doses of rTNF-a and again a maximum stimulation was quickly reached. In this case, the maximum stimulation was lower than the maximum found for IFN-a/B. When an optimal dose of rlL-2 was used, rTNF-a was seen to have no significant effect (unpublished data). The reason for these differences can not easily be attributed to any single factor given the range of animals and tissues used.
However, the degree of similarity between the finding
presented here and those reported by Ostensen et.al. (23) tends to indicate that TFN-a has an immunomodulatory function for NK activity. We found no consistent effect of rlL-1 on lung NK activity. This is different from the finding of Dinarello et al (20) that IL-1 alone is a potent
247
NATURAL KILLER ACTIVITY
activator of PBL NK activity in humans. Dempsey et al (21) using purified IL-1 on human PBL NK, reported an effect only in the presence of T cell
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growth factor (TCGF) or IFN. However, we have found that NK cells do not respond to rlL-1 even in combination with rlL-2. It is possible that the discrepency is a result of some contamination of the native proteins used in the Dempsey report. This seems unlikely because Dinarello et al. (20) have found an enhancement after treatment with recombinant 11-1 and 11-2. We feel that it is more likely a reflection of the difference in effector cell origin. In conclusion, we have characterized the responsiveness of lung interstitial lymphocyte NK activity to a battery of BRM that have been reported to enhance NK activity in other compartments. We have found some discrepencies in response of lung NK activity when compared to the published reports and we suggest that these differences result from organassociated alterations in the NK cell. These data provide insights into the regulation of natural immunity in the lung that should be considered in the development of rational therapeutic approaches. ACKNOWLEDGEMENTS
This work was supported by a grant from the National Cancer Institute of Canada. W. Lauzon is an Ontario Graduate Scholar. Dr. 1. Lemaire is a scholar of MRC (Medical Research Council of Canada). We thank Mrs. Carole blonde for excellent secretarial assistance. FOOTNOTES ‘Abbreviations:
NK, natural killer; BRM, biological response modifiers;
LL, lung lymphocytes;
LGL, large granular lymphocytes;
MNC,
mononuclear cells; NNLL, nylon wool nonadherent lung lymphocytes; rlL1, recombinant interleukin-1; rlL-2, recombinant interleukin-2; rTNF-a, recombinant tumor necrosis factor-a; IFN-a/O, interferon-a/B; rlFN-y, recombinant interferon-y; 51Cr,chromium-51; LU, lytic unit; SI, stimulatory index.
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LAUZON AND LEMAIRE
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