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Age-dependent macrophage functions in New Zealand Black mice
CELLULAR
IMMUNOLOGY
45, 309-317 (1979)
Age-Dependent Macrophage Functions New Zealand Black Mice MENASHE The Laurenberg
Centerfbr
Tumor
in
BAR-ELI AND RUTH GALLILY and General Immunology, Hebrew, School, Jerusalem, Israel Received
October
University-Hadassah
Medical
30. 1978
Various functions of macrophage derived from young (2-month-old) and old (14- to 17-month-old) New Zealand Black (NZB) mice with autoimmune disease were studied and compared with macrophage functions of age-matched BALB/c mice. Macrophages from young and old NZB mice demonstrated elevated levels of P-glucuronidase, cathepsin D, lysozyme, and DNase compared with those from age-matched BALB/c. DNase activity in the macrophages of NZB mice significantly increased with age. Macrophages from young and old NZB mice had greater phagocytic capacity for both ‘Y-labeled Shigella j?exneri and Staphylococcus albus than did BALB/c macrophages. NZB macrophages from both young and old mice had higher bactericidal activity against S. albus than those from age-matched BALBic mice. The number of macrophage/granulocyte colony-forming cells (CFC) in both bone marrow and spleen was markedly higher in young and old NZB mice than in BALBic mice. Colony-stimulating factor (CSF) released by macrophages derived from NZB mice had higher CFC activity than that released from macrophages of age-matched BALBic mice. In NZB mice, the CSF activity significantly increased with age. It is suggested that potentiation of macrophage number and activity compensates for the deficiency of T cell functions in NZB mice with autoimmune disease.
INTRODUCTION
The NZB strain of mouse has been used by immunologists as an experimental model for the study of the immunopathology of autoimmune disease. NZB mice are presumed to be genetically predisposed to spontaneous development of a wide range of autoantibodies including antibodies to nucleic acids (I), erythrocytes autoantibodies (2), and natural thymocytoxic antibodies (3). The nature of the abnormal activity of the immunologic mechanism has yet to be fully elucidated. NZB mice are known to be hyperresponsive in humoral reaction to protein antigens (4, 5), but are also known to have depressed cellular immunity. Early in life, they manifest immune hyperresponsiveness (5,6) as well as a defect in suppressor cell function (7). Later in life, the T cell function is depressed, including T cell-dependent antibody production (4), skin allograft rejection (8), induction of graft-versus-host disease (9), and response to T cell mitogens such as phytohemagglutinin (10). Although the important role of macrophages in defense and immune reactions is well documented, little is known about their functions in NZB mice. In the present investigation, we studied enzyme levels, phagocytosis, and batericidal activities. In 309 0008-8749/79/080309-09$02.00/0 Copyright Ail rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
310
BAR-ELI
AND
GALLILY
addition, we determined the number of macrophage/granulocyte progenitors in bone marrow and in spleen. Our aim was twofold: We hoped to find out, first whether changes in macrophage activities occurred during spontaneous development of autoimmune disease and, second, whether these changes might explain some characteristics of autoimmune disease in NZB mice. Our findings demonstrated that macrophages derived from either young (Zmonth-old) or old (14to 17-month-old) NZB mice were more active in various functions than those derived from age-matched BALB/c mice. Several macrophage activities showed markedly higher values in older NZB mice than in younger animals of the same strain. MATERIALS
AND METHODS
Female NZB and BALB/c mice were obtained from the Experimental Animal Unit of the Weizmann Institute of Science, Rehovot, Israel. For each experiment, we selected mice of the same sex and age (within the range of 2 to 17 months) from both strains of mouse. Harvesting and culture of macrophages. Cells were obtained by washing the peritoneal cavities of donor animals with 5 ml RPMI-1640 medium. The yield per normal mouse was about 3 to 5 x 106, 30 to 40% of which were macrophages. The cells were suspended in the same medium supplemented with 2% fetal calf serum (FCS) (Microbiological Associates, Bethesda, Md.) and then cultivated in 30-mm plastic petri dishes (Nunc, Denmark) at 1 to 2 x lo6 cells per dish or in 6-mm-well tissue culture microtiter plates (Nunc, Denmark) at 3 x lo5 cells per well. After 2 to 3 hr of incubation at 37°C in a humidified atmosphere containing 5% COz, the nonadhering cells were removed by intensive rinsing with phosphate-buffered saline (PBS), and fresh medium was added. More than 90% of the remaining adherent cells were defined as macrophages by both morphologic and phagocytic criteria. The number of cells adherent to the 30-mm petri dishes was determined by counting these cells (1 l), while the number of cells adherent to the 6-mm wells was determined by a modification of the Nq5Cr0,-uptake method as described by More et al. (12). Enzyme assays. Lysates of macrophage monolayers were prepared as previously described (13, 14) and assayed for activity of four hydrolytic enzymes: (i) acid phosphatase by hydrolysis of p-nitrophenyl phosphate; (ii) j3-glucuronidase by hydrolysis of p-nitrophenyl-/3-D-glucuronide (Sigma Chemical Co.) (15); (iii) cathepsin D by hydrolysis of 8% hemoglobin (16); and (iv) lysozyme activity by hydrolysis of Micrococcus lysodeikticus (17). DNase activity was assayed by a modification of Kunitz’s method (18). The assay was based on the increased uv absorption of 260 nm, during the course of the depolymerization of DNA (Sigma) by DNase. The enzyme activity unit was defined as the activity which increases the absorbency by 0.001 per ml at 25°C. Phagocytic assay. Alcohol- or glutaraldehyde (GA)-killed bacteria were fixed with 0.25% GA and iodinated with lz51-labeled Na (carrier free, Amersham, England) (19); 2 mCi isotope was used for the labeling of 400 mg bacteria. The final intensity of labeling for both Shigella flexneri and Staphylococcus albus was 50 bacteria per cpm. 1.25 x 10’ labeled bacteria (2.5 x lo5 cpm) were added to the macrophage cultures in tissue culture microtiter plates (6-mm well diameter, lo5 Animals.
MACROPHAGE
FUNCTIONS
IN NZB
MICE
311
cells/well), giving a ratio of 100 bacteria to 1 cell. The cultures were incubated for various periods of time, then washed, and the wells were cut apart. Phagocytosis was assessed by counting each well in a Packard 5110 scintillation counter (20). Bactericidal assay. A saline suspension of S. albus in the log phase of growth (2 1) was added to macrophage cultures in microplates at a ratio of 100 bacteria to 1 cell. After incubation for 30 min at 37°C the nonphagocytosed bacteria were removed by intensive rinsing, and the macrophage monolayers were incubated for an additional 30 and 60 min. The cells were then washed and lysed for 3 min with 0.1% Triton X- 100. The number of viable staphylococci was determined by plating appropriate dilutions of the lysate in brain-heart agar. The colonies were counted after incubation of the agar plates for 24 hr at 37°C. Cell cloning. Cloning of bone marrow and spleen cells in double-layer agar culture was performed according to Apte et al. (22). The source of colonystimulating factor (CSF) was serum from BALB/c mice bled 6 hr after i.v. injection of lipopolysaccharide (LPS) (10 pg/mouse), diluted 1:4 and added to the cultures at a final concentration of 10%. This diluted serum served as a conditioned medium (CM). Bone marrow cells ( 105) or spleen cells ( 106) were cloned in soft agar medium (0.37%) supplemented with 10% CSF, plated over firm agar (0.5%). Triplicate plates were incubated in 5% CO, at 37°C.for 8 days, and the number of colonies (>40 cells per colony) in the upper layer was determined. It has been shown that each colony growing in soft agar represents a colony-forming cell (CFC) (23). By this technique we determined the number of macrophage/granulocyte progenitors cells, and it was expressed as the number of CFC. Synthesis ofcolony-stimulatingfactor (CSF). Peritoneal cells (5 x 105/ml/plate) were cultured for 2 hr in 30-mm petri dishes in RPMI- 1640 medium supplemented with 5% FCS. Following intensive rinsing, the adherent cells (90 to 95% of which were macrophages) were further incubated for 4 days in serum-free RPM1 medium with the addition of LPS (50 pg/ml/plate). The supernatant fluids were then collected, centrifuged (175g for 10 min), and served as conditioned medium (CM) for CFC assay. The CFC assay was performed on bone marrow cells of young BALBic mice as previously described. RESULTS Lysosomal enzymes. The activities of acid phosphatase, /3-glucuronidase, cathepsin D, and lysozyme were determined in macrophage monolayers consisting of cells derived from four age groups of unstimulated NZB and BALB/c mice. It was found (Figs. la-d) that the activity of acid phosphatase in macrophages derived from age-matched groups of NZB and BALB/c mice was not significantly different (P > 0.1). The activity of ,@glucuronidase was higher by about twofold only in NZB macrophages from young (2- to 3-month-old) and old (16-month-old) mice when compared with BALB/c macrophages from the same age groups (P < 0.001). Similarly, lysozyme activity in the cells was higher by twofold in NZB macrophages from young (2- to 3-month-old) and old (IQmonth-old) mice than in BALB/c macrophages. However, the level of cathepsin D was higher by 1.3- to 2.5-fold in NZB macrophages from the four age groups when compared with the matched groups of BALB/c macrophages (P < 0.005). It was also noted (Fig. 1) that
312
BAR-ELI
a
Acid
Age
c
b
Dhosohatase
(months)
Catheosin
Age
AND GALLILY
(months)
NZB
0
BALE/c
m
j3 - glucuronidase
Age
D
(months)
Lvsozvme
Age
(months)
FIG. 1. Specific enzyme activity of (a) acid phosphatase, (b) P-glucuronidase, (c) cathepsin D, and (d) lysozyme in macrophages derived from various age groups of NZB (0) and BALB/c (m) mice. (Each point represents the mean of six to nine determinations *SD).
macrophages from old NZB mice possessed a significantly higher activity of acid phosphatase than did cells of young NZB mice (P < O.OOl), whereas the activities of P-glucuronidase and cathepsin D showed little difference in macrophages between the old and young NZB mice. DNase activity. The activity of DNase was determined in macrophages derived from various age groups of unstimulated NZB and BALB/c mice. It was found (Fig. 2) that DNase activity was higher by 9-fold in macrophages from l- to 3-month-old NZB mice than in macrophages from BALB/c mice of the same age (P < 0.001). The activity of DNase was higher by 2%fold in macrophages from old (12- to 16-month-old) NZB mice than in microphages from old BALB/c mice (P < 0.001). It was also noted (Fig. 2) that macrophages from old NZB mice possessed significantly higher DNase activity than those from young NZB mice (P < 0.01). Uptake of labeled bacteria. The uptake of S. flexneri and S. albus by young, adult, and old NZB and BALB/c macrophage monolayers was determined after various periods of incubation. As can be seen in Figs. 3 and 4, the NZB macrophages derived from 2-, 6-, and 16-month-old mice demonstrated a higher capacity to
MACROPHAGE
FUNCTIONS
IN NZB MICE
313
DNase NZB
0
BALE/C
m
al6. 5. 4. 3-
&
2I. 1-3
a
12Age
i
1-3
a
12-16
(months)
FIG. 2. DNase activity in macrophages derived from various age groups of NZB (0) and BALB/c (a) mice. (Each bar represents the mean of three to nine determinations 2 standard error.)
phagocytize both S. flexneri and S. albus than BALB/c macrophages from mice of the same age groups (P < 0.005) at 15, 30, and 60 min. These results were also obtained at a 1:50 and a 1:25 bacteria to cell ratio (unpublished data). It should be noted that the phagocytic ability of NZB macrophages derived from 6- and 16-month-old mice increased significantly from that of cells from 2-month-old mice (P < 0.005). On the other hand, the phagocytic activity of BALB/c cells showed little change with age, and a decrease was even noted in the older BALB/c mice. Bactericidal activity. Survival of phagocytized S. albus was assessed 30 and 60 min following their engulfment by macrophages from various age groups of NZB and BALB/c mice. As shown in Table 1, macrophages derived from young and old
FIG. 3. Uptake of 1251-Iabeled Shigelln jkrneri by macrophages derived from 2-month-old (0). 6-month-old (0), and 16month-old (A) mice of NZB (a) and BALBic (b) mice. (A representative experiment of three performed; each point represents the mean of triplicate determinations.)
314
BAR-ELI
AND GALLILY
.
2
o
6 months
months
.
16months
Fro. 4. Uptake of rZ51-labeled S. a/bus by macrophages derived from 2-month-old (O), 6-month-old (0), and ldmonth-old (A) mice of NZB (a) and BALBlc (b) mice. (A representative experiment of three performed; each point represents the mean of triplicate determinations.)
NZB mice had a higher bactericidal capacity than BALB/c macrophages from the same age groups. The number of intracellular bacteria decreased in macrophages of NZB mice during 60 min, while an increase in the number of bacteria was noted in macrophages derived from BALB/c mice. Colony-forming cells (CFC) response. The number of colony-forming cells (CFC) was determined in normal and thioglycolate-stimulated mice by cloning bone marrow and spleen cells from two age groups of NZB and BALB/c mice (Table 2). The number of CFC in bone marrow of young and old normal NZB mice was higher by up to 1.7- and 2.3-fold, respectively, than the number of CFC from normal bone TABLE Killing of Staphylococcus albus
Macrophage donors Expt no.
Strain
Age (months)
1
by Macrophages from Young and Old NZB and BALB/c (Bactericidal Activity)
0 min”
30 min
60 min
Difference during the first 30-min interval cm
No. of viable bacteria/ macrophage after
Mice
Difference during 60-min interval (%I
Relative increase in bactericidal activity* (W
1
NZB NZB BALBlc BALBlc
2 17 2 17
15.8 17.2 15.3 14.0
13.7 6.5 14.2 11.0
11.0 4.9 25.2 17.9
- 13.3 -62.6 -7.2 -21.5
-30.4 -71.6 64.7 27.8
95.1 99.4
2
NZB NZB BALBlc BALBlc
2 17 2 17
13.4 7.2 6.0 5.0
10.3 3.3 6.4 4.2
12.1 4.0 10.4 9.0
-23.2 -54.2 6.6 -16.0
-9.8 -44.5 73.3 80.0
83.1 124.5
a Number of engulfed bacteria. b Compared with BALB/c mice of the same age group.
MACROPHAGE
FUNCTIONS TABLE
2
Number of CFC in Normal and Thioglycolate-Stimulated
Young and Old NZB and BALBic
Normal” Expt no.
Mice
Age (months)
Colonies/lOG spleen cells
31.5
IN NZB MICE
Mice
Thioglycolate”
Colonies/105 bone marrow cells
ColoniesilO” spleen cells
Colonies/IO” bone marrow cells
1
NZB NZB BALBic BALBlc
2 12 2 12
421 10 c 2 220 351
982 144t 7Ok 105k
7 2 0 1
8t2 17 t 3 821 221
97t 8 136 k 10 852 4 85t 5
2
NZB NZB BALB/c BALBlc
2 12 2 12
33 85 10 10
175 k 28 1802 0 1002 7 785 5
320 32 +- 3 3+-l 521
113 178 92 93
-t ? + f
5 7 2 3
t 12 + 30 ?Y31 f 18
n The values in the table are means of tetraplicates _f SD.
marrow of the corresponding BALB/c mice. Spleen cells of young and old NZB mice also showed a much higher number of CFC (by up to three- and eightfold) than did the spleen cells of age-matched BALB/c mice. Usually, the CFC counts of both spleen and bone marrow increase significantly with age in NZB but not in BALB/c mice. Following thioglycolate stimulation, the CFC number in the bone marrow and the spleen of old NZB was always higher than that in old BALB/c mice. S.ynthesis of colony-stimulating factor (CSF). In vitro synthesis of CSF by normal macrophages from two age groups of NZB and BALB/c mice was assessed in the following manner. Conditioned medium (CM) from these cells was added to bone marrow of 2-month-old BALB/c mice in soft agar culture. The resulting colony counts showed that CSF produced by macrophages from 2- and ICmonth-old NZB mice gave 1.4 and 1.8 times more CFC than that from macrophages of matched groups of BALB/c mice, respectively (Table 3). DISCUSSION To provide some insight into the relationship autoimmune disease, where T cell deficiency TABLE Synthesis of Colony-Stimulating
between macrophage functions and was described, we studied several
3
Factor (CSF) by Macrophages from NZB and BALB/c Mice
Origin of CSF Strain
Age (months)
NZB NZB BALBlc BALBlc ” Bone marrow cells of 2-month-old
2 14 2 14 BALB/c mice.
Number of colonies per lo5 cells” 156 2 217 t 114 t 1202
11 11 10 2
316
BAR-ELI
AND
GALLILY
activities of macrophages derived from an autoimmune strain of mouse (NZB) compared with macrophages from a nonautoimmune strain of mouse (BALB/c). There was no significant difference in the activity of acid phosphatase between macrophages from NZB mice and those from age-matched BALB/c mice. The level of /3-glucuronidase was found to be higher by twofold in the NZB macrophages from young and old mice when compared with BALBlc macrophages from mice of the same age groups. The activities of cathespin D and lysozyme were found to be approximately twofold higher in the NZB macrophages than in BALB/c cells from mice of the same age groups. Similarly, the activity of the DNase enzyme in macrophages from NZB mice was markedly higher than that in macrophages from BALB/c mice of the same age group, and it increased with the age of NZB macrophage donors. It is suggested that the activity of DNase was potentiated by an increased number of damaged cells in NZB mice following the appearance of autoantibodies to nucleic acid (1). This enzyme probably plays a significant role in degrading DNA of the engulfed damaged cells. The uptake of 125-I labeled S. JEexneri and 1251-labeled S. albus by macrophages derived from 2-, 6-, and 16-month-old NZB mice was significantly higher than by macrophages of age-matched groups of BALB/c mice. In addition, a marked potentiation of phagocytic activity was noted in macrophages from 6- and 16-month-old NZB mice compared with cells from 2-month-old animals. A similar result was obtained by Thomas and Weir (24) using 1251-labeled bovine serum albumin as an antigen. Our data also shown that macrophage bactericidal activity against S. albus was significantly greater in cells from young and old NZB mice than in those from age-matched BALB/c mice. The low number of surviving intracellular bacteria in macrophages of old NZB mice suggests that the bactericidal activity of NZB macrophages increased with age. The techniques described here may not be adequate to rule out the binding of bacteria on macrophage surfaces. Although microscopic examination suggested that many of the phagocytized bacteria were localized within the macrophages, we could not exclude the possibility that some of the organisms were instead firmly attached to their surface. In two experiments, we tried to overcome this difficulty by adding lysostaphin (100 U/ml for 10 min at 37”C), which is known for its rapid killing of staphylococci, to our macrophage monolayers immediately after terminating their incubation with test bacteria. After incubating our cultures with lysostaphin for 10 min at 37”C, the findings were identical to those seen in the untreated cells. Our results demonstrated that two macrophage functions, phagocytosis and antibacterial activity, crucial in the nonspecific antimicrobial defense mechanism, were potentiated in old NZB mice carrying autoimmune disease. Thus, the decreased functions of T cells in old NZB mice (4, 8, 10, 25) did not decrease the antibactericidal activity of their macrophages, although potentiation of intracellular bactericidal activity of macrophages is usually attributed to macrophage activation by T cells (26). The following assumptions might explain our results: (i) A subset of the T cell population that activates macrophages still resides in old NZB mice; (ii) macrophages might be activated by B cell mediators; and (iii) potentiation of macrophage antibactericidal activity is an autonomous feature of these cells. The number of macrophage/granulocyte progenitors or colony-forming cells (CFC) was determined in bone marrow and spleen cells from unstimulated and thioglycolate-stimulated NZB and BALB/c mice of two age groups. Assuming that
MACROPHAGE
FUNCTIONS
IN NZB
317
MICE
each colony is produced from one stem cell (23), we found that the number of CFC in bone marrow and spleen from unstimulated normal NZB mice was higher than that in age-matched BALB/c mice by two- to eightfold. The number of CFC in both spleen and bone marrow of NZB mice usually increased significantly with age by 2.5- and 1.5-fold, respectively. In BALB/c mice, we noted no increase and sometimes even observed a decrease with age. The higher number of macrophage/granulocyte progenitor cells in NZB mice, especially in the older mice, provides the animal with a potential source of cells that participate in defense functions required for the animal survival. In addition, the increase in vifro synthesis of CSF by macrophages from old NZB mice might also supply old NZB mice with an active stimulus for multiplication of macrophage progenitor cells. It is suggested that the increased number of CFC, as well as the potentiation of macrophage activities, especially in old NZB mice, develops to compensate for the deficiency of T cell function, thus assisting in NZB surveillance. ACKNOWLEDGMENT This study
was supported
by research
grants
from
Concern
Foundation
REFERENCES 1. Siegel. B.C.. Accinni, L., Andres, G. A., Beiser, R. M.. Chritian. C. L., Erlanger. K. C., J. E~rprp. Mrd. 130, 203, 1969. 9, 21.5, 1968. 2. Howie. J. B., and Helyer, B. J., Advan. Immune/. 3. Shirai. T., and Mellors, R. C., Proc. Nat. Acud. Sci. 68, 1412, 197 I. 4. Staples, P. J., and Talal, N.. J. Exp. Med. 129, 123, 1969. 5. Playfair, J. H. L., Immunology 15, 35, 1968. 6. Gazdar, A. F.. Beitzel, W., and Talal, N., C/in. Exp. fmmunol. 8, 501. 1971. 7. Dauphinee, M. J., and Talal, N., J. Immunol. 114, 1713, 1975. 8. Gelfand, M. D., and Steinberg, A. D., J. Immunol. 110, 1652, 1973. 9. Gerber, N. L., Hardin, J. A., Chused, T. M., and Steinberg, A. D., J. Immunol. IO. Leventhal, B. G., and Talal, N.. J. Immunol. 104, 918, 1970. Il. Geiger, B.. and Gallily, R., Clin. Exp. Immunol. 16, 643, 1974. 12. More, R., Yron, I., Ben Sasson. Z., and Weiss, D. W., Cell. Immunol. 15, 382. 18, 317, 1975. 13. Bar-Eli, M., and Gallily, R., J. ReticuloendotheI. SW. 14. Gallily R., and Eliahu, H., fmmuno/og?: 26, 603. 1974. 15. Beck, C., Mahadeven. S., Brightwell, R.. Dillard. C. J., and Tappel, A. L., Biophys. 128, 369, 1968. 16. Barrett, A. I., Biochem. J. 104, 601, 1967. 17. Lahav, M.. Neeaman, N., Adler, E., and Ginsburg, I., J. InfE<.i. 0i.5. 129, 528. 18. Kunitz, M., J. &II. Pi~ysi~l. 33, 349, 1950. 19. Carpenter, R. R., .I. Immunol. 96, 992, 1966. 33, 121, 1977. 20. Schroit, A. J.. and Gallily, R., Immunology Immun. 18, 405, 1977. 21. Gallily, R., Douchan, Z., and Weiss, D. W., Injkr. 22. Apte. R. N.. Chanita, F., Hertogs, and Pluznik, D. H., J. Immunol. 118, 1435, 23. Pluznik. D. H.. and Sachs. L., E-VP. Cell Res. 43, 553. 1966. 12, 263, 1972. 24. Thomas, H. I. J., and Weir, D. M., C/in. E,rp. Immunol. 25. Talal, N., Progr. C/in. Immunol. 2, 201. 1975. 26. Mackaness, G. B., J. Exp. Med. 129, 973. 1969.
F. F.. and Hsu.
113,
161X.
1974.
1975.
Arch.
1974.
1977.
Biot,/rr,m.
IMMUNOLOGY
45, 309-317 (1979)
Age-Dependent Macrophage Functions New Zealand Black Mice MENASHE The Laurenberg
Centerfbr
Tumor
in
BAR-ELI AND RUTH GALLILY and General Immunology, Hebrew, School, Jerusalem, Israel Received
October
University-Hadassah
Medical
30. 1978
Various functions of macrophage derived from young (2-month-old) and old (14- to 17-month-old) New Zealand Black (NZB) mice with autoimmune disease were studied and compared with macrophage functions of age-matched BALB/c mice. Macrophages from young and old NZB mice demonstrated elevated levels of P-glucuronidase, cathepsin D, lysozyme, and DNase compared with those from age-matched BALB/c. DNase activity in the macrophages of NZB mice significantly increased with age. Macrophages from young and old NZB mice had greater phagocytic capacity for both ‘Y-labeled Shigella j?exneri and Staphylococcus albus than did BALB/c macrophages. NZB macrophages from both young and old mice had higher bactericidal activity against S. albus than those from age-matched BALBic mice. The number of macrophage/granulocyte colony-forming cells (CFC) in both bone marrow and spleen was markedly higher in young and old NZB mice than in BALBic mice. Colony-stimulating factor (CSF) released by macrophages derived from NZB mice had higher CFC activity than that released from macrophages of age-matched BALBic mice. In NZB mice, the CSF activity significantly increased with age. It is suggested that potentiation of macrophage number and activity compensates for the deficiency of T cell functions in NZB mice with autoimmune disease.
INTRODUCTION
The NZB strain of mouse has been used by immunologists as an experimental model for the study of the immunopathology of autoimmune disease. NZB mice are presumed to be genetically predisposed to spontaneous development of a wide range of autoantibodies including antibodies to nucleic acids (I), erythrocytes autoantibodies (2), and natural thymocytoxic antibodies (3). The nature of the abnormal activity of the immunologic mechanism has yet to be fully elucidated. NZB mice are known to be hyperresponsive in humoral reaction to protein antigens (4, 5), but are also known to have depressed cellular immunity. Early in life, they manifest immune hyperresponsiveness (5,6) as well as a defect in suppressor cell function (7). Later in life, the T cell function is depressed, including T cell-dependent antibody production (4), skin allograft rejection (8), induction of graft-versus-host disease (9), and response to T cell mitogens such as phytohemagglutinin (10). Although the important role of macrophages in defense and immune reactions is well documented, little is known about their functions in NZB mice. In the present investigation, we studied enzyme levels, phagocytosis, and batericidal activities. In 309 0008-8749/79/080309-09$02.00/0 Copyright Ail rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
310
BAR-ELI
AND
GALLILY
addition, we determined the number of macrophage/granulocyte progenitors in bone marrow and in spleen. Our aim was twofold: We hoped to find out, first whether changes in macrophage activities occurred during spontaneous development of autoimmune disease and, second, whether these changes might explain some characteristics of autoimmune disease in NZB mice. Our findings demonstrated that macrophages derived from either young (Zmonth-old) or old (14to 17-month-old) NZB mice were more active in various functions than those derived from age-matched BALB/c mice. Several macrophage activities showed markedly higher values in older NZB mice than in younger animals of the same strain. MATERIALS
AND METHODS
Female NZB and BALB/c mice were obtained from the Experimental Animal Unit of the Weizmann Institute of Science, Rehovot, Israel. For each experiment, we selected mice of the same sex and age (within the range of 2 to 17 months) from both strains of mouse. Harvesting and culture of macrophages. Cells were obtained by washing the peritoneal cavities of donor animals with 5 ml RPMI-1640 medium. The yield per normal mouse was about 3 to 5 x 106, 30 to 40% of which were macrophages. The cells were suspended in the same medium supplemented with 2% fetal calf serum (FCS) (Microbiological Associates, Bethesda, Md.) and then cultivated in 30-mm plastic petri dishes (Nunc, Denmark) at 1 to 2 x lo6 cells per dish or in 6-mm-well tissue culture microtiter plates (Nunc, Denmark) at 3 x lo5 cells per well. After 2 to 3 hr of incubation at 37°C in a humidified atmosphere containing 5% COz, the nonadhering cells were removed by intensive rinsing with phosphate-buffered saline (PBS), and fresh medium was added. More than 90% of the remaining adherent cells were defined as macrophages by both morphologic and phagocytic criteria. The number of cells adherent to the 30-mm petri dishes was determined by counting these cells (1 l), while the number of cells adherent to the 6-mm wells was determined by a modification of the Nq5Cr0,-uptake method as described by More et al. (12). Enzyme assays. Lysates of macrophage monolayers were prepared as previously described (13, 14) and assayed for activity of four hydrolytic enzymes: (i) acid phosphatase by hydrolysis of p-nitrophenyl phosphate; (ii) j3-glucuronidase by hydrolysis of p-nitrophenyl-/3-D-glucuronide (Sigma Chemical Co.) (15); (iii) cathepsin D by hydrolysis of 8% hemoglobin (16); and (iv) lysozyme activity by hydrolysis of Micrococcus lysodeikticus (17). DNase activity was assayed by a modification of Kunitz’s method (18). The assay was based on the increased uv absorption of 260 nm, during the course of the depolymerization of DNA (Sigma) by DNase. The enzyme activity unit was defined as the activity which increases the absorbency by 0.001 per ml at 25°C. Phagocytic assay. Alcohol- or glutaraldehyde (GA)-killed bacteria were fixed with 0.25% GA and iodinated with lz51-labeled Na (carrier free, Amersham, England) (19); 2 mCi isotope was used for the labeling of 400 mg bacteria. The final intensity of labeling for both Shigella flexneri and Staphylococcus albus was 50 bacteria per cpm. 1.25 x 10’ labeled bacteria (2.5 x lo5 cpm) were added to the macrophage cultures in tissue culture microtiter plates (6-mm well diameter, lo5 Animals.
MACROPHAGE
FUNCTIONS
IN NZB
MICE
311
cells/well), giving a ratio of 100 bacteria to 1 cell. The cultures were incubated for various periods of time, then washed, and the wells were cut apart. Phagocytosis was assessed by counting each well in a Packard 5110 scintillation counter (20). Bactericidal assay. A saline suspension of S. albus in the log phase of growth (2 1) was added to macrophage cultures in microplates at a ratio of 100 bacteria to 1 cell. After incubation for 30 min at 37°C the nonphagocytosed bacteria were removed by intensive rinsing, and the macrophage monolayers were incubated for an additional 30 and 60 min. The cells were then washed and lysed for 3 min with 0.1% Triton X- 100. The number of viable staphylococci was determined by plating appropriate dilutions of the lysate in brain-heart agar. The colonies were counted after incubation of the agar plates for 24 hr at 37°C. Cell cloning. Cloning of bone marrow and spleen cells in double-layer agar culture was performed according to Apte et al. (22). The source of colonystimulating factor (CSF) was serum from BALB/c mice bled 6 hr after i.v. injection of lipopolysaccharide (LPS) (10 pg/mouse), diluted 1:4 and added to the cultures at a final concentration of 10%. This diluted serum served as a conditioned medium (CM). Bone marrow cells ( 105) or spleen cells ( 106) were cloned in soft agar medium (0.37%) supplemented with 10% CSF, plated over firm agar (0.5%). Triplicate plates were incubated in 5% CO, at 37°C.for 8 days, and the number of colonies (>40 cells per colony) in the upper layer was determined. It has been shown that each colony growing in soft agar represents a colony-forming cell (CFC) (23). By this technique we determined the number of macrophage/granulocyte progenitors cells, and it was expressed as the number of CFC. Synthesis ofcolony-stimulatingfactor (CSF). Peritoneal cells (5 x 105/ml/plate) were cultured for 2 hr in 30-mm petri dishes in RPMI- 1640 medium supplemented with 5% FCS. Following intensive rinsing, the adherent cells (90 to 95% of which were macrophages) were further incubated for 4 days in serum-free RPM1 medium with the addition of LPS (50 pg/ml/plate). The supernatant fluids were then collected, centrifuged (175g for 10 min), and served as conditioned medium (CM) for CFC assay. The CFC assay was performed on bone marrow cells of young BALBic mice as previously described. RESULTS Lysosomal enzymes. The activities of acid phosphatase, /3-glucuronidase, cathepsin D, and lysozyme were determined in macrophage monolayers consisting of cells derived from four age groups of unstimulated NZB and BALB/c mice. It was found (Figs. la-d) that the activity of acid phosphatase in macrophages derived from age-matched groups of NZB and BALB/c mice was not significantly different (P > 0.1). The activity of ,@glucuronidase was higher by about twofold only in NZB macrophages from young (2- to 3-month-old) and old (16-month-old) mice when compared with BALB/c macrophages from the same age groups (P < 0.001). Similarly, lysozyme activity in the cells was higher by twofold in NZB macrophages from young (2- to 3-month-old) and old (IQmonth-old) mice than in BALB/c macrophages. However, the level of cathepsin D was higher by 1.3- to 2.5-fold in NZB macrophages from the four age groups when compared with the matched groups of BALB/c macrophages (P < 0.005). It was also noted (Fig. 1) that
312
BAR-ELI
a
Acid
Age
c
b
Dhosohatase
(months)
Catheosin
Age
AND GALLILY
(months)
NZB
0
BALE/c
m
j3 - glucuronidase
Age
D
(months)
Lvsozvme
Age
(months)
FIG. 1. Specific enzyme activity of (a) acid phosphatase, (b) P-glucuronidase, (c) cathepsin D, and (d) lysozyme in macrophages derived from various age groups of NZB (0) and BALB/c (m) mice. (Each point represents the mean of six to nine determinations *SD).
macrophages from old NZB mice possessed a significantly higher activity of acid phosphatase than did cells of young NZB mice (P < O.OOl), whereas the activities of P-glucuronidase and cathepsin D showed little difference in macrophages between the old and young NZB mice. DNase activity. The activity of DNase was determined in macrophages derived from various age groups of unstimulated NZB and BALB/c mice. It was found (Fig. 2) that DNase activity was higher by 9-fold in macrophages from l- to 3-month-old NZB mice than in macrophages from BALB/c mice of the same age (P < 0.001). The activity of DNase was higher by 2%fold in macrophages from old (12- to 16-month-old) NZB mice than in microphages from old BALB/c mice (P < 0.001). It was also noted (Fig. 2) that macrophages from old NZB mice possessed significantly higher DNase activity than those from young NZB mice (P < 0.01). Uptake of labeled bacteria. The uptake of S. flexneri and S. albus by young, adult, and old NZB and BALB/c macrophage monolayers was determined after various periods of incubation. As can be seen in Figs. 3 and 4, the NZB macrophages derived from 2-, 6-, and 16-month-old mice demonstrated a higher capacity to
MACROPHAGE
FUNCTIONS
IN NZB MICE
313
DNase NZB
0
BALE/C
m
al6. 5. 4. 3-
&
2I. 1-3
a
12Age
i
1-3
a
12-16
(months)
FIG. 2. DNase activity in macrophages derived from various age groups of NZB (0) and BALB/c (a) mice. (Each bar represents the mean of three to nine determinations 2 standard error.)
phagocytize both S. flexneri and S. albus than BALB/c macrophages from mice of the same age groups (P < 0.005) at 15, 30, and 60 min. These results were also obtained at a 1:50 and a 1:25 bacteria to cell ratio (unpublished data). It should be noted that the phagocytic ability of NZB macrophages derived from 6- and 16-month-old mice increased significantly from that of cells from 2-month-old mice (P < 0.005). On the other hand, the phagocytic activity of BALB/c cells showed little change with age, and a decrease was even noted in the older BALB/c mice. Bactericidal activity. Survival of phagocytized S. albus was assessed 30 and 60 min following their engulfment by macrophages from various age groups of NZB and BALB/c mice. As shown in Table 1, macrophages derived from young and old
FIG. 3. Uptake of 1251-Iabeled Shigelln jkrneri by macrophages derived from 2-month-old (0). 6-month-old (0), and 16month-old (A) mice of NZB (a) and BALBic (b) mice. (A representative experiment of three performed; each point represents the mean of triplicate determinations.)
314
BAR-ELI
AND GALLILY
.
2
o
6 months
months
.
16months
Fro. 4. Uptake of rZ51-labeled S. a/bus by macrophages derived from 2-month-old (O), 6-month-old (0), and ldmonth-old (A) mice of NZB (a) and BALBlc (b) mice. (A representative experiment of three performed; each point represents the mean of triplicate determinations.)
NZB mice had a higher bactericidal capacity than BALB/c macrophages from the same age groups. The number of intracellular bacteria decreased in macrophages of NZB mice during 60 min, while an increase in the number of bacteria was noted in macrophages derived from BALB/c mice. Colony-forming cells (CFC) response. The number of colony-forming cells (CFC) was determined in normal and thioglycolate-stimulated mice by cloning bone marrow and spleen cells from two age groups of NZB and BALB/c mice (Table 2). The number of CFC in bone marrow of young and old normal NZB mice was higher by up to 1.7- and 2.3-fold, respectively, than the number of CFC from normal bone TABLE Killing of Staphylococcus albus
Macrophage donors Expt no.
Strain
Age (months)
1
by Macrophages from Young and Old NZB and BALB/c (Bactericidal Activity)
0 min”
30 min
60 min
Difference during the first 30-min interval cm
No. of viable bacteria/ macrophage after
Mice
Difference during 60-min interval (%I
Relative increase in bactericidal activity* (W
1
NZB NZB BALBlc BALBlc
2 17 2 17
15.8 17.2 15.3 14.0
13.7 6.5 14.2 11.0
11.0 4.9 25.2 17.9
- 13.3 -62.6 -7.2 -21.5
-30.4 -71.6 64.7 27.8
95.1 99.4
2
NZB NZB BALBlc BALBlc
2 17 2 17
13.4 7.2 6.0 5.0
10.3 3.3 6.4 4.2
12.1 4.0 10.4 9.0
-23.2 -54.2 6.6 -16.0
-9.8 -44.5 73.3 80.0
83.1 124.5
a Number of engulfed bacteria. b Compared with BALB/c mice of the same age group.
MACROPHAGE
FUNCTIONS TABLE
2
Number of CFC in Normal and Thioglycolate-Stimulated
Young and Old NZB and BALBic
Normal” Expt no.
Mice
Age (months)
Colonies/lOG spleen cells
31.5
IN NZB MICE
Mice
Thioglycolate”
Colonies/105 bone marrow cells
ColoniesilO” spleen cells
Colonies/IO” bone marrow cells
1
NZB NZB BALBic BALBlc
2 12 2 12
421 10 c 2 220 351
982 144t 7Ok 105k
7 2 0 1
8t2 17 t 3 821 221
97t 8 136 k 10 852 4 85t 5
2
NZB NZB BALB/c BALBlc
2 12 2 12
33 85 10 10
175 k 28 1802 0 1002 7 785 5
320 32 +- 3 3+-l 521
113 178 92 93
-t ? + f
5 7 2 3
t 12 + 30 ?Y31 f 18
n The values in the table are means of tetraplicates _f SD.
marrow of the corresponding BALB/c mice. Spleen cells of young and old NZB mice also showed a much higher number of CFC (by up to three- and eightfold) than did the spleen cells of age-matched BALB/c mice. Usually, the CFC counts of both spleen and bone marrow increase significantly with age in NZB but not in BALB/c mice. Following thioglycolate stimulation, the CFC number in the bone marrow and the spleen of old NZB was always higher than that in old BALB/c mice. S.ynthesis of colony-stimulating factor (CSF). In vitro synthesis of CSF by normal macrophages from two age groups of NZB and BALB/c mice was assessed in the following manner. Conditioned medium (CM) from these cells was added to bone marrow of 2-month-old BALB/c mice in soft agar culture. The resulting colony counts showed that CSF produced by macrophages from 2- and ICmonth-old NZB mice gave 1.4 and 1.8 times more CFC than that from macrophages of matched groups of BALB/c mice, respectively (Table 3). DISCUSSION To provide some insight into the relationship autoimmune disease, where T cell deficiency TABLE Synthesis of Colony-Stimulating
between macrophage functions and was described, we studied several
3
Factor (CSF) by Macrophages from NZB and BALB/c Mice
Origin of CSF Strain
Age (months)
NZB NZB BALBlc BALBlc ” Bone marrow cells of 2-month-old
2 14 2 14 BALB/c mice.
Number of colonies per lo5 cells” 156 2 217 t 114 t 1202
11 11 10 2
316
BAR-ELI
AND
GALLILY
activities of macrophages derived from an autoimmune strain of mouse (NZB) compared with macrophages from a nonautoimmune strain of mouse (BALB/c). There was no significant difference in the activity of acid phosphatase between macrophages from NZB mice and those from age-matched BALB/c mice. The level of /3-glucuronidase was found to be higher by twofold in the NZB macrophages from young and old mice when compared with BALBlc macrophages from mice of the same age groups. The activities of cathespin D and lysozyme were found to be approximately twofold higher in the NZB macrophages than in BALB/c cells from mice of the same age groups. Similarly, the activity of the DNase enzyme in macrophages from NZB mice was markedly higher than that in macrophages from BALB/c mice of the same age group, and it increased with the age of NZB macrophage donors. It is suggested that the activity of DNase was potentiated by an increased number of damaged cells in NZB mice following the appearance of autoantibodies to nucleic acid (1). This enzyme probably plays a significant role in degrading DNA of the engulfed damaged cells. The uptake of 125-I labeled S. JEexneri and 1251-labeled S. albus by macrophages derived from 2-, 6-, and 16-month-old NZB mice was significantly higher than by macrophages of age-matched groups of BALB/c mice. In addition, a marked potentiation of phagocytic activity was noted in macrophages from 6- and 16-month-old NZB mice compared with cells from 2-month-old animals. A similar result was obtained by Thomas and Weir (24) using 1251-labeled bovine serum albumin as an antigen. Our data also shown that macrophage bactericidal activity against S. albus was significantly greater in cells from young and old NZB mice than in those from age-matched BALB/c mice. The low number of surviving intracellular bacteria in macrophages of old NZB mice suggests that the bactericidal activity of NZB macrophages increased with age. The techniques described here may not be adequate to rule out the binding of bacteria on macrophage surfaces. Although microscopic examination suggested that many of the phagocytized bacteria were localized within the macrophages, we could not exclude the possibility that some of the organisms were instead firmly attached to their surface. In two experiments, we tried to overcome this difficulty by adding lysostaphin (100 U/ml for 10 min at 37”C), which is known for its rapid killing of staphylococci, to our macrophage monolayers immediately after terminating their incubation with test bacteria. After incubating our cultures with lysostaphin for 10 min at 37”C, the findings were identical to those seen in the untreated cells. Our results demonstrated that two macrophage functions, phagocytosis and antibacterial activity, crucial in the nonspecific antimicrobial defense mechanism, were potentiated in old NZB mice carrying autoimmune disease. Thus, the decreased functions of T cells in old NZB mice (4, 8, 10, 25) did not decrease the antibactericidal activity of their macrophages, although potentiation of intracellular bactericidal activity of macrophages is usually attributed to macrophage activation by T cells (26). The following assumptions might explain our results: (i) A subset of the T cell population that activates macrophages still resides in old NZB mice; (ii) macrophages might be activated by B cell mediators; and (iii) potentiation of macrophage antibactericidal activity is an autonomous feature of these cells. The number of macrophage/granulocyte progenitors or colony-forming cells (CFC) was determined in bone marrow and spleen cells from unstimulated and thioglycolate-stimulated NZB and BALB/c mice of two age groups. Assuming that
MACROPHAGE
FUNCTIONS
IN NZB
317
MICE
each colony is produced from one stem cell (23), we found that the number of CFC in bone marrow and spleen from unstimulated normal NZB mice was higher than that in age-matched BALB/c mice by two- to eightfold. The number of CFC in both spleen and bone marrow of NZB mice usually increased significantly with age by 2.5- and 1.5-fold, respectively. In BALB/c mice, we noted no increase and sometimes even observed a decrease with age. The higher number of macrophage/granulocyte progenitor cells in NZB mice, especially in the older mice, provides the animal with a potential source of cells that participate in defense functions required for the animal survival. In addition, the increase in vifro synthesis of CSF by macrophages from old NZB mice might also supply old NZB mice with an active stimulus for multiplication of macrophage progenitor cells. It is suggested that the increased number of CFC, as well as the potentiation of macrophage activities, especially in old NZB mice, develops to compensate for the deficiency of T cell function, thus assisting in NZB surveillance. ACKNOWLEDGMENT This study
was supported
by research
grants
from
Concern
Foundation
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