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Porcine alveolar macrophages discriminate between self and nonself in lectin-mediated cellular cytotoxicity
CELLULAR
IMMUNOLOGY
Porcine
68, 368-376 (1982)
Alveolar Macrophages Discriminate between Nonself in Lectin-Mediated Cellular Cytotoxicity’ ROBERTROTHLEINANDYOONBERM Sloan-Kettering
Self and
KIM
Institute for Cancer Research, Rye, New York 10580
Received January
7, 1982; accepted February
12, 1982
The pulmonary alveolar macrophages (AM) from specific pathogen-free Minnesota miniature swine were tested for their ability to discriminate between xenogeneic and autologous red blood cell (RBC) targets in lectin-mediated cellular cytotoxicity measured by an 18-hr “Cr-release assay. It was found that the porcine AM would lyse xenogeneic chicken and sheep RBC but not autologous pig RBC in the presence of PHA. It was also shown that pig RBC was susceptible to lysis by the AM in a PHA-mediated assay if the effector AM were from rabbits. It is postulated that a nonspecific signal in conjunction with foreign recognition is able to trigger the AM lytic mechanism.
INTRODUCTION It is well established that macrophages can be activated to be cytotoxic to bacterial, red blood cell (RBC),’ and tumor targets in vitro (l-5). Although the mechanism of activation has begun to be elucidated, the means by which they recognize targets is still poorly understood. It has been shown that by in vivo treatment with Bacillus Calmette-G&in (6, 7) or by in vitro treatment with mitogens (8-1 l), T-cell products and/or lipopolysaccharides ( 12- 14), or target-specific antibody ( 15- 18), macrophages can be made cytotoxic to bacterial, tumor, and RBC targets. Also, it was reported that blood monocytes will lyse RBC targets by culturing the peripheral blood mononuclear cells for 1 week prior to the cytotoxicity assay (19). It is known that activated or elicited macrophages can specifically lyse tumor but not normal targets (20, 2 1). Also, Cabilly and Gallily found that mouse peritoneal exudate macrophages can distinguish between allogeneic and xenogeneic fibroblast targets in a cytotoxic assay (22), and Muchmore et al. reported that macrophages can distinguish between RBC of different species with the specificity being conferred by a nonimmunoglobulin serum factor (23). ’ This investigation was supported in part by USPHS Grants CA-25384 and CA-08748 from the National Cancer Institute and HD-12097 from the National Institute of Child Health and Human Development. Portions of this work will be submitted by R. Rothlein in partial fulfillment of the rcquirements for the Doctor of Philosophy degree at Cornell University Graduate School of Medicine Sciences. * Abbreviations used: AM, pulmonary alveolar macrophages; RBC, red blood cells; CRBC, chicken RBC, PRBC, pig RBC, SRBC, sheep RBC; RRBC, rabbit RBC; PHA, phytohemagglutinin; Con A, concanavalin A, PWM, pokeweed mitogen; BSS, balanced salt solution. 368 OOOS-8749/82/060368-09$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BY ALVEOLAR
MACROPHAGES
369
Although most of these cytotoxic studies have been performed using macrophages from the blood or peritoneal cavity, some studies have shown that pulmonary alveolar macrophages (AM) also have cytotoxic capabilities (9, 24). In this paper we describe an assay system in which porcine AM are capable of discriminating between xenogeneic and autologous RBC in the presence of phytohemagglutinin (PHA) in a chromium-release cytotoxicity assay. MATERIALS
AND METHODS
Animals, Young adult animals from a herd of specific pathogen-free Minnesota miniature swine maintained in a facility with filtered air (Hepa filter) and fed an autoclaved diet with chlorinated water were used in these experiments (25). Also, young adult miniature New Zealand white rabbits were used. Alveolar macrophages. AM were collected as previously described (26). Briefly, 300 ml of phosphate-buffered saline (PBS), pH 7.2, was injected intratracheally into swine sacrificed by CO* anesthesia. The PBS was then withdrawn and the lavage cells were washed three times with balanced salt solution (BSS). Rabbit lungs were lavaged in a similar fashion using three separate lavages of 20 ml each, Preparation of pig anti-chicken red blood cell serum. One milliliter of 20% washed chicken RBC (CRBC) was injected intraperitoneally into a swine on three occasions at l-week intervals. One week following the final injection the serum was collected, heat-inactivated, aliquoted, and stored frozen until used. Labeling of target cells. RBC were labeled with “Cr by incubating lo7 RBC in 100 ~1 of BSS plus 100 &i of Na”CrO, (sp act 250-500 mCi/mmol, New England Nuclear, No. NEZ-030s) in 100 ~1for 2 hr at 37°C with constant shaking. The labeled RBC were then washed three times with BSS and resuspended to lo6 cells/ml with RPM1 1640 with 10% heat-inactivated fetal bovine serum and 1% Pen-Strep and 1% glutamine (complete medium). Cytotoxicity assay. Fifty microliters of complete medium containing appropriately diluted pig anti-CRBC serum, mitogen, or nothing was added to appropriate wells of a flat-bottomed, 96-well microtest plate (Falcon No. 3001, Oxnard, Calif.) AM, IO5 in 50 ~1 of complete medium were then added to each well and the plates were incubated at 37°C. After 1 hr, lo5 labeled targets in 100 ~1 of complete medium were added to each well, and the plates were incubated at 37°C. The assay was terminated by collecting 100 ~1 of supernatants at appropriate times and measuring their radioactivity in a well-type gamma counter as described elsewhere (27). Cytotoxic activity was calculated as
% specific release (lysis) =
cpm experimental - cpm SR x 100, cpm MR - cpm SR
where spontaneous release (SR) was defined as the counts per minute (cpm) released from targets incubated with complete medium alone, and maximum release (MR) was the cpm after lysis of the RBC targets with 1% Triton X-100. Values are expressed as means of triplicate determinations. Throughout the experiments MR were higher than 97% of total isotope uptake and the mean SR for chicken red blood cells, porcine red blood cells, sheep red blood cells, and rabbit red blood cells were 3.3 f 2.0, 3.5 -+ 0.1, 4.2 f 0.5, and 8.2 -t 2.4%, respectively.
370
ROTHLEIN
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RESULTS Comparison of Various Concentrations of Mitogens in Mediating CRBC Lysis by the Porcine AM Table 1 shows the results of a representative experiment giving the percentage of CRBC lysed by porcine AM in the presence of concanavalin A (Con A), pokeweed mitogen (PWM), or PHA at various concentrations in an 18-hr ‘iCr-release assay at an effector-to-target ratio of 1:1. It can be seen that there was little or no lysing of the CRBC targets when either Con A or PWM was used as a mediator. However, PHA was capable of mediating lysis by the AM at a concentration of 0.25 pg/ml, the lowest mitogen concentration tested. The optimum concentration for PHA-mediated lysis was between 2.5 and 5.0 pg/ml and there was a gradual decrease in lysis at higher PHA concentrations. Comparison of PHA and Antibody-Mediating
CRBC Lysis
Table 2 shows the results that compared the optimum PHA-mediated CRBC lysis with the optimum CRBC lysis mediated by pig anti-CRBC serum in the 18hr chromium-release assay with porcine AM as effector cells. It can be seen that the percentages of specific 51Cr release were 64.1 + 8.9% when PHA was the mediator, 52.8 f 5.4% when the assay was mediated by pig anti-CRBC serum, and 1.5 f 1.4% when no mediator was present in the assay. Note that in the 12 pigs tested, the percentage of lysis in the antibody-mediated system never exceeded that of the PHA-mediated system. Also, there is a strong correlation between the two mediators in that when lysis mediated by PHA was high, so was the lysis mediated by the antiserum. Ability of the Porcine AM to Discriminate between Xenogeneic and Autologous RBC Table 3 shows the results of PHA-mediated %r-release assaysusing xenogeneic CRBC, SRBC, and autologous PRBC targets. It can be seen that the AM showed very little spontaneous cytotoxicity toward any of the RBC targets. However, when PHA was added to the assay there were an average of greater than 70% lysis of TABLE 1 Effect of Various Concentrations of Mitogens on Mediating the Cytotoxicity of Porcine Alveolar Macroohaees to S’Cr-Labeled Chicken Red Blood Cell Targets Percentage cytotoxicity’ Mitogen dose (ridmU
Con A
PHA
PWM
0 0.25 2.5 5.0 25 50
0.0 -0.3 -0.2 -0.6 -0.1 1.0
0.0 53.0 73.0 75.0 61.8 55.6
0.0 1.0 -0.5 -0.8 -1.1 -1.0
a Effector-to-target cell ratio of 1:l assayed after an 1%hr incubation at 37°C.
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MACROPHAGES
TABLE 2 Comparison of Antibody- and PHA-Mediated Cytotoxicity of Porcine Alveolar Macrophages to “Cr-Labeled Chicken Red Blood Cell Targets Percentage cytotoxicity” Animal No.
None
PHAb
Pig anti-CRBC’
SPF 3137-3 SPF 4036-4 SPF 4056-5 SPF 4058-4 SPF 3117-7 SPF 4059-l SPF 4067-4 SPF 3152-8 SPF 4029-4 SPF 4060-7 SPF 3089-5 SPF 4047-6
0.2 0.3 3.6 2.0 4.5 0.8 0.7 1.9 -0.2 2.3 I.5 0.5
63.7 55.7 70.5 74.8 52.9 67.3 71.1 54.0 59.0 76.0 52.7 71.5
55.1 48.3 63.1 55.5 49.6 53.2 59.3 46.3 49.4 53.6 44.6 55.3
64.1 f 8.9
52.8 2 5.4
Mean + SD
1.5 z!z1.4
a Effector-to-target cell ratio of 1:1 assayed after an 18-hr incubation at 37°C. ’ PHA was at a concentration of 2.5 pg/ml in the assay. ‘Pig anti-CRBC serum was diluted 1:400 in the assay.
the CRBC targets, 18% lysis of the SRBC targets, and essentially no lysis of the autologous PRBC targets. The optimum concentration of PHA used in the assays involving SRBC targets was 25 rg/ml, which was 10 times greater than that found to be optimum for CRBC targets. There was no lysis of PRBC at any PHA concentration. TABLE 3 Ability of the Porcine Alveolar Macrophage to Distinguish between Xenogeneic and Autologous Red Blood Cell Targets in the PHA-Mediated Cytotoxicity Assay System Percentage cytotoxicity” Animal No.
PHA*
CRBC
SRBC
PRBC
SPF 4056-S
+
3.6 73.7
0.0 16.5
0.1 0.9
SPF 4058-4
+
2.0 76.0
0.2 25.7
0.9 0.1
SPF 4047-6
+
0.5 71.5
1.8 19.2
1.0 1.8
SPF 4091-t
+
0.0 68.9
0.4 9.1
0.0 0.0
-
a Effector-to-target cell ratio of 1:l assayed after an 18-hr incubation at 37°C. b The optimum dosesof PHA used in this assay were 2.5 rg/ml for CRBC targets and 25 rg/ml for SRBC and PRBC.
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FIG. 1. Kinetics of RBC lysis by AM in the PHA-mediated cytotoxicity assay. Percentage specific “Cr release from CRBC (0) and autologous PRBC (0) assayed at various time intervals after incubation at 37°C with PHA (10 pg/ml). Effector-to-target cell ratio of 1:l.
Kinetics of Lysis of CRBC and PRBC in the PHA-Mediated
Cytotoxicity Assay
Figure 1 shows that maximum lysis of CRBC by AM in the presence of PHA was achieved within 18 hr after the initiation of the assay. However, there was no lysis of the autologous PRBC assayed for as long as 48 hr. This observation eliminates the possibility that the PRBC is being lysed by the porcine AM at a rate too slow to be detected in our 18-hr assay. Ability of Rabbit AM to Discriminate between Xenogeneic and Autologous RBC Since the PRBC were not lysed in the PHA-mediated assay system when porcine AM were effector cells, it was important to determine if this target was resistant to macrophage lysis, if PHA was a target-specific mediator, and/or if it was some self-recognition ability of the autologous effector cell that prevented lysis of the PRBC. Table 4 shows the results of experiments using rabbit AM in the PHATABLE 4 Ability of the Rabbit Alveolar Macrophage to Distinguish between Xenogeneic and Autologous Red Blood Cell Targets in the PHA-Mediated Cytotoxicity Assay System Percentage cytotoxicity’ CRBC
SRBC
PRBC
RRBC
Rabbit 651
-0.1 56.9
ND ND
0.3 16.7
1.6 1.2
Rabbit 7 14
2.2 55.1
3.5 26.2
18.3 52.4
-1.6 -0.5
Rabbit 716
0.6 57.4
0.3 16.9
0.1 35.4
-1.5 -2.1
Rabbit 718
1.7 68.1
0.2 22.1
0.4 41.7
-1.7 -1.4
Animal No.
PHAb
DEffector-to-target cell ratio of 1:l assayed after an 18-hr incubation at 37°C. * The optimum dosesof PHA used in this assay were 2.5 @g/ml for CRBC targets and 25 rg/ml for SRBC, PRBC, and RRBC.
SELF/NONSELF
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BY ALVEOLAR TABLE
Comparison
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MACROPHAGES
5
of PHA-Mediated Lysis of Chicken Red Blood Cells and Autologous Pig Red Blood Cells by Porcine Alveolar Macrophages
and Allogeneic
Percentage cytotoxicity”
CRBC
Autologous PRBC
Allogeneic PRBC
Animal No.
PHAb
SPF 3086-7
+
0.4 38.5
-0.6 1.2
0.0 -0.2
SPF 3111-3
+
4.5 61.4
-0.2 1.9
-0.4 -0.1
SPF 4102-6
+
5.3 59.9
-0.5 0.2
-0.4 0.2
SPF 4121-1
+
1.8 69.3
-0.2 0.5
-0.6 1.8
SPF 4125-3
+
-0.9 12.4
-0.3 0.3
-1.3 1.9
’ Effector-to-target cell ratio of 1:l assayed after an 18-hr incubation at 37°C. * The optimum doses of PHA used in this assay were 2.5 pg/ml for CRBC and 25 pg/ml for PRBC.
mediated assay. Like the pig AM, the rabbit AM shows little spontaneous cytotoxicity toward any RBC tested. However, when PHA is added (25 pg/ml) there were about 60% lysis of CRBC, 20% lysis of SRBC, 43% lysis of PRBC, and no lysis of the autologous rabbit RBC (RRBC). This shows that the PRBC are not resistant to AM lysis in a PHA-mediated system and suggests that AM are capable of recognizing self. AM Lack of Ability
to Distinguish
between Allogeneic
and Autologous
PRBC
It can be seen in Table 5 that although there is 61.5 + 13.7% lysis of CRBC by AM in the presence of PHA, there is no lysis of either allogeneic or autologous PRBC. This suggests that the recognition by AM for self/nonself is broadly species specific or that all pigs lack a specific antigen necessary to trigger the porcine AM lytic mechanism. Target Selectivity
of the Porcine AM in a Mixed-Target
PHA-Mediated
Assay
Table 6 shows the results of experiments designed to measure the selectivity of the alveolar macrophage to lyse xenogeneic but not autologous RBC targets in a mixed-target PHA-mediated assay system. Varying ratios of chromium-labeled CRBC and unlabeled PRBC or unlabeled CRBC and chromium-labeled PRBC were added to the PHA-mediated assay. The total number of labeled targets always equaled lo5 cells which yielded an effector-to-target ratio of 1:1. It can be seen that the percentage of CRBC lysis remained constant despite the varying ratios of CRBC:PRBC. It can also be seen that even though the macrophages were actively lysing the CRBC, there was no lysis of the autologous PRBC targets,
374
ROTHLEIN
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TABLE 6 Ability of the Porcine Alveolar Macrophage to Selectively Lyse Xenogeneic but Not Autologous Red Blood Cell Targets in a Mixed-Target PHA-Mediated Assay System Percentage cytotoxicity”
CRBC
CRBC only
56.1
CRBC:PRBC 3:1 2:2 1:3
63.2 62.0 61.6
PRBC only
-
Expt 3
Expt 2
Expt 1 CRBC:PRBC ratiob
PRBC
CRBC
PRBC
59.3 -2.3 -0.3 -0.4 -0.4
56.1 56.6 65.7 -
0.6 -0.4 -0.6 -0.1
CRBC
PRBC
61.8
-
64.5 70.3 70.8 -
-1.2 0.3 -0.1 0.0
’ Effector-to-target cell (total) ratio of 1:1 assayed after an 18-hr incubation at 37°C with 25 pg/ml of PHA for specific lysis of “Cr-labeled targets. * Varying ratios of 5’Cr-CRBC and unlabeled PRBC or unlabeled CRBC and “Cr-PRBC were mixed so that the final number of total red blood cells was constant at 10’ cells/well.
suggesting that macrophages triggered to lyse RBC targets can discriminate between the xenogeneic CRBC target and the autologous PRBC target. DISCUSSION We have observed that PHA mediates cytotoxicity of AM toward xenogeneic but not autologous or allogeneic RBC in an 1%hr assay. We believe that this observation represents a self/nonself recognition system by. the AM and not merely a crosslinking of the RBC to AM by PHA or a resistance of the PRBC to the AM lytic mechanism. This hypothesis is supported by the observations that other agents capable of agglutinating and crosslinking RBC, such as Con A, do not promote lysis of RBC from any species even though the concentration employed in the cytotoxicity assay was well above the minimum concentration necessary for agglutination to occur, and that PHA was capable of agglutinating PRBC almost as efficiently as CRBC (unpublished data), yet promoted no lysis of the autologous target by the AM. The data in Tables 3-5 further support the idea of self/nonself recognition by the AM since autologous PRBC will not be lysed by porcine AM which are capable of lysing xenogeneic CRBC and SRBC in the presence of PHA and that the rabbit AM will lyse PRBC as well as CRBC and SRBC but not autologous RRBC. These data suggest that PRBC resistance to the AM lytic mechanism was not responsible for our observations sinee the rabbit AM were perfectly capable of lysing PRBC and that both the porcine and rabbit AM are capable of recognizing and lysing xenogeneic but not autologous RBC in the presence of PHA. Also, Table 5 suggests that the self/nonself recognition mechanism of the AM is relatively broad since xenogeneic but not allogeneic or autologous RBC can trigger PHA-mediated lysis by AM. The observation that macrophages are capable of discriminating between xenogeneic and autologous RBC is in agreement with Muchmore et al. (23). However, their system requires the addition of
SELF/NONSELF
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375
a heat-labile serum factor while our PHA-mediated system does not. In fact, the only serum in our assay is heat-inactivated fetal bovine serum, which does not promote lysis of either xenogeneic or autologous RBC in the absence of PHA. On the other hand, it is possible that AM may synthesize this heat-labile factor in the PHA-mediated system. To eliminate the possibility that autologous RBC are being lysed at a slower rate than xenogeneic targets, we extended our 18-hr assay to as long as 48 hr. Figure 1 shows that although maximum killing occurs by 18 hr with CRBC targets, there is no killing of PRBC targets up to 48 hr. It is unlikely that contaminating lymphocytes in the AM preparation are responsible for the PHA-mediated cytotoxicity and specificity since we have shown that in the lung lavage fluid of adult SPF Minnesota miniature swine there were greater than 90% AM and less than 5% lymphocytes (26). This would yield a maximum lymphocyte-to-target ratio of 1:20, which is well below that found to be effective in a PHA-mediated cytotoxicity assay with lymphocyte effecters (8). Furthermore, it was found that CRBC but not PRBC lysis did occur when only adherent lavage fluid cells devoid of lymphocytes were used as effecters and that no lysis occurred when peripheral blood lymphocytes were used (unpublished data). It was reported that lymphocytes are capable of lysing autologous RBC in the presence of PHA (28); however, data in Tables 3-5 and Fig. 1 show that this is not the case in our assay, further suggesting that lymphocytes do not play a role in the PHAmediated cytotoxicity assay used in this report. Finally, the results of the mixed-target PHA-mediated assay system (Table 6) suggest not only that the AM can distinguish between self and nonself, but also that this recognition is localized on the AM membrane. When labeled CRBC were mixed with unlabeled PRBC, there was a constant percentage of CRBC being lysed despite the ratio of CRBC:PRBC, and when unlabeled CRBC were mixed with labeled PRBC, there was no bystander lysis of the PRBC. This shows that cells which are actively lysing xenogeneic RBC will not lyse autologous cells, suggesting that both the recognition and lytic mechanism of the AM take place on localized sites of the AM membrane. In conclusion, we propose that the AM are capable of discriminating between self and nonself and that foreign recognition in conjunction with some other nonspecific signal, such as PHA, is able to trigger the AM lytic mechanism. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Green, G. M., and Kass, E. H., .I. Exp. Med. 119, 167, 1964. Cohen, A. B., and Kline, M. J., J. Clin. Invest. 50, 1390, 1971. Evans, R., and Alexander, P., Nature (London) 236, 168, 1972. Melsom, H., and Seljelid, R., J. Exp. Med. 137, 807, 1973. Tagliabue, A., Mantovani, A., Kilgallen, M., Herberman, R. B., and McCoy, J. L., J. Immunol. 122, 2363, 1979. Cleveland, P. R., Meltzer, M. S., and Zbar, B., J. Nat. Cancer Insr. 52, 1887, 1974. Hibbs, J. B., Jr., Science 184, 469, 1974. Muchmore, A. V., Nelson, D. L., Kirchner, H., and Blaese, R. M., Cell. Immunol. 19, 78, 1975. Hunninghake, G. W., and Fauci, A. S., Cell. Immunol. 26, 98, 1976. Toh, K., Sato, N., and Kikuchi, K., J. Rericuloendothel. Sot. 25, 17, 1979. Kurisu, M., Yamazaki, M., and Mizuno, D., Cancer Res. 40, 3798, 1980. Nacy, C. A., and Meltzer, M. S., J. Immunol. 123, 2544, 1979. Meltzer, M. S., J. Immunol. 127, 179, 1981.
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Sone, S., and Fidler, I. J., J. Reticuloendothel. Sot. 27, 269, 1980. Holm, G., Engwall, S., Hammarstriim, S., and Natvig, J. B., Scund. J. Immunol. 3, 173, 1974. Ralph, P., and Nakoinz, I., J. Immunol. 119, 950, 1977. Kurisu, M., Dohi, N., Yamazaki, M., and Mizuno, D., J. Reticuloendothel. Sot. 28, 237, 1980. Koren, H. S., Anderson, S. J., and Adams, D. O., Cell. Zmmunol. 57, 51, 198 1. Muchmore, A. V., Decker, J. M., and Blaese, R. M., J. Zmmunol. 119, 1680, 1977. Hihbs, J. B., Jr., Lambert, L. H., and Remington, J. S., Science 177, 998, 1972. Adams, D. O., J. Immunol. 124, 286, 1980. Cabilly, S., and Gallily, R., Cell. Immunol. 29, 54, 1977. Muchmore, A. V., Decker, J. M., and Blaese, R. M., J. Immunol. 122, 1146, 1979. Keller, R., &it. J. Cancer 37, 732, 1978. Kim, Y. B., In “Immunodeficiency in Man and Animals” (D. Bergsma, R. A. Good, and J. Finstad, Eds.), Birth Defects: Original Article Series, Vol. XI, No. 1, pp. 5499557. Sinauer, Sunderland, Mass., 1975. 26. Rothlein, R., Gallily, R., and Kim, Y. B., J. Reticuloendothel. Sot. 30, 483, 1981. 27. Kim, Y. B., Huh, N. D., Koren, H. S., and Amos, B., J. Immunol. 125, 755, 1980. 28. Pearlmann, P., Pearlmann, H., and Holm, G., Science 160, 306, 1968.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
IMMUNOLOGY
Porcine
68, 368-376 (1982)
Alveolar Macrophages Discriminate between Nonself in Lectin-Mediated Cellular Cytotoxicity’ ROBERTROTHLEINANDYOONBERM Sloan-Kettering
Self and
KIM
Institute for Cancer Research, Rye, New York 10580
Received January
7, 1982; accepted February
12, 1982
The pulmonary alveolar macrophages (AM) from specific pathogen-free Minnesota miniature swine were tested for their ability to discriminate between xenogeneic and autologous red blood cell (RBC) targets in lectin-mediated cellular cytotoxicity measured by an 18-hr “Cr-release assay. It was found that the porcine AM would lyse xenogeneic chicken and sheep RBC but not autologous pig RBC in the presence of PHA. It was also shown that pig RBC was susceptible to lysis by the AM in a PHA-mediated assay if the effector AM were from rabbits. It is postulated that a nonspecific signal in conjunction with foreign recognition is able to trigger the AM lytic mechanism.
INTRODUCTION It is well established that macrophages can be activated to be cytotoxic to bacterial, red blood cell (RBC),’ and tumor targets in vitro (l-5). Although the mechanism of activation has begun to be elucidated, the means by which they recognize targets is still poorly understood. It has been shown that by in vivo treatment with Bacillus Calmette-G&in (6, 7) or by in vitro treatment with mitogens (8-1 l), T-cell products and/or lipopolysaccharides ( 12- 14), or target-specific antibody ( 15- 18), macrophages can be made cytotoxic to bacterial, tumor, and RBC targets. Also, it was reported that blood monocytes will lyse RBC targets by culturing the peripheral blood mononuclear cells for 1 week prior to the cytotoxicity assay (19). It is known that activated or elicited macrophages can specifically lyse tumor but not normal targets (20, 2 1). Also, Cabilly and Gallily found that mouse peritoneal exudate macrophages can distinguish between allogeneic and xenogeneic fibroblast targets in a cytotoxic assay (22), and Muchmore et al. reported that macrophages can distinguish between RBC of different species with the specificity being conferred by a nonimmunoglobulin serum factor (23). ’ This investigation was supported in part by USPHS Grants CA-25384 and CA-08748 from the National Cancer Institute and HD-12097 from the National Institute of Child Health and Human Development. Portions of this work will be submitted by R. Rothlein in partial fulfillment of the rcquirements for the Doctor of Philosophy degree at Cornell University Graduate School of Medicine Sciences. * Abbreviations used: AM, pulmonary alveolar macrophages; RBC, red blood cells; CRBC, chicken RBC, PRBC, pig RBC, SRBC, sheep RBC; RRBC, rabbit RBC; PHA, phytohemagglutinin; Con A, concanavalin A, PWM, pokeweed mitogen; BSS, balanced salt solution. 368 OOOS-8749/82/060368-09$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
SELF/NONSELF
RECOGNITION
BY ALVEOLAR
MACROPHAGES
369
Although most of these cytotoxic studies have been performed using macrophages from the blood or peritoneal cavity, some studies have shown that pulmonary alveolar macrophages (AM) also have cytotoxic capabilities (9, 24). In this paper we describe an assay system in which porcine AM are capable of discriminating between xenogeneic and autologous RBC in the presence of phytohemagglutinin (PHA) in a chromium-release cytotoxicity assay. MATERIALS
AND METHODS
Animals, Young adult animals from a herd of specific pathogen-free Minnesota miniature swine maintained in a facility with filtered air (Hepa filter) and fed an autoclaved diet with chlorinated water were used in these experiments (25). Also, young adult miniature New Zealand white rabbits were used. Alveolar macrophages. AM were collected as previously described (26). Briefly, 300 ml of phosphate-buffered saline (PBS), pH 7.2, was injected intratracheally into swine sacrificed by CO* anesthesia. The PBS was then withdrawn and the lavage cells were washed three times with balanced salt solution (BSS). Rabbit lungs were lavaged in a similar fashion using three separate lavages of 20 ml each, Preparation of pig anti-chicken red blood cell serum. One milliliter of 20% washed chicken RBC (CRBC) was injected intraperitoneally into a swine on three occasions at l-week intervals. One week following the final injection the serum was collected, heat-inactivated, aliquoted, and stored frozen until used. Labeling of target cells. RBC were labeled with “Cr by incubating lo7 RBC in 100 ~1 of BSS plus 100 &i of Na”CrO, (sp act 250-500 mCi/mmol, New England Nuclear, No. NEZ-030s) in 100 ~1for 2 hr at 37°C with constant shaking. The labeled RBC were then washed three times with BSS and resuspended to lo6 cells/ml with RPM1 1640 with 10% heat-inactivated fetal bovine serum and 1% Pen-Strep and 1% glutamine (complete medium). Cytotoxicity assay. Fifty microliters of complete medium containing appropriately diluted pig anti-CRBC serum, mitogen, or nothing was added to appropriate wells of a flat-bottomed, 96-well microtest plate (Falcon No. 3001, Oxnard, Calif.) AM, IO5 in 50 ~1 of complete medium were then added to each well and the plates were incubated at 37°C. After 1 hr, lo5 labeled targets in 100 ~1 of complete medium were added to each well, and the plates were incubated at 37°C. The assay was terminated by collecting 100 ~1 of supernatants at appropriate times and measuring their radioactivity in a well-type gamma counter as described elsewhere (27). Cytotoxic activity was calculated as
% specific release (lysis) =
cpm experimental - cpm SR x 100, cpm MR - cpm SR
where spontaneous release (SR) was defined as the counts per minute (cpm) released from targets incubated with complete medium alone, and maximum release (MR) was the cpm after lysis of the RBC targets with 1% Triton X-100. Values are expressed as means of triplicate determinations. Throughout the experiments MR were higher than 97% of total isotope uptake and the mean SR for chicken red blood cells, porcine red blood cells, sheep red blood cells, and rabbit red blood cells were 3.3 f 2.0, 3.5 -+ 0.1, 4.2 f 0.5, and 8.2 -t 2.4%, respectively.
370
ROTHLEIN
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RESULTS Comparison of Various Concentrations of Mitogens in Mediating CRBC Lysis by the Porcine AM Table 1 shows the results of a representative experiment giving the percentage of CRBC lysed by porcine AM in the presence of concanavalin A (Con A), pokeweed mitogen (PWM), or PHA at various concentrations in an 18-hr ‘iCr-release assay at an effector-to-target ratio of 1:1. It can be seen that there was little or no lysing of the CRBC targets when either Con A or PWM was used as a mediator. However, PHA was capable of mediating lysis by the AM at a concentration of 0.25 pg/ml, the lowest mitogen concentration tested. The optimum concentration for PHA-mediated lysis was between 2.5 and 5.0 pg/ml and there was a gradual decrease in lysis at higher PHA concentrations. Comparison of PHA and Antibody-Mediating
CRBC Lysis
Table 2 shows the results that compared the optimum PHA-mediated CRBC lysis with the optimum CRBC lysis mediated by pig anti-CRBC serum in the 18hr chromium-release assay with porcine AM as effector cells. It can be seen that the percentages of specific 51Cr release were 64.1 + 8.9% when PHA was the mediator, 52.8 f 5.4% when the assay was mediated by pig anti-CRBC serum, and 1.5 f 1.4% when no mediator was present in the assay. Note that in the 12 pigs tested, the percentage of lysis in the antibody-mediated system never exceeded that of the PHA-mediated system. Also, there is a strong correlation between the two mediators in that when lysis mediated by PHA was high, so was the lysis mediated by the antiserum. Ability of the Porcine AM to Discriminate between Xenogeneic and Autologous RBC Table 3 shows the results of PHA-mediated %r-release assaysusing xenogeneic CRBC, SRBC, and autologous PRBC targets. It can be seen that the AM showed very little spontaneous cytotoxicity toward any of the RBC targets. However, when PHA was added to the assay there were an average of greater than 70% lysis of TABLE 1 Effect of Various Concentrations of Mitogens on Mediating the Cytotoxicity of Porcine Alveolar Macroohaees to S’Cr-Labeled Chicken Red Blood Cell Targets Percentage cytotoxicity’ Mitogen dose (ridmU
Con A
PHA
PWM
0 0.25 2.5 5.0 25 50
0.0 -0.3 -0.2 -0.6 -0.1 1.0
0.0 53.0 73.0 75.0 61.8 55.6
0.0 1.0 -0.5 -0.8 -1.1 -1.0
a Effector-to-target cell ratio of 1:l assayed after an 1%hr incubation at 37°C.
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MACROPHAGES
TABLE 2 Comparison of Antibody- and PHA-Mediated Cytotoxicity of Porcine Alveolar Macrophages to “Cr-Labeled Chicken Red Blood Cell Targets Percentage cytotoxicity” Animal No.
None
PHAb
Pig anti-CRBC’
SPF 3137-3 SPF 4036-4 SPF 4056-5 SPF 4058-4 SPF 3117-7 SPF 4059-l SPF 4067-4 SPF 3152-8 SPF 4029-4 SPF 4060-7 SPF 3089-5 SPF 4047-6
0.2 0.3 3.6 2.0 4.5 0.8 0.7 1.9 -0.2 2.3 I.5 0.5
63.7 55.7 70.5 74.8 52.9 67.3 71.1 54.0 59.0 76.0 52.7 71.5
55.1 48.3 63.1 55.5 49.6 53.2 59.3 46.3 49.4 53.6 44.6 55.3
64.1 f 8.9
52.8 2 5.4
Mean + SD
1.5 z!z1.4
a Effector-to-target cell ratio of 1:1 assayed after an 18-hr incubation at 37°C. ’ PHA was at a concentration of 2.5 pg/ml in the assay. ‘Pig anti-CRBC serum was diluted 1:400 in the assay.
the CRBC targets, 18% lysis of the SRBC targets, and essentially no lysis of the autologous PRBC targets. The optimum concentration of PHA used in the assays involving SRBC targets was 25 rg/ml, which was 10 times greater than that found to be optimum for CRBC targets. There was no lysis of PRBC at any PHA concentration. TABLE 3 Ability of the Porcine Alveolar Macrophage to Distinguish between Xenogeneic and Autologous Red Blood Cell Targets in the PHA-Mediated Cytotoxicity Assay System Percentage cytotoxicity” Animal No.
PHA*
CRBC
SRBC
PRBC
SPF 4056-S
+
3.6 73.7
0.0 16.5
0.1 0.9
SPF 4058-4
+
2.0 76.0
0.2 25.7
0.9 0.1
SPF 4047-6
+
0.5 71.5
1.8 19.2
1.0 1.8
SPF 4091-t
+
0.0 68.9
0.4 9.1
0.0 0.0
-
a Effector-to-target cell ratio of 1:l assayed after an 18-hr incubation at 37°C. b The optimum dosesof PHA used in this assay were 2.5 rg/ml for CRBC targets and 25 rg/ml for SRBC and PRBC.
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FIG. 1. Kinetics of RBC lysis by AM in the PHA-mediated cytotoxicity assay. Percentage specific “Cr release from CRBC (0) and autologous PRBC (0) assayed at various time intervals after incubation at 37°C with PHA (10 pg/ml). Effector-to-target cell ratio of 1:l.
Kinetics of Lysis of CRBC and PRBC in the PHA-Mediated
Cytotoxicity Assay
Figure 1 shows that maximum lysis of CRBC by AM in the presence of PHA was achieved within 18 hr after the initiation of the assay. However, there was no lysis of the autologous PRBC assayed for as long as 48 hr. This observation eliminates the possibility that the PRBC is being lysed by the porcine AM at a rate too slow to be detected in our 18-hr assay. Ability of Rabbit AM to Discriminate between Xenogeneic and Autologous RBC Since the PRBC were not lysed in the PHA-mediated assay system when porcine AM were effector cells, it was important to determine if this target was resistant to macrophage lysis, if PHA was a target-specific mediator, and/or if it was some self-recognition ability of the autologous effector cell that prevented lysis of the PRBC. Table 4 shows the results of experiments using rabbit AM in the PHATABLE 4 Ability of the Rabbit Alveolar Macrophage to Distinguish between Xenogeneic and Autologous Red Blood Cell Targets in the PHA-Mediated Cytotoxicity Assay System Percentage cytotoxicity’ CRBC
SRBC
PRBC
RRBC
Rabbit 651
-0.1 56.9
ND ND
0.3 16.7
1.6 1.2
Rabbit 7 14
2.2 55.1
3.5 26.2
18.3 52.4
-1.6 -0.5
Rabbit 716
0.6 57.4
0.3 16.9
0.1 35.4
-1.5 -2.1
Rabbit 718
1.7 68.1
0.2 22.1
0.4 41.7
-1.7 -1.4
Animal No.
PHAb
DEffector-to-target cell ratio of 1:l assayed after an 18-hr incubation at 37°C. * The optimum dosesof PHA used in this assay were 2.5 @g/ml for CRBC targets and 25 rg/ml for SRBC, PRBC, and RRBC.
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Comparison
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5
of PHA-Mediated Lysis of Chicken Red Blood Cells and Autologous Pig Red Blood Cells by Porcine Alveolar Macrophages
and Allogeneic
Percentage cytotoxicity”
CRBC
Autologous PRBC
Allogeneic PRBC
Animal No.
PHAb
SPF 3086-7
+
0.4 38.5
-0.6 1.2
0.0 -0.2
SPF 3111-3
+
4.5 61.4
-0.2 1.9
-0.4 -0.1
SPF 4102-6
+
5.3 59.9
-0.5 0.2
-0.4 0.2
SPF 4121-1
+
1.8 69.3
-0.2 0.5
-0.6 1.8
SPF 4125-3
+
-0.9 12.4
-0.3 0.3
-1.3 1.9
’ Effector-to-target cell ratio of 1:l assayed after an 18-hr incubation at 37°C. * The optimum doses of PHA used in this assay were 2.5 pg/ml for CRBC and 25 pg/ml for PRBC.
mediated assay. Like the pig AM, the rabbit AM shows little spontaneous cytotoxicity toward any RBC tested. However, when PHA is added (25 pg/ml) there were about 60% lysis of CRBC, 20% lysis of SRBC, 43% lysis of PRBC, and no lysis of the autologous rabbit RBC (RRBC). This shows that the PRBC are not resistant to AM lysis in a PHA-mediated system and suggests that AM are capable of recognizing self. AM Lack of Ability
to Distinguish
between Allogeneic
and Autologous
PRBC
It can be seen in Table 5 that although there is 61.5 + 13.7% lysis of CRBC by AM in the presence of PHA, there is no lysis of either allogeneic or autologous PRBC. This suggests that the recognition by AM for self/nonself is broadly species specific or that all pigs lack a specific antigen necessary to trigger the porcine AM lytic mechanism. Target Selectivity
of the Porcine AM in a Mixed-Target
PHA-Mediated
Assay
Table 6 shows the results of experiments designed to measure the selectivity of the alveolar macrophage to lyse xenogeneic but not autologous RBC targets in a mixed-target PHA-mediated assay system. Varying ratios of chromium-labeled CRBC and unlabeled PRBC or unlabeled CRBC and chromium-labeled PRBC were added to the PHA-mediated assay. The total number of labeled targets always equaled lo5 cells which yielded an effector-to-target ratio of 1:1. It can be seen that the percentage of CRBC lysis remained constant despite the varying ratios of CRBC:PRBC. It can also be seen that even though the macrophages were actively lysing the CRBC, there was no lysis of the autologous PRBC targets,
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TABLE 6 Ability of the Porcine Alveolar Macrophage to Selectively Lyse Xenogeneic but Not Autologous Red Blood Cell Targets in a Mixed-Target PHA-Mediated Assay System Percentage cytotoxicity”
CRBC
CRBC only
56.1
CRBC:PRBC 3:1 2:2 1:3
63.2 62.0 61.6
PRBC only
-
Expt 3
Expt 2
Expt 1 CRBC:PRBC ratiob
PRBC
CRBC
PRBC
59.3 -2.3 -0.3 -0.4 -0.4
56.1 56.6 65.7 -
0.6 -0.4 -0.6 -0.1
CRBC
PRBC
61.8
-
64.5 70.3 70.8 -
-1.2 0.3 -0.1 0.0
’ Effector-to-target cell (total) ratio of 1:1 assayed after an 18-hr incubation at 37°C with 25 pg/ml of PHA for specific lysis of “Cr-labeled targets. * Varying ratios of 5’Cr-CRBC and unlabeled PRBC or unlabeled CRBC and “Cr-PRBC were mixed so that the final number of total red blood cells was constant at 10’ cells/well.
suggesting that macrophages triggered to lyse RBC targets can discriminate between the xenogeneic CRBC target and the autologous PRBC target. DISCUSSION We have observed that PHA mediates cytotoxicity of AM toward xenogeneic but not autologous or allogeneic RBC in an 1%hr assay. We believe that this observation represents a self/nonself recognition system by. the AM and not merely a crosslinking of the RBC to AM by PHA or a resistance of the PRBC to the AM lytic mechanism. This hypothesis is supported by the observations that other agents capable of agglutinating and crosslinking RBC, such as Con A, do not promote lysis of RBC from any species even though the concentration employed in the cytotoxicity assay was well above the minimum concentration necessary for agglutination to occur, and that PHA was capable of agglutinating PRBC almost as efficiently as CRBC (unpublished data), yet promoted no lysis of the autologous target by the AM. The data in Tables 3-5 further support the idea of self/nonself recognition by the AM since autologous PRBC will not be lysed by porcine AM which are capable of lysing xenogeneic CRBC and SRBC in the presence of PHA and that the rabbit AM will lyse PRBC as well as CRBC and SRBC but not autologous RRBC. These data suggest that PRBC resistance to the AM lytic mechanism was not responsible for our observations sinee the rabbit AM were perfectly capable of lysing PRBC and that both the porcine and rabbit AM are capable of recognizing and lysing xenogeneic but not autologous RBC in the presence of PHA. Also, Table 5 suggests that the self/nonself recognition mechanism of the AM is relatively broad since xenogeneic but not allogeneic or autologous RBC can trigger PHA-mediated lysis by AM. The observation that macrophages are capable of discriminating between xenogeneic and autologous RBC is in agreement with Muchmore et al. (23). However, their system requires the addition of
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a heat-labile serum factor while our PHA-mediated system does not. In fact, the only serum in our assay is heat-inactivated fetal bovine serum, which does not promote lysis of either xenogeneic or autologous RBC in the absence of PHA. On the other hand, it is possible that AM may synthesize this heat-labile factor in the PHA-mediated system. To eliminate the possibility that autologous RBC are being lysed at a slower rate than xenogeneic targets, we extended our 18-hr assay to as long as 48 hr. Figure 1 shows that although maximum killing occurs by 18 hr with CRBC targets, there is no killing of PRBC targets up to 48 hr. It is unlikely that contaminating lymphocytes in the AM preparation are responsible for the PHA-mediated cytotoxicity and specificity since we have shown that in the lung lavage fluid of adult SPF Minnesota miniature swine there were greater than 90% AM and less than 5% lymphocytes (26). This would yield a maximum lymphocyte-to-target ratio of 1:20, which is well below that found to be effective in a PHA-mediated cytotoxicity assay with lymphocyte effecters (8). Furthermore, it was found that CRBC but not PRBC lysis did occur when only adherent lavage fluid cells devoid of lymphocytes were used as effecters and that no lysis occurred when peripheral blood lymphocytes were used (unpublished data). It was reported that lymphocytes are capable of lysing autologous RBC in the presence of PHA (28); however, data in Tables 3-5 and Fig. 1 show that this is not the case in our assay, further suggesting that lymphocytes do not play a role in the PHAmediated cytotoxicity assay used in this report. Finally, the results of the mixed-target PHA-mediated assay system (Table 6) suggest not only that the AM can distinguish between self and nonself, but also that this recognition is localized on the AM membrane. When labeled CRBC were mixed with unlabeled PRBC, there was a constant percentage of CRBC being lysed despite the ratio of CRBC:PRBC, and when unlabeled CRBC were mixed with labeled PRBC, there was no bystander lysis of the PRBC. This shows that cells which are actively lysing xenogeneic RBC will not lyse autologous cells, suggesting that both the recognition and lytic mechanism of the AM take place on localized sites of the AM membrane. In conclusion, we propose that the AM are capable of discriminating between self and nonself and that foreign recognition in conjunction with some other nonspecific signal, such as PHA, is able to trigger the AM lytic mechanism. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Green, G. M., and Kass, E. H., .I. Exp. Med. 119, 167, 1964. Cohen, A. B., and Kline, M. J., J. Clin. Invest. 50, 1390, 1971. Evans, R., and Alexander, P., Nature (London) 236, 168, 1972. Melsom, H., and Seljelid, R., J. Exp. Med. 137, 807, 1973. Tagliabue, A., Mantovani, A., Kilgallen, M., Herberman, R. B., and McCoy, J. L., J. Immunol. 122, 2363, 1979. Cleveland, P. R., Meltzer, M. S., and Zbar, B., J. Nat. Cancer Insr. 52, 1887, 1974. Hibbs, J. B., Jr., Science 184, 469, 1974. Muchmore, A. V., Nelson, D. L., Kirchner, H., and Blaese, R. M., Cell. Immunol. 19, 78, 1975. Hunninghake, G. W., and Fauci, A. S., Cell. Immunol. 26, 98, 1976. Toh, K., Sato, N., and Kikuchi, K., J. Rericuloendothel. Sot. 25, 17, 1979. Kurisu, M., Yamazaki, M., and Mizuno, D., Cancer Res. 40, 3798, 1980. Nacy, C. A., and Meltzer, M. S., J. Immunol. 123, 2544, 1979. Meltzer, M. S., J. Immunol. 127, 179, 1981.
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Sone, S., and Fidler, I. J., J. Reticuloendothel. Sot. 27, 269, 1980. Holm, G., Engwall, S., Hammarstriim, S., and Natvig, J. B., Scund. J. Immunol. 3, 173, 1974. Ralph, P., and Nakoinz, I., J. Immunol. 119, 950, 1977. Kurisu, M., Dohi, N., Yamazaki, M., and Mizuno, D., J. Reticuloendothel. Sot. 28, 237, 1980. Koren, H. S., Anderson, S. J., and Adams, D. O., Cell. Zmmunol. 57, 51, 198 1. Muchmore, A. V., Decker, J. M., and Blaese, R. M., J. Zmmunol. 119, 1680, 1977. Hihbs, J. B., Jr., Lambert, L. H., and Remington, J. S., Science 177, 998, 1972. Adams, D. O., J. Immunol. 124, 286, 1980. Cabilly, S., and Gallily, R., Cell. Immunol. 29, 54, 1977. Muchmore, A. V., Decker, J. M., and Blaese, R. M., J. Immunol. 122, 1146, 1979. Keller, R., &it. J. Cancer 37, 732, 1978. Kim, Y. B., In “Immunodeficiency in Man and Animals” (D. Bergsma, R. A. Good, and J. Finstad, Eds.), Birth Defects: Original Article Series, Vol. XI, No. 1, pp. 5499557. Sinauer, Sunderland, Mass., 1975. 26. Rothlein, R., Gallily, R., and Kim, Y. B., J. Reticuloendothel. Sot. 30, 483, 1981. 27. Kim, Y. B., Huh, N. D., Koren, H. S., and Amos, B., J. Immunol. 125, 755, 1980. 28. Pearlmann, P., Pearlmann, H., and Holm, G., Science 160, 306, 1968.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.