Int. J. lmmunopharmac., Vol. I, pp. 197-203 Pergamon Press Ltd. 1979, Printed in Great Britain.
EFFECTS OF LATEX PARTICLES ON IMMUNE RESPONSES OF THE MURINE HOST AND ON MACROPHAGES IN VITRO* (I) TOMII(O TANAKA,t KAVO TAKAYA,I" TAKEHIKO KUNIMOTO,,'I:and HIROYASU BABA~ ~Department of Pharmacology and :t Department of Chemotherapy, National Cancer Center Research Institute, Tsukiji 5-l-l, Chuo-ku Tokyo 104, Japan (Received 18 January 1979 and in final form 17 May 1979)
Abstract--Previously we reported that the antitumor effect of BCG was found to be negated when mice were injected intraperitoneally with latex particles. The same results were found when mice were injected with anti-macrophage serum (AMS). However, neither latex particles nor AMS had any influence on the effectiveness of Propionibacterium aches. In order to assess the consequences in vivo of latex particle administration in normal mice, we determined, at varying intervals, the effect of latex particles on inflammatory cell accumulation in the peritoneal cavity, the effect on delayed hypersensitivity-type skin reaction induced by phytohemagglutinin, and the effect on carbon clearance. Results were as follows: (I) the cell accumulation in an inflammatory site was lowered by as much as 30o70 5 days after the latex administration and the low level persisted on day 10. (2) The inflammatory skin reaction to phytohemagglutinin in mice injected with latex particles was also depressed to less than 50°70 of normal for 14 days. (3) The phagocytic capacity of the mice treated with latex decreased to about 50o70 of normal for 10 days. The effect of latex particles on lysozyme excretion from macrophages cultured in vitro is also reported. After an excessive phagocytic load of latex particles, macrophages detached from the plastic and the number of remaining cells was one-third of that of control cultures. The lysozyme excretion was totally suppressed for the first 2 days, but was 10GT0of control values by day 6. Latex particles appear to be useful for evaluating the rSle of macrophages in immunological studies, since various functions of macrophages in vivo and in vitro, were blocked for a long period without toxicity.
Subsequent to the development of the guinea-pig model for cancer immunotherapy (Zbar, Bernstein & Rapp, 1971; Zbar, Bernstein, Tanaka & Rapp, 1970), we examined the therapeutic efficacy of intradermal inoculation of tumor cell-BCG vaccine in syngeneic mice (Tanaka & Saito, 1975; Tanaka & Sasaki, 1974; Tanaka & Tokunaga, 1971; Tokunaga, Kataoka, Nakamura, Y a m a m o t o & Tanaka, 1973). Additionally, since the antitumor activity of Propionibacterium aches (P. aches) had also been reported (Fujita & Saino, 1971; Hattori & Mori, 1973; Kato, Nakagawa, A k i m o t o , Fujita, Tanaka & Kimura, 1976), we established the therapeutic efficacy of P. aches-tumor cell vaccine. More recently, we made a comparative study of the cellular basis of BCGmediated and P. aches-mediated tumor killing by the use of anti-thymocyte serum (ATS), anti-macrophage serum (AMS) and latex particles (Tanaka, Kato, Nakagawa, Fujita & Kimura, 1978) which are taken up by and may cause damage to, macrophages (Allison & Hart, 1968; Zisman, Hirsch & Allison, 1970). The results showed that intraperitoneal administration of latex particles (40 rag) 2 h before
vaccine inoculation, as in the case of A M S treatment, negated the immunotherapeutic efficacy of BCG (both viable and nonviable). However, neither treatment affected the antitumor efficacy of P. acnes. The study reported here was designed to determine whether intraperitoneal latex particles would be an effective treatment for blocking various host immune responses in normal mice, that is, to assess the in vivo consequences of administering latex particles by: (l) counting the number of peritoneal exudate cells accumulated after intraperitoneai injection of phytohemagglutinin (PHA), (2) measuring the increment of the footpad thickness elicited by P H A injection as a delayed hypersensitivity-type reaction and (3) examining the phagocytic capacity of mice at varying intervals (generally, on days l, 5 and 10) after the injection of latex particles. We also examined whether latex particles would cause any alteration of macrophage function in vitro, such as secretion of lysozyme which is a cell-specific marker for macrophages and polymorphonuclear cells. For this purpose, the lysozyme secretion of macrophages cultured in vitro was measured after 2
* Research supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan. 197
T. TanaKa et al.
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and 16 h exposure to latex particles, and was compared to that of control macrophages. EXPERIMENTAL PROCEDURES
Animals. Young adult syngeneic mice (SWM/Ms, C57BL/6, and C3H/HeN) were used. Latex particles. A suspension of uniform polystyrene particles (diameter, 1.0 ~m), obtained f r o m the Dow Chemical Co., U.S.A., was sterilized at 65°C for 40 min and mice were injected intraperitoneally in doses of 4, 10, 40 or 100 mg per adult mouse. Phytohemagglutinin. Purified PHA (PHA-P) was purchased from Difco Laboratories, U.S.A. The lyophilized powder was reconstituted to 10 mg/ml in water, kept cold, and diluted with normal saline solution for use.
Assay for leucocyte accumulation in peritoneal cavity (Snyderman, Pike, Blaylock & Weinstein, 1976). The diluted PHA-P (50 ~g/0.5 ml/mouse) was injected intraperitoneally in order to induce a delayed hypersensitivity-type of inflammatory reaction. Forty-eight hours after the injection, mice were sacrificed by cervical dislocation. Peritoneal cells were collected by intraperitoneal injection of 5 ml of Hanks' balanced salt solution (BSS) containing 5 unit/ml of sodium heparin. These cells were combined with two other washings of 3 ml Hanks' BSS. Peritoneal fluid was withdrawn through a small incision in the abdominal wall and centrifuged for 10 min at 1000 r.p.m, at 4°C. The cell pellet was then resuspended in Hanks' BSS without heparin and the total cell number was counted using a hemocytometer. The number of animals per group varied from 3 to 6.
Assay for delayed hypersensitivity-type skin reaction. PHA-P (50 /~g/0.05 ml/mouse) was injected into a rear footpad and, as a control, physiological saline (0.05 mi) was injected into the other rear footpad. The increment in footpad thickness was measured with a Peacock dial gauge (Ozaki Co., Tokyo) before and 20-24 h after PHA-P injection, since the time for maximum footpad swelling was 20-24 h after the PHA-P injection and returned to near normal size in 72 h. The mean increase in thickness in control footpad was little or no change. The number of mice per group varied from 2 to 6 and results were expressed as the mean increment in footpad thickness (in ram) for a group. Assay of phagocytic capacity (Watanuki, Dohi, Saito, Miura & Suzuki, 1965). Phagocytic capacity was tested by carbon clearance, using 20 mg/ml Pelikan Ink (C 11/ 1431 a, GUnther Wagner PelikanWerke, Hannover, West Germany), injected into the tail vein in a dose of 1 ml/100 g body weight. Blood
was drawn from the retro-orbital sinus at 2, 5 and 8 rain after carbon injection; 0.02 ml of each blood sample was pipetted into 1.5 ml of 0.1% of Na2CO ~ solution; optical density was read with a spectrophotometer at 660 nm. Under these conditions, the rate of clearance (K) is an expotential function of time, according to the equation K=(log C I - l o g C2)/(T2- T I) where C~ and C 2 represent carbon concentrations in the blood at time T~ and T2, respectively. Two- four animals per group were used. Assay of lysozyme activity (Gordon, Todd & Cohn, 1974; Parry, Chandan & Shahani, 1965). Lysozyme activity was determined by measuring the initial rate of lysis of a suspension of Micrococcus lysodeikticus (0.15 mg/ml in M/15 phosphate buffer, pH 6.25). The decrease of absorbance at 450 nm was recorded every 20 s for 3 min and the slope of the initial linear portion was compared to the standard curve prepared with known concentrations of egg white lysozyme. Phagocytosis in vitro. A known number of cells (1.2×106 cells/dish) lavaged from the peritoneal cavity of normal mice (SWM/Ms) were allowed to adhere to plastic dishes for 2 h at 37°C. After the dishes were washed vigorously with Hanks' BSS to remove nonadherent cells, the remaining monolayers of macrophages (2.4× 105/dish) were cultivated in RPMI-1640 medium supplemented with 10o70 fetal bovine serum. Latex particles were added at a concentration of 0.1°70. Within a 2 h period of phagocytosis, more than 95o7o of the macrophages had ingested the particles. Excess latex was removed after 2 and after 16 h, and the cells were cultivated in a fresh medium at 37°C in a COz-incubator. Aliquots of the medium were removed at intervals and lysozyme activity was determined. RESULTS
Effect of latex particles on PHA-induced inflammatory cell accumulation in the peritoneal cavity of normal mice Table I shows the kinetics of inflammatory cell accumulation in the peritoneal cavity of normal mice injected with 50/~g of PHA. Because of this result and others (Snyderman, et al., 1976), we made the total cell count at 48 h after PHA injection. As shown in Table 2, 5 days after the latex injection, accumulation of peritoneal cells in the cavity in response to PHA decreased to about one-third of normal, and the low value persisted for more than l0 days. However, on day 1 the cell number increased markedly to 3 - 4 times the normal counts. To find the optimal dose of latex particles for inhibiting cell accumulation or cell mobilization in the
Effects of Latex Particles on Immune Responses inflammatory site without any toxicity to animals, we compared the effect in various doses of latex particles on peritoneal cell accumulation to PHA-induced inflammation. Results summarized in Table 3 show that 40/~g was an effective dose and was considered optimal because 100 mg seemed toxic to the animals in terms of weight loss. It is noteworthy that the decrease on day 5 and 10 did not occur, if particles were used in a dose less than 40 mg.
Effect of latex particles on PHA-induced delayed hypersensitivity-type skin reactions in normal mice This test falls within the same category as the former experiment, only the site of the inflammatory reaction to PHA differs, being subcutaneous rather than intraperitoneal. As shown in Table 4, in contrast to the first experiment, on the day of latex particle injection and l day after the injection, the delayed hypersensitivity-type skin reaction to PHA was reduced to less than half of that observed in normal mice and this reduction did not change on day 5, I l and 14.
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Effect of latex particles on phagocytic capacity in normal mice As shown in Table 5, intraperitoneal administration of latex particles in mice reduced their phagocytic capacity (carbon clearance) to 40e/0 of normal on day 1, and then this low capacity persisted for 10 days. These results indicate that latex particle administration inhibits not only circulating macrophage function but also the overall capacity of the reticuloendothelial system (RES).
Effect of latex particles on lysozyme activity secreted in cultured medium and morphology of murine macrophages To determine the fate of macrophages after a phagocytic load of latex particles, macrophages cultured in vitro were allowed to ingest the particles for 2 or 16 h, and the morphology and secretion of lysozyme into the medium were examined for 7 days after the particle ingestion. The number of adherent cells was not influenced by the 2 h ingestion of latex particles. The latex-phagocytized cells firmly adhered
Table 1. Kinetics of PHA-induced inflammatory cell accumulation in the peritoneal cavity of normal mice
No. of peritoneal cells accumulated in the cavity (x 10~S.D.) at indicated times after intraperitoneal injection of PHA
Hours
0 2.3±0.4
7 3.7±1.2
24 4.3±3.5
48 4.2±0.6
72 3.5±0.6
96 2.3±1.5
The values represent the mean (±S.D.) number of peritoneal cells recovered from the cavity of 5-6 mice at each indicated time after an initial injection (i.p.) of 50 ~g of PHA.
Table 2. Effect of varying intervals of latex particles on PHA-induced inflammatory cell accumulation in mice
Sex
Age (weeks)
No. of mice
SWM/Ms SWM/Ms
~ ~
11 8
4 3
C57BL/6
~
11
5
C3H/HeN
~
8
3-6
Mice strain
Average no. of peritoneal cells (x 106±S.D.) Treated mice, Normal mice day after particles injection
Day 1 Day 5 Day 10 3.8±!.0 15.7__.4.1 1.1±1.0 !.0 4.9* (2.9)* 13.9" (13.5)* 1.5" (I.0)* 1.1" (0.4)* 4.3-+0.9 16.4-+3.3 2.9-+I. 1 1.2~0.3 3.2~1.3 19.8-+5.1 1.7-+0.8 1.0-+0.1
On day 0, 40 mg of latex panicles (diam. 1 pm) were injected intraperitoneally, and at indicated intervals mice were injected (i.p.) with 50/~g of PHA. Forty-eight hours after PHA injection mice were sacrificed and cells in washings of the peritoneal cavity were counted. Figures in parentheses are values from mice without PHA injection. * Pooled samples were used.
200
1-. |A.~,AKA ef a[. Table 3.
Mice strain
Effect of various doses of latex particles on PHA-induced inflammatory cell accumulation in m~ce
Dose of No. of particles mice (mg/mouse)
Sex
Age (weeks)
C3H/HeN
!~
9
3
SWM/Ms
~
8
2
Average no. of peritoneal cells ( × 106) Day after particles injection
Day I 4.1 (2.7) 10.9 1 I. 1 13.2 -+ ---
0 10 40 100 0 4
C3H/HeN
0
6
2
SWM/Ms
~
5
3
40 0 4 40 0 10 40 100
Day 5 -(3.1) 6.5 2.4 1.4 4.4 (2.9) 5.8
--
---5.9 (3.6) - - (22.5) 16.6 (22.4) --- (23.0)
Day 10 4.3 (3.4) 6.9 0.7 0.6 +---
1.4
--
2.4 4.7 0.7 4.6 -1.2 --
--...... -----+-
(4.0) (3.6) (2.3)
Latex particles of indicated doses were injected intraperitoneally (i.p.). On day 0, and at indicated intervals mice were injected (i.p.) with 50 tag of PHA. Mice were sacrificed 48 h after PHA injection, and cells in pooled washings of peritoneal cavity were counted. Figures in parentheses are values from mice without PHA stimulation.
Table 4.
Mice st rail1
SWM/Ms
Effect at varying intervals of latex particles on PHA-induced delayed hypersensitivity-type skin reaction in mice
Sex
~
Mean (n = 3 or 4) Day after-particles injection increment in thickness of footpads (mm_+S.D.J treated mice/normal mice (%)
Age (weeks)
9
Day 0 (4 h) 0.137_+0.063
Day 5 0.153_+0.012
0.470_+0.085
0.565_+0.021
(29)
C3H/HeN
~
7-8
Day 1 0.173_+0.034
Day 6 0.140__.0.053
Day I I 0.125_+0.017
Day 17 0.330-+0.054
0.395_+0.007
0.300~_0.028
0.348_+0.057
0.485_+0.035
(44)
SWM/Ms
"J
8
(27)
(47)
(35)
(68)
Day 7 0.223_+0.077
Day 14 0.150-+0.037
Day 21 0.213_+0.083
0.395_+0.064
0.310-&-_0.028
0.260_+0.014
(49)
(48)
(82)
On day 0, 50 ~g of P H A in 0.05 ml were injected into a right footpad of normal mice and of mice treated with 40 mg of latex particles. Values in parentheses indicate percentage of increment in footpad thickness of treated mice compared to that of control mice.
201
Effects of Latex Particles on Immune Responses to the plastic, resisting vigorous washing with saline, and were well spread out for 7 days. However, cells which had ingested latex secreted a lesser amount of lysozyme into the medium than normal macrophages. The secretion accelerated after 3 days in culture and reached the same level as that of control cells after 7 days, as shown in Fig. la. When adherent cells were allowed to ingest the particles for 16 h, most latex-phagocytized cells became rounded and were detached from the plastic by gentle agitation. The number of remaining adherent cells was less than one-third of that of the control culture. The few remaining cells were well spread out. Secretion of lysozyme by latex-loaded macrophages, on a per cell basis, was totally suppressed for 2 days and, although a slight increase of lysozyme concentration in the medium was observed later, the concentration on the 6th day was 10°70 o f that of the control cells (Fig. lb). A negligible amount of lysozyme activity was detected in the culture medium of the detached cells. Similar results were obtained with macrophages from C 3 H / H e N mice. Table 5.
Mice strain
Sex
SWM/Ms
~
8-12
C3H/HeN
~
9
~
/
tO
.u
_1
I 2
4
6
0
~,-..----~
?
2
6
4
Incubation time, doys Fig. 1. Lysozyme secretion by latex-loaded macrophages. Macrophage monolayers of normal SWM/Ms mice (2.4x 105 cells/60 mm dish) were allowed to ingest latex particles for 2 h (a) or 16 h (b) at 37°C. After the excess latex was removed, macrophages were cultivated in fresh RPMI 1640 medium and lysozyme activity in the medium was determined at the indicated intervals. Q ; control culture, O; latex-loaded culture.
Effect of varying intervals of latex particles on phagocytic activity in mice
Age (weeks)
SWM/Ms
/
I0-
8
Average of phagocytic index (K) Mice treated with latex particles (070). Day after particles injection
Normal mice
0.06305 ±0.0115 0.09615 ±0.0173 0.1978 ±0.0158 0.1518 ±0.0245 0.1265 _+0.0625
Day 1 0.05058 ±0.00378
0.08515 ±0.02043
(80)
Day 5 0.04280 ±0.00443 0.03342 ±0.00656
Day 10 (67) (34)
0.0743 ±0.0165
(77)
0.04790 ±0.01068
(37)
(43) 0.05943 ±0.02197
(39)
Pelikan C1 !/1431 ink (20 mg/ml/100 g of body weight) was injected intravenously. The assessed average rate of clearance (K) of carbon particles from the peripheral blood of 2 - 4 normal mice and of mice treated with an injection (i.p.) of 40 mg of latex particles is shown for days I, 5 and 10. * Values in parentheses indicate percentage of K of treated mice compared to K of control mice. K = (log C~ - log C2)/(T 2 - Tj). See Experimental Procedures. DISCUSSION Previously we showed that when mice were treated intraperitoneally with latex particles, the effect o f BCG-mediated tumor killing was completely negated as in the case o f treatment by A M S (Tanaka, Nakagawa, Kato, Yoshimura, Fujita & Kimura, 1977). Agents such as silica (Allison & Hart, 1968; Zisman,
1970) and Trypan Blue (Hibbs, 1975) are known to diminish the functional capacities of macrophages. In the present experiment we tested the use of latex particles in vivo and in vitro, in order to determine whether this agent is as effective an antimacrophage agent as the others. First, we tested the consequences of latex particles administered in vivo and secondly, the consequences in vitro. et al.,
202
~. ~ANAKA et a~.
Since PHA stimulates lymphocyte blastogenesis and chemotactic lymphokine production by unsensitized lymphocytes in vitro, it was hypothesized that injection of PHA into animals might produce a delayed hypersensitivity-type of inflammatory reaction in vivo (Boetcher & Meltzer, 1975). As shown in Table 3, the accumulation of peritoneal cells in response to PHA, was strongly reduced (to about 30°7o of normal on day 5, and even less on day 10) by latex particle treatment. The significance of the reduced cell accumulation in mice treated with latex particles remains obscure, but it may be assumed that after excessive ingestion of these undigestible particles: (1) the macrophages themselves might be ruptured (this correlates well with our in vitro findings), (2) the reduction of macrophage chemotactic movement response to lymphokine may have limited the entry of macrophages into peritoneal cavity, (3) in contrast, lymphocytes may be indirectly affected by a substance released from the damaged macrophages, as in the case of silica treatment, which first increased and then reduced the Con A responsiveness of spleen cells (Levy & Wheelock, 1975). The early increase may have been due to release of lymphocyte activating factor. The later decrease may have been caused by refractory state in the lymphocyte population, attributed to the effect of suppressor lymphocytes. Recently the accumulation of inflammatory cells in the peritoneal cavity induced by the intraperitoneal injection of tumor cells or normal spleen cells was reported (Evans, Booth & Spencer, 1978). This inflammatory response peaked around days 5-7, and it is noteworthy that control mice did not respond to these stimuli. In the case of injection of latex particles, accumulation of cells in the peritoneal cavity was significafitly increased on day 1, and strongly reduced on days 5 and 10, either with or without PHA stimulation (Tables 2 and 3). Therefore it would be desirable to check the kinetics of peritoneal cell accumulation after latex particle injection without PHA stimulation. Also, the effect of intravenous injection of latex particles on the inflammatory response should be determined in conjunction with other routes of administration for comparison. Such a study now in progress, also includes an examination of the histopathology of the spleen and liver at varying intervals after the injection of latex particles. Masses of particles partly covering these organs and the peritoneum were obvious. The organs had adhered to each other and to the peritoneum through the heavily attached particles. Therefore, we changed the site for inducing inflammation to the footpads. PHA was injected into footpads and produced the delayed hypersensitivity-type skin reaction. As seen in Table 4, the skin reaction in mice treated with
particles followed a course similar to that of the former experiment, except that there was no increase on day 1, and the depressive reaction caused by latex particles persisted more than 2 weeks. It is important to note that latex particles, at the optimal dose for blocking inflammatory response, do not seem to be as toxic as silica and do not result in loss of body weight in animals. At lower dosage (4 or 10 mg per mouse), latex particles are consistently stimulatory (Table 3). Such divergent observations on the stimulative and depressive effects of latex particles on delayed hypersensitivity reactions are probably related to dosage. Previously we had shown (Tanaka et al., 1977) that BCG-mediated tumor destruction was nullified by the administration of latex particles. The use of latex particles negated not only BCG-mediated tumor killing but also the delayed hypersensitivity response to PPD (purified protein derivatives) similar to the use of ATS and cortisone (Chung, Zbar & Rapp, 1973). Although the exact function of the large number of macrophages and lymphocytes in inflammatory sites is not clear, latex particles seem to be a very useful agent for assessing macrophage participation in the immune response. The effect of latex particles on phagocytic capacity in vivo was also suppressive as shown in -Fable 5. Latex particle-induced RES blockade persisted for more than 5 days, suggesting that these particles not only saturated but also destroyed RES macrophages. Having demonstrated, in vivo, the effects of latex particles on fixed and unfixed macrophages, we finally studied the effect of latex particles in vitro on cultured peritoneal macrophages. Macrophages cultured in vitro were allowed to ingest particles for 2 and 16 h, and their morphology and the secretion of lysozyme were examined. When cells were allowed to ingest the particles for 16 h most latex-loaded cells become rounded and detached from the plastic. Secretion of lysozyme by those macrophages on the 6th day of culture was reduced to less than 10% of that of control cells (Fig. 1B), but these results did not occur when cells were allowed to ingest the particles for 2 h, suggesting that the effect of latex partides on macrophage function in vitro is dose dependent, as in the case of the in vivo experiments (Table 3). These results indicate that macrophages lose their ability to adhere to plastic surfaces and to secrete lysozyme when there is a heavy phagocytic load of latex particles. Tight adherence to plastic is characteristic of macrophages and it is known that lysozyme is secreted constantly by macrophages regardless of the culture conditions. Gordon and colleagues (Gordon et al., 1974) have shown that lysozyme secretion is not drastically changed by phagocytic stimuli. The rounding and detaching
Effects of Latex Particles on Immune Responses f r o m the plastic a n d the decreased secretion o f lysozyme observed with m a c r o p h a g e s that have phagocytized latex particles m a y be a reflection o f p r o f o u n d i m p a i r m e n t o f their f u n c t i o n leading to the loss o f viability. Results o f this experiment show that latex particles lessened the delayed hypersensitivity-type reaction to
203
P H A for a relatively long period o f time (at least 2 weeks). These findings indicate that latex particles may be a useful experimental tool for evaluating the r f l e of m a c r o p h a g e s by eliminating or suppressing their f u n c t i o n in vivo for extended periods without toxicity.
REFERENCES ALLISON, A. C. & HART, PD'A. (1968). Potentiation by silica of the growth of Mycobacterium tuberculosis in macrophage cultures. Br. J. exp. Path., 49, 465-476. BOETCHER, D. A. & MELTZER, M. S. (1975). Mouse mononuclear cell chemotaxis: description of system. J. hath. Cancer Inst., 54, 795-799. CHUNG, E., ZBAR, B. & RAPe, H. J. (1973). Tumor regression mediated by Mycobacterium boris (strain BCG)--effects of isonicotinic acid hydrazide, cortisone acetate and anti-thymocyte serum. J. natn. Cancer Inst., 51,241-250. EVANS, R., BOOTH, C. G. & SPENCER, F. (1978). Lack of correlation between in vivo rejection of syngeneic fibrosarcomas and in vitro non-specific macrophage cytotoxicity. Br. J. Cancer, 38, 583-590. FUJITA, H. & SAINO, Y. (1971). Isolation of anaerobic Corynebacterium from human bone marrows and its protective activity against sarcoma 180. J. Jap. stomat. Soc., 38, 294-299. (In Japanese.) GORDON, S., TODD. J. &COHN, Z. A. (1974). In vitro synthesis and secretion of lysozyme by mononuclear phagocytes. J. exp. Med., 139, 1228-1248. HATTORI, T. & MORI, A. (1973). Antitumor activity of anaerobic Corynebacterium isolated from the human bone marrow. Gann, 64, 15-27. Hinas, J. B. (1975). Activated macrophages as cytotoxic effector cells. Transplantation, 19, 77-81. KATO, A., NAKAGAWA, H., AKIMOTO, n . , FUJITA, H., TANAKA, T. & KIMURA, K. (1976). Antitumor effects of Corynebacterium liquefaciens. Gan-to-Kagakuryoho, 3, 677-686. (In Japanese.) LEVY, M. H. & WrtEELOCK, E. F. (1975). Effects of intravenous silica on immune and non-immune functions of the murine host. J. lmmun., 115, 41-48. PARRY,R. M., JR., CHANDAN,R. C. & SHAHANI,K. M. (1965). A rapid and sensitive assay of muramidase. Proc. Soc. exp. Biol. Med., 119, 384--386. SNYDERMAN, R., PIKE, M. C., BLAYCOCK, B. L. & WEINSTEIN, P. (1976). Effects of neoplasms on inflammation: depression of macrophage accumulation after tumor implantation. J. Immun., 116, 585--589. TANAKA, T., KATO, A., NAKAGAWA,H., FUJITA, H. & KIMURA, K. (1978). Immunotherapy of cancer: therapeutic efficacy of the vaccine containing anaerobic Corynebacterium or BCG and tumor cells in mice. In Gann Monographs on Cancer Research, No. 21 (eds. YAMAMURA, Y., KITAGAWA, M., and AZUMA, I.), pp. 93--107. Japan Scientific Societies, Tokyo. TANAKA, T., NAKAGAWA, H., KATO, A., YOSHIMURA, M., FUJITA, H. & KIMURA, K. (1977). Effect of anti-thymocyte serum, anti-macrophage serum, and latex particles on the therapeutic efficacy of BCG or Corynebacterium liquefaciens (Propionibacterium acnes C7) in syngeneic mice. Gann, 68, 45-52. TANAKA, T. & SAITO, T. (1975). Immunotherapy of cancer: induction of tumor immunity with a mixture of tumor cellBCG, and the effect of intratumor injection of BCG and of nonliving BCG preparation. Gann, 66, 631-640. TANAKA, T, & SASAKI, N. (1974). Effect of immunotherapy, chemotherapy and surgery on tumor immunity in mice. Gann, 65, 395--402. TANAKA, T. & TOKUNAGA, T. (1971). Suppression of tumor growth and induction of specific tumor immunity by intradermal inoculation of a mixture of living tumor cells and live Mycobacterium bovis in syngeneic mice. Gann, 62, 433-434. TOKUNAGA, T., KATAOKA, T., NAKAMURA, R. M., YAMAMOTO, S. & TANAKA, T. (1973). Tumor immunity induced by BCG-tumor cell mixtures in syngeneic mice. Jap. J. reed. Sci. Biol., 26° 71--85. WATANUKI,T., DOHI, C., SAITO, T., MIURA, Y. & SUZUKI,Y. (1965). Studies of carbon clearance assay in mice. Nippon Monaikei Gakkai Kaishi, 5, 295-305. (In Japanese.) ZaAR, B., BERNSTEIN, I. & RAt,p, H. J. (1971). Suppression of tumor growth at the site of injection of living Bacillus Calmette-Guerin. J. natn. Cancer Inst., 46, 831-839. ZaAR, B., BERNSTEIN, I., TANAKA, T. & RAPe, H. J. (1970). Tumor immunity produced by the intradermal inoculation of living tumor cells and living Mycobacterium boris (strain BCG). Science, 170, 1217-1218. ZISMAN, B., HIRSCH, M. S. & ALLISON, A. C. (1970). Selective effects of anti-macrophage serum, silica and antilymphocyte serum on pathogenesis of herpes virus infection of young adult mice. J. lmmun., 104, 1155-1159.
EFFECTS OF LATEX PARTICLES ON IMMUNE RESPONSES OF THE MURINE HOST AND ON MACROPHAGES IN VITRO* (I) TOMII(O TANAKA,t KAVO TAKAYA,I" TAKEHIKO KUNIMOTO,,'I:and HIROYASU BABA~ ~Department of Pharmacology and :t Department of Chemotherapy, National Cancer Center Research Institute, Tsukiji 5-l-l, Chuo-ku Tokyo 104, Japan (Received 18 January 1979 and in final form 17 May 1979)
Abstract--Previously we reported that the antitumor effect of BCG was found to be negated when mice were injected intraperitoneally with latex particles. The same results were found when mice were injected with anti-macrophage serum (AMS). However, neither latex particles nor AMS had any influence on the effectiveness of Propionibacterium aches. In order to assess the consequences in vivo of latex particle administration in normal mice, we determined, at varying intervals, the effect of latex particles on inflammatory cell accumulation in the peritoneal cavity, the effect on delayed hypersensitivity-type skin reaction induced by phytohemagglutinin, and the effect on carbon clearance. Results were as follows: (I) the cell accumulation in an inflammatory site was lowered by as much as 30o70 5 days after the latex administration and the low level persisted on day 10. (2) The inflammatory skin reaction to phytohemagglutinin in mice injected with latex particles was also depressed to less than 50°70 of normal for 14 days. (3) The phagocytic capacity of the mice treated with latex decreased to about 50o70 of normal for 10 days. The effect of latex particles on lysozyme excretion from macrophages cultured in vitro is also reported. After an excessive phagocytic load of latex particles, macrophages detached from the plastic and the number of remaining cells was one-third of that of control cultures. The lysozyme excretion was totally suppressed for the first 2 days, but was 10GT0of control values by day 6. Latex particles appear to be useful for evaluating the rSle of macrophages in immunological studies, since various functions of macrophages in vivo and in vitro, were blocked for a long period without toxicity.
Subsequent to the development of the guinea-pig model for cancer immunotherapy (Zbar, Bernstein & Rapp, 1971; Zbar, Bernstein, Tanaka & Rapp, 1970), we examined the therapeutic efficacy of intradermal inoculation of tumor cell-BCG vaccine in syngeneic mice (Tanaka & Saito, 1975; Tanaka & Sasaki, 1974; Tanaka & Tokunaga, 1971; Tokunaga, Kataoka, Nakamura, Y a m a m o t o & Tanaka, 1973). Additionally, since the antitumor activity of Propionibacterium aches (P. aches) had also been reported (Fujita & Saino, 1971; Hattori & Mori, 1973; Kato, Nakagawa, A k i m o t o , Fujita, Tanaka & Kimura, 1976), we established the therapeutic efficacy of P. aches-tumor cell vaccine. More recently, we made a comparative study of the cellular basis of BCGmediated and P. aches-mediated tumor killing by the use of anti-thymocyte serum (ATS), anti-macrophage serum (AMS) and latex particles (Tanaka, Kato, Nakagawa, Fujita & Kimura, 1978) which are taken up by and may cause damage to, macrophages (Allison & Hart, 1968; Zisman, Hirsch & Allison, 1970). The results showed that intraperitoneal administration of latex particles (40 rag) 2 h before
vaccine inoculation, as in the case of A M S treatment, negated the immunotherapeutic efficacy of BCG (both viable and nonviable). However, neither treatment affected the antitumor efficacy of P. acnes. The study reported here was designed to determine whether intraperitoneal latex particles would be an effective treatment for blocking various host immune responses in normal mice, that is, to assess the in vivo consequences of administering latex particles by: (l) counting the number of peritoneal exudate cells accumulated after intraperitoneai injection of phytohemagglutinin (PHA), (2) measuring the increment of the footpad thickness elicited by P H A injection as a delayed hypersensitivity-type reaction and (3) examining the phagocytic capacity of mice at varying intervals (generally, on days l, 5 and 10) after the injection of latex particles. We also examined whether latex particles would cause any alteration of macrophage function in vitro, such as secretion of lysozyme which is a cell-specific marker for macrophages and polymorphonuclear cells. For this purpose, the lysozyme secretion of macrophages cultured in vitro was measured after 2
* Research supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan. 197
T. TanaKa et al.
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and 16 h exposure to latex particles, and was compared to that of control macrophages. EXPERIMENTAL PROCEDURES
Animals. Young adult syngeneic mice (SWM/Ms, C57BL/6, and C3H/HeN) were used. Latex particles. A suspension of uniform polystyrene particles (diameter, 1.0 ~m), obtained f r o m the Dow Chemical Co., U.S.A., was sterilized at 65°C for 40 min and mice were injected intraperitoneally in doses of 4, 10, 40 or 100 mg per adult mouse. Phytohemagglutinin. Purified PHA (PHA-P) was purchased from Difco Laboratories, U.S.A. The lyophilized powder was reconstituted to 10 mg/ml in water, kept cold, and diluted with normal saline solution for use.
Assay for leucocyte accumulation in peritoneal cavity (Snyderman, Pike, Blaylock & Weinstein, 1976). The diluted PHA-P (50 ~g/0.5 ml/mouse) was injected intraperitoneally in order to induce a delayed hypersensitivity-type of inflammatory reaction. Forty-eight hours after the injection, mice were sacrificed by cervical dislocation. Peritoneal cells were collected by intraperitoneal injection of 5 ml of Hanks' balanced salt solution (BSS) containing 5 unit/ml of sodium heparin. These cells were combined with two other washings of 3 ml Hanks' BSS. Peritoneal fluid was withdrawn through a small incision in the abdominal wall and centrifuged for 10 min at 1000 r.p.m, at 4°C. The cell pellet was then resuspended in Hanks' BSS without heparin and the total cell number was counted using a hemocytometer. The number of animals per group varied from 3 to 6.
Assay for delayed hypersensitivity-type skin reaction. PHA-P (50 /~g/0.05 ml/mouse) was injected into a rear footpad and, as a control, physiological saline (0.05 mi) was injected into the other rear footpad. The increment in footpad thickness was measured with a Peacock dial gauge (Ozaki Co., Tokyo) before and 20-24 h after PHA-P injection, since the time for maximum footpad swelling was 20-24 h after the PHA-P injection and returned to near normal size in 72 h. The mean increase in thickness in control footpad was little or no change. The number of mice per group varied from 2 to 6 and results were expressed as the mean increment in footpad thickness (in ram) for a group. Assay of phagocytic capacity (Watanuki, Dohi, Saito, Miura & Suzuki, 1965). Phagocytic capacity was tested by carbon clearance, using 20 mg/ml Pelikan Ink (C 11/ 1431 a, GUnther Wagner PelikanWerke, Hannover, West Germany), injected into the tail vein in a dose of 1 ml/100 g body weight. Blood
was drawn from the retro-orbital sinus at 2, 5 and 8 rain after carbon injection; 0.02 ml of each blood sample was pipetted into 1.5 ml of 0.1% of Na2CO ~ solution; optical density was read with a spectrophotometer at 660 nm. Under these conditions, the rate of clearance (K) is an expotential function of time, according to the equation K=(log C I - l o g C2)/(T2- T I) where C~ and C 2 represent carbon concentrations in the blood at time T~ and T2, respectively. Two- four animals per group were used. Assay of lysozyme activity (Gordon, Todd & Cohn, 1974; Parry, Chandan & Shahani, 1965). Lysozyme activity was determined by measuring the initial rate of lysis of a suspension of Micrococcus lysodeikticus (0.15 mg/ml in M/15 phosphate buffer, pH 6.25). The decrease of absorbance at 450 nm was recorded every 20 s for 3 min and the slope of the initial linear portion was compared to the standard curve prepared with known concentrations of egg white lysozyme. Phagocytosis in vitro. A known number of cells (1.2×106 cells/dish) lavaged from the peritoneal cavity of normal mice (SWM/Ms) were allowed to adhere to plastic dishes for 2 h at 37°C. After the dishes were washed vigorously with Hanks' BSS to remove nonadherent cells, the remaining monolayers of macrophages (2.4× 105/dish) were cultivated in RPMI-1640 medium supplemented with 10o70 fetal bovine serum. Latex particles were added at a concentration of 0.1°70. Within a 2 h period of phagocytosis, more than 95o7o of the macrophages had ingested the particles. Excess latex was removed after 2 and after 16 h, and the cells were cultivated in a fresh medium at 37°C in a COz-incubator. Aliquots of the medium were removed at intervals and lysozyme activity was determined. RESULTS
Effect of latex particles on PHA-induced inflammatory cell accumulation in the peritoneal cavity of normal mice Table I shows the kinetics of inflammatory cell accumulation in the peritoneal cavity of normal mice injected with 50/~g of PHA. Because of this result and others (Snyderman, et al., 1976), we made the total cell count at 48 h after PHA injection. As shown in Table 2, 5 days after the latex injection, accumulation of peritoneal cells in the cavity in response to PHA decreased to about one-third of normal, and the low value persisted for more than l0 days. However, on day 1 the cell number increased markedly to 3 - 4 times the normal counts. To find the optimal dose of latex particles for inhibiting cell accumulation or cell mobilization in the
Effects of Latex Particles on Immune Responses inflammatory site without any toxicity to animals, we compared the effect in various doses of latex particles on peritoneal cell accumulation to PHA-induced inflammation. Results summarized in Table 3 show that 40/~g was an effective dose and was considered optimal because 100 mg seemed toxic to the animals in terms of weight loss. It is noteworthy that the decrease on day 5 and 10 did not occur, if particles were used in a dose less than 40 mg.
Effect of latex particles on PHA-induced delayed hypersensitivity-type skin reactions in normal mice This test falls within the same category as the former experiment, only the site of the inflammatory reaction to PHA differs, being subcutaneous rather than intraperitoneal. As shown in Table 4, in contrast to the first experiment, on the day of latex particle injection and l day after the injection, the delayed hypersensitivity-type skin reaction to PHA was reduced to less than half of that observed in normal mice and this reduction did not change on day 5, I l and 14.
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Effect of latex particles on phagocytic capacity in normal mice As shown in Table 5, intraperitoneal administration of latex particles in mice reduced their phagocytic capacity (carbon clearance) to 40e/0 of normal on day 1, and then this low capacity persisted for 10 days. These results indicate that latex particle administration inhibits not only circulating macrophage function but also the overall capacity of the reticuloendothelial system (RES).
Effect of latex particles on lysozyme activity secreted in cultured medium and morphology of murine macrophages To determine the fate of macrophages after a phagocytic load of latex particles, macrophages cultured in vitro were allowed to ingest the particles for 2 or 16 h, and the morphology and secretion of lysozyme into the medium were examined for 7 days after the particle ingestion. The number of adherent cells was not influenced by the 2 h ingestion of latex particles. The latex-phagocytized cells firmly adhered
Table 1. Kinetics of PHA-induced inflammatory cell accumulation in the peritoneal cavity of normal mice
No. of peritoneal cells accumulated in the cavity (x 10~S.D.) at indicated times after intraperitoneal injection of PHA
Hours
0 2.3±0.4
7 3.7±1.2
24 4.3±3.5
48 4.2±0.6
72 3.5±0.6
96 2.3±1.5
The values represent the mean (±S.D.) number of peritoneal cells recovered from the cavity of 5-6 mice at each indicated time after an initial injection (i.p.) of 50 ~g of PHA.
Table 2. Effect of varying intervals of latex particles on PHA-induced inflammatory cell accumulation in mice
Sex
Age (weeks)
No. of mice
SWM/Ms SWM/Ms
~ ~
11 8
4 3
C57BL/6
~
11
5
C3H/HeN
~
8
3-6
Mice strain
Average no. of peritoneal cells (x 106±S.D.) Treated mice, Normal mice day after particles injection
Day 1 Day 5 Day 10 3.8±!.0 15.7__.4.1 1.1±1.0 !.0 4.9* (2.9)* 13.9" (13.5)* 1.5" (I.0)* 1.1" (0.4)* 4.3-+0.9 16.4-+3.3 2.9-+I. 1 1.2~0.3 3.2~1.3 19.8-+5.1 1.7-+0.8 1.0-+0.1
On day 0, 40 mg of latex panicles (diam. 1 pm) were injected intraperitoneally, and at indicated intervals mice were injected (i.p.) with 50/~g of PHA. Forty-eight hours after PHA injection mice were sacrificed and cells in washings of the peritoneal cavity were counted. Figures in parentheses are values from mice without PHA injection. * Pooled samples were used.
200
1-. |A.~,AKA ef a[. Table 3.
Mice strain
Effect of various doses of latex particles on PHA-induced inflammatory cell accumulation in m~ce
Dose of No. of particles mice (mg/mouse)
Sex
Age (weeks)
C3H/HeN
!~
9
3
SWM/Ms
~
8
2
Average no. of peritoneal cells ( × 106) Day after particles injection
Day I 4.1 (2.7) 10.9 1 I. 1 13.2 -+ ---
0 10 40 100 0 4
C3H/HeN
0
6
2
SWM/Ms
~
5
3
40 0 4 40 0 10 40 100
Day 5 -(3.1) 6.5 2.4 1.4 4.4 (2.9) 5.8
--
---5.9 (3.6) - - (22.5) 16.6 (22.4) --- (23.0)
Day 10 4.3 (3.4) 6.9 0.7 0.6 +---
1.4
--
2.4 4.7 0.7 4.6 -1.2 --
--...... -----+-
(4.0) (3.6) (2.3)
Latex particles of indicated doses were injected intraperitoneally (i.p.). On day 0, and at indicated intervals mice were injected (i.p.) with 50 tag of PHA. Mice were sacrificed 48 h after PHA injection, and cells in pooled washings of peritoneal cavity were counted. Figures in parentheses are values from mice without PHA stimulation.
Table 4.
Mice st rail1
SWM/Ms
Effect at varying intervals of latex particles on PHA-induced delayed hypersensitivity-type skin reaction in mice
Sex
~
Mean (n = 3 or 4) Day after-particles injection increment in thickness of footpads (mm_+S.D.J treated mice/normal mice (%)
Age (weeks)
9
Day 0 (4 h) 0.137_+0.063
Day 5 0.153_+0.012
0.470_+0.085
0.565_+0.021
(29)
C3H/HeN
~
7-8
Day 1 0.173_+0.034
Day 6 0.140__.0.053
Day I I 0.125_+0.017
Day 17 0.330-+0.054
0.395_+0.007
0.300~_0.028
0.348_+0.057
0.485_+0.035
(44)
SWM/Ms
"J
8
(27)
(47)
(35)
(68)
Day 7 0.223_+0.077
Day 14 0.150-+0.037
Day 21 0.213_+0.083
0.395_+0.064
0.310-&-_0.028
0.260_+0.014
(49)
(48)
(82)
On day 0, 50 ~g of P H A in 0.05 ml were injected into a right footpad of normal mice and of mice treated with 40 mg of latex particles. Values in parentheses indicate percentage of increment in footpad thickness of treated mice compared to that of control mice.
201
Effects of Latex Particles on Immune Responses to the plastic, resisting vigorous washing with saline, and were well spread out for 7 days. However, cells which had ingested latex secreted a lesser amount of lysozyme into the medium than normal macrophages. The secretion accelerated after 3 days in culture and reached the same level as that of control cells after 7 days, as shown in Fig. la. When adherent cells were allowed to ingest the particles for 16 h, most latex-phagocytized cells became rounded and were detached from the plastic by gentle agitation. The number of remaining adherent cells was less than one-third of that of the control culture. The few remaining cells were well spread out. Secretion of lysozyme by latex-loaded macrophages, on a per cell basis, was totally suppressed for 2 days and, although a slight increase of lysozyme concentration in the medium was observed later, the concentration on the 6th day was 10°70 o f that of the control cells (Fig. lb). A negligible amount of lysozyme activity was detected in the culture medium of the detached cells. Similar results were obtained with macrophages from C 3 H / H e N mice. Table 5.
Mice strain
Sex
SWM/Ms
~
8-12
C3H/HeN
~
9
~
/
tO
.u
_1
I 2
4
6
0
~,-..----~
?
2
6
4
Incubation time, doys Fig. 1. Lysozyme secretion by latex-loaded macrophages. Macrophage monolayers of normal SWM/Ms mice (2.4x 105 cells/60 mm dish) were allowed to ingest latex particles for 2 h (a) or 16 h (b) at 37°C. After the excess latex was removed, macrophages were cultivated in fresh RPMI 1640 medium and lysozyme activity in the medium was determined at the indicated intervals. Q ; control culture, O; latex-loaded culture.
Effect of varying intervals of latex particles on phagocytic activity in mice
Age (weeks)
SWM/Ms
/
I0-
8
Average of phagocytic index (K) Mice treated with latex particles (070). Day after particles injection
Normal mice
0.06305 ±0.0115 0.09615 ±0.0173 0.1978 ±0.0158 0.1518 ±0.0245 0.1265 _+0.0625
Day 1 0.05058 ±0.00378
0.08515 ±0.02043
(80)
Day 5 0.04280 ±0.00443 0.03342 ±0.00656
Day 10 (67) (34)
0.0743 ±0.0165
(77)
0.04790 ±0.01068
(37)
(43) 0.05943 ±0.02197
(39)
Pelikan C1 !/1431 ink (20 mg/ml/100 g of body weight) was injected intravenously. The assessed average rate of clearance (K) of carbon particles from the peripheral blood of 2 - 4 normal mice and of mice treated with an injection (i.p.) of 40 mg of latex particles is shown for days I, 5 and 10. * Values in parentheses indicate percentage of K of treated mice compared to K of control mice. K = (log C~ - log C2)/(T 2 - Tj). See Experimental Procedures. DISCUSSION Previously we showed that when mice were treated intraperitoneally with latex particles, the effect o f BCG-mediated tumor killing was completely negated as in the case o f treatment by A M S (Tanaka, Nakagawa, Kato, Yoshimura, Fujita & Kimura, 1977). Agents such as silica (Allison & Hart, 1968; Zisman,
1970) and Trypan Blue (Hibbs, 1975) are known to diminish the functional capacities of macrophages. In the present experiment we tested the use of latex particles in vivo and in vitro, in order to determine whether this agent is as effective an antimacrophage agent as the others. First, we tested the consequences of latex particles administered in vivo and secondly, the consequences in vitro. et al.,
202
~. ~ANAKA et a~.
Since PHA stimulates lymphocyte blastogenesis and chemotactic lymphokine production by unsensitized lymphocytes in vitro, it was hypothesized that injection of PHA into animals might produce a delayed hypersensitivity-type of inflammatory reaction in vivo (Boetcher & Meltzer, 1975). As shown in Table 3, the accumulation of peritoneal cells in response to PHA, was strongly reduced (to about 30°7o of normal on day 5, and even less on day 10) by latex particle treatment. The significance of the reduced cell accumulation in mice treated with latex particles remains obscure, but it may be assumed that after excessive ingestion of these undigestible particles: (1) the macrophages themselves might be ruptured (this correlates well with our in vitro findings), (2) the reduction of macrophage chemotactic movement response to lymphokine may have limited the entry of macrophages into peritoneal cavity, (3) in contrast, lymphocytes may be indirectly affected by a substance released from the damaged macrophages, as in the case of silica treatment, which first increased and then reduced the Con A responsiveness of spleen cells (Levy & Wheelock, 1975). The early increase may have been due to release of lymphocyte activating factor. The later decrease may have been caused by refractory state in the lymphocyte population, attributed to the effect of suppressor lymphocytes. Recently the accumulation of inflammatory cells in the peritoneal cavity induced by the intraperitoneal injection of tumor cells or normal spleen cells was reported (Evans, Booth & Spencer, 1978). This inflammatory response peaked around days 5-7, and it is noteworthy that control mice did not respond to these stimuli. In the case of injection of latex particles, accumulation of cells in the peritoneal cavity was significafitly increased on day 1, and strongly reduced on days 5 and 10, either with or without PHA stimulation (Tables 2 and 3). Therefore it would be desirable to check the kinetics of peritoneal cell accumulation after latex particle injection without PHA stimulation. Also, the effect of intravenous injection of latex particles on the inflammatory response should be determined in conjunction with other routes of administration for comparison. Such a study now in progress, also includes an examination of the histopathology of the spleen and liver at varying intervals after the injection of latex particles. Masses of particles partly covering these organs and the peritoneum were obvious. The organs had adhered to each other and to the peritoneum through the heavily attached particles. Therefore, we changed the site for inducing inflammation to the footpads. PHA was injected into footpads and produced the delayed hypersensitivity-type skin reaction. As seen in Table 4, the skin reaction in mice treated with
particles followed a course similar to that of the former experiment, except that there was no increase on day 1, and the depressive reaction caused by latex particles persisted more than 2 weeks. It is important to note that latex particles, at the optimal dose for blocking inflammatory response, do not seem to be as toxic as silica and do not result in loss of body weight in animals. At lower dosage (4 or 10 mg per mouse), latex particles are consistently stimulatory (Table 3). Such divergent observations on the stimulative and depressive effects of latex particles on delayed hypersensitivity reactions are probably related to dosage. Previously we had shown (Tanaka et al., 1977) that BCG-mediated tumor destruction was nullified by the administration of latex particles. The use of latex particles negated not only BCG-mediated tumor killing but also the delayed hypersensitivity response to PPD (purified protein derivatives) similar to the use of ATS and cortisone (Chung, Zbar & Rapp, 1973). Although the exact function of the large number of macrophages and lymphocytes in inflammatory sites is not clear, latex particles seem to be a very useful agent for assessing macrophage participation in the immune response. The effect of latex particles on phagocytic capacity in vivo was also suppressive as shown in -Fable 5. Latex particle-induced RES blockade persisted for more than 5 days, suggesting that these particles not only saturated but also destroyed RES macrophages. Having demonstrated, in vivo, the effects of latex particles on fixed and unfixed macrophages, we finally studied the effect of latex particles in vitro on cultured peritoneal macrophages. Macrophages cultured in vitro were allowed to ingest particles for 2 and 16 h, and their morphology and the secretion of lysozyme were examined. When cells were allowed to ingest the particles for 16 h most latex-loaded cells become rounded and detached from the plastic. Secretion of lysozyme by those macrophages on the 6th day of culture was reduced to less than 10% of that of control cells (Fig. 1B), but these results did not occur when cells were allowed to ingest the particles for 2 h, suggesting that the effect of latex partides on macrophage function in vitro is dose dependent, as in the case of the in vivo experiments (Table 3). These results indicate that macrophages lose their ability to adhere to plastic surfaces and to secrete lysozyme when there is a heavy phagocytic load of latex particles. Tight adherence to plastic is characteristic of macrophages and it is known that lysozyme is secreted constantly by macrophages regardless of the culture conditions. Gordon and colleagues (Gordon et al., 1974) have shown that lysozyme secretion is not drastically changed by phagocytic stimuli. The rounding and detaching
Effects of Latex Particles on Immune Responses f r o m the plastic a n d the decreased secretion o f lysozyme observed with m a c r o p h a g e s that have phagocytized latex particles m a y be a reflection o f p r o f o u n d i m p a i r m e n t o f their f u n c t i o n leading to the loss o f viability. Results o f this experiment show that latex particles lessened the delayed hypersensitivity-type reaction to
203
P H A for a relatively long period o f time (at least 2 weeks). These findings indicate that latex particles may be a useful experimental tool for evaluating the r f l e of m a c r o p h a g e s by eliminating or suppressing their f u n c t i o n in vivo for extended periods without toxicity.
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