- Email: [email protected]
Role of macrophages in delayed hypersensitivity
CELLI'LAH
I~~MLYSOLOGY
Role
269-274
2,
of
Macrophages
I. Induction
EMIL Department
(1971)
R.
in
with
Hypersensitivity
Macrophage-Bound
UNANUE
of Experimental 476 Prosfiect
Delayed
’ AND JOSEPH
Pathology, Scrip@ Street, La /olln, Received
Janzcnvy
Antigen
D.
1
FELD&IAN
Clinic and Research California 92037
Foundation,
22, 1971
Macrophage-bound antigen inoculated into footpads of rats induced a state of delayed hypersensitivity (DH) equivalent to that induced by free antigen but less than that induced by antigen incorporated into complete Freund’s adjuvant. Antigen bound to macrophages was more immunogenic than free antigen and elicited antibody responses in all injected rats. Macrophages injected intradermally into rats immunized with antigen in complete Freund’s adjuvant excited an acute inflammatory reaction consisting of numerous polymorphonuclear leucocytes and abscesses. In the same animals free antigen produced typical DH lesions, and antigen bound to macrophages produced a mixture of polymorphonuclear and mononuclear cell infiltrates. Intradermal deposit of macrophages or macrophages with antigen increased the number of basophils in areas of inflammation. Basophils did not vary in number or distribution in DH lesions elicited by free antigen at 14 or 28 days after inoculation.
INTRODUCTION During induction of an immune response macrophages appear to play a role of variable importance ( 1). These phagocytic cells take up a variety of antigens, which, depending upon their physicochemical state, may be retained at the surface membranes of the macrophages and/or endocytosed and catabolized (reviewed in 1). Association of some antigens with macrophages favors immunogenicity (2), i.e., doses of antigens that would ordinarily not elicit antibody in viva will do so when the antigens are bound to macrophages (3, 4). Macrophage-bound antigen may also, augment manyfold the production of antibody (3, 4). In in vitro systems, macrophages appear to be needed for lymphocytes to proliferate (5, 6) or to synthesize antibody in the presence of antigen (7, 8). The participation of macrophages in delayed hypersensitivity (DH) has not been fully explo’red. although it is well known that these cells form a major part of the 1 This is Publication No. 467 from the Department of Experimental Pathology, Clinic and Research Foundation, La Jolla, Calif. This work was supported by a United Public Health Grant -41-07007 and Atomic Energy Commission Contract -4T (04-3) -779. 2 Supported by a Senior Present Address : Department
Scripps States
Fellowship of the iZmerican Cancer Society, California Division. of Pathology, Harvard Medical School, Boston, Mass. 02115.
270
UNANUE
AND
FELDMAN
inflammatory DH skin lesion (9). In this paper we report on the role of macrophages in the induction of DH and in the following paper the effect of anti-macrophage antibodies oa the expression of DH. MATERIALS
AND
METHODS
Inbred Lewis (Le) or (Lewis X Buffalo)F, (LBfF,) rats, (both strains purchased from Simonsen Laboratories, Gilroy, Calif., or Microbiological Associates, Inc., Walkersville, Md.) weighing 250-300 g, were used as donors of macrophages and as test animals for the induction of DH. Macrophages were harvested from the peritoneal cavities of rats injected intraperitoneally 3 days before with 3 ml of peptone (Difco Laboratories, Detroit, Mich.) . They were incubated in suspension for 1 hr at 37” with appropriate quantities of heat-aggregated 1311-labeled human serum albumin ( lSII-HSA) (Reheis Chemical Co., Division of Armour Pharmaceutical Co., Los Angeles, Calif.) and then were washed 3 times with balanced salt solution.3 An aliquot of cells was counted in a gamma ray spectrometer. In different experiments about 1 X lo7 cells bound from 1 to 10 PLg of antigen as calculated from radioactive counts. Groups of Le or LBfF, rats were inoculated in their footpads with sufficient syngeneic cells (generally 4-5 X 107) to deliver 5-50 pg of antigen, and 14 or 28 days later they were skin tested with 10 pg of soluble HSA (nonaggregated) in 0.1 ml of saline. Erythema and induration of skin test sites were measured in millimeters at 6 and 24 hr, and rats were bled at 24 hr for detection of circulating antibody by the Farr technique (10). The percentage of 1311-HSA bound at a 1:2 dilution of sera was determined. No attempt was made to establish the dilution of sera binding 33% of the antigen. For comparison, other groups of rats were simultaneously prepared by injecting into the footpads equivalent amounts of free antigen or antigen in a water and oil emulsion containing 1 mg of dried M. tuberc&sis (H37Ra) (Difco Laboratories, Detroit, Mich.) per ml of emulsion (CFA). Several groups of rats were given 5-50 pg of free or macrophage-bound antigen subcutaneously, intravenously, and intraperitoneally. They were skin tested between 6 and 28 days later. Induction of DH with antigen administered by these routes was infrequently achieved, and when successful, skin reactions were weakly positive. The results of these groups will therefore not be reported. In another series of experiments, the efficacy of antigen bound to viable macrophages in eliciting positive skin tests was compared with the efficacy of free antigen. Groups of rats were inoculated in their footpads with 1 mg of antigen in CFA. Fourteen days later each rat was injected intradermally in three different sites with 5 X lo6 syngeneic macrophages carrying 10 PLg of antigen, 5 X lo6 syngeneic macrophages without antigen, and 10 pg of free antigen. A control group, uninoculated, was skin tested simultaneously with the three different preparations. Skin test sites from all rats were measured grossly at 24 hr and removed. After fixation the biopsy was sliced into four l-mm wide strips and these were sectioned, stained with hematoxylin-eosin and toluidine blue, and examined microscopically. 3 Nonaggregated albumins, both human but were taken up by macrophages in small
and bovine, were amounts and were
tested in preliminary experiments therefore difficult to use.
MACROPHAGES
Each test site was evaluated cellular infiltration in each number of sections exhibiting To determine the number magnifications) were scanned subcutaneous fibrofatty tissue single high power field (HPF)
IN
DELAYED
HYPERSENSITIVITY
271
I
histologically on a scale of l-4 based on the extent of section, the amount of cellular infiltration, and the an inflammatory response. of basophils, 10 contiguous high power fields (250 in the superficial dermis, the deep dermis, and the of each section. The average number of basophils per was calculated. RESULTS
Macrophage-bound antigen, injected into the footpads of rats, was capable of inducing DH (Table 1). Generally, the skin test reactions were macroand microscopically similar in rats given free or bound antigen. Also, there was a poor
TABLE Su~x~ay
OF EXPERIMENTS IX WHICH RATS WERE INOCULATED WITH AGGRBGATITI HSA TO VIABLE MACROPHAGES, FREE, OR EMULSED IN COMPLETE FREUND'SADJUVANT Aggregated
Macrophage No. x
1
cells 106
A. 14 Days 4
HSA
bound amount (Pd after
footpad 10
Free amount bg)
CFA amount
10 50 50 50 B. 28 Dal-5 4
after
footpad
Induration (mm)
Ab in serum (range of values)
‘X3,6,7
1,2,1,2
0,3,3,5
1,1,1,2
Neg.
15,16,18
4.4,4
+
(>60)
2,3,4,7
2.2.2,l
+
(21-26)
2,5,6,7
1,1,2.2
Neg.
8,10,10,12,13
4,4,4.4,4
Not
3,4,4,5,5 6,6,7,10,11
2,3,4,1,1 3,3,1..1.3
+
wmw
1,2.1,2.2 1,1,1.1,1
Neg.
1,2,2,3,1 1,2&r 1
+
(31-40)
4,6,7,11 0,2,2,3,4
1,1,2,1,2
+
(O-0.4)
4,5,6,7
1,2,2,1
4
+
(l-13)
done
injection
10
10
0,3,4,5,5 4
Histology
injection
10
4
BIXJND
‘W,2,3,3
50
50
n Gross measurements are matched to corresponding histologic evaluation. b Percentage of HSA bound at a 1:2 dilution of sera. c Acute polymorphonuclear leucocyte inflammation and abscess.
(8-28)
6
272
UNANUE
AND
FELDMAN
correlation between the gross induration and the histologic evaluation, although the largest lesions showed the most severe microscopic infiltrations (see Table 1, CFA groups). In one group prepared with 10 pg of macrophage-bound antigen and tested 28 days later, 6 of 10 reactions were rated histologically 3 and 4; in the comparable groups of 10 rats inoculated in their footpads with free antigen, none developed a lesion that was rated 3 or 4. Detectable differences between lesions of other groups of rats were not consistently observed. Inoculation of either 10 or 50 pg of HSA incorporated into CFA induced a high degree of DH as manifested by the vigorous skin reactions in test sites 14 days later. Control rats inoculated with 10 and 50 pg of antigen in CFA and tested 28 days later were not included in Table 1 because their skin lesions were apparently mixtures of both Arthus and DH reactions. As can be seen in Table 1, antibody was found in the sera of all groups of animals that were given macrophage-bound antigen or antigen in CFA, either 14 or 28 days after injection. In one group of rats injected with 50 pg of free heat-aggregated antigen five of nine rats had antibody in their sera at low concentrations, while all of nine given macrophage-bound antigen had antibody in their sera. In Table 2 are shown the results of skin testing with macrophage-bound antigen, macrophages alone, and free antigen. In order to communicate more accurately the effect of macrophages on the skin test reactions, lesions were divided into small (3-5 mm of induration) and large (6-12 mm of induration). Macrophage-bound antigen elicited larger macroscopic lesions at 6 and 24 hr than did free antigen or macrophages without antigen, and the histologic lesions were composed of mixtures of numerous polymorphonuclear and mononuclear leucocytes and of abscesses. Free antigen elicited typical DH responses, composed chiefly of mononuclear cells; they were generally smaller than those induced by
TABLE RESULTS
IN RATS (I TESTED MACROPHAGES,
INTKADERMALLY AND WITH LIVE
Induration
2
WITH 10 pg OF HSA, FREE OR BOUND MACKOPHAGES LACKING ANTIGEN
(mm)
6 Hr
Experimental Mac + Ag Mac Ag Controls Mac Mac &
+ Ag
a Unimmunized
TO LIVE
Histology
24 Hr
3-s
6-12
3-5
6-12
2/11
9/11
s/11
6/11
2/9 8/11
7/9 3/11
7/9 8/l 1
2/9 3/11
PMN’s and mononuclear cells, abscesses Chiefly PMNs, absczssx Chiefly memo ~uclxu c-lls
o/4
4/4 4/4 o/4
2/4
2/4 o/4 o/4
PMN’s and abscesses PMN’s and abscesses No reaction
o/4 4/4 controls
and rats
immunized
4/4 4/4 3 weeks
previously
with
1 mg of HSA
in CF4.
MACROPHAGES
IN
DELAYED
HYPERSENSITI\‘ITY
I
273
i7lacropl?age-bo~Ind antigen. Skin reactions to macrophages that were syngeneic in the tested rats were also smaller than those produced by macrophage-bound antigen and were composed of numerous polymorphonuclear leucocytes and abscesses. A study of the number and distribution of hasophils in test sites was prompted by the recent observations on skin reactions to hapten protein conjugates (11, 12). Test sites were examined at 14 days when basophil response was reported to be maximal in guinea pig skin reactions and at 28 days when basophil response was absent or minimal. ?To significant differences were noted among the different groups of rats listed in Table 1. Basophils ranged from 5-lO/HPF in the deep dermis and subcutaneous tissues and from 2-3/HPF in the superficial dermis beneath the epithelium. The number and distribution were similar in lesions elicited at 14 or 25 days in all groups and in skin unaffected by an inflammatory response. The amount of macroscopic induration and the quality and quantity of cellular infiltration did not alter the coutits of basophils. By contrast, in the groups of rats listed in Table 2 basophils were increased to between 30-70/HPF in lesions elicited by macrophages or macrophages with antigens but not by free antigen. DISCUSSION We conclude from the results of this study that macrophage-bound antigen was capable of inducing DH. By implication, the antigen was bound to cells in such a fashion, presumably on their surfaces, as to be available to the host and to he immunogenic, i.e., not all of it was destroyed by intracellular digestion. That macrophages. in this sytem, might enhance the immunogenicity of antigen for the induction of DH was not decisively determined. In one set of experiments the skin test reactions were detectably more severe in rats inoculatd with antigen 11ound to macrophages than in rats inoculated with free antigen. On the other hand, macrophage-bound antigen was consistently more immunogenic than free antigen in the production of antibody, a result that corroborated and confirmed the enhancing effect of macrophages in other species (2, 4). It would be fruitless speculation to discuss how macrophages might augment the immunogenicity of antigen for the induction of DH, especially since the results were not decisive. The presence or absence of antibody in serum was not related to the development, size, and severity of DH reactions. Nor would it be possible to ascertain whether macrophages by binding antigen might help in the induction of DH in living animals that are suitaMy inoculated. It has been shown that macrophages, treated with adjuvants, augmented in vim the immune response to protein antigens ( 13). Macrophages were irritating in the skins of syngeneic hosts. producing severe acute polymorphonuclear leucocytic inflammation. Xntigen bound to macrophages elicited the most severe reactions in properly prepared hosts and the reactions appeared to be a summation of the effects produced by macrophages alone and by antigen alone, i.e., a DH response was added to the acute nonspecific inflammation. In areas of acute inflammation kindled by the intradermal injection of macrophages or macrophages with antigen, there was a striking increase of basophils. l\*hether this increase was due directly to the presence of macrophages or their cel-
274
UNANUE
AND
FELDMAN
Mar contents, or due to the appearance of chemical mediators in response to macrophages, was not determined.
provided
by the host
REFERENCES 1. Unanue, E. R., and Cerottini, J.-C., Semin. Hematol. 7, 225, 1970. 2. Frei, P. C., Benacerraf, B., and Thorbecke, G. J., Proc. Nat. Acad. Sci. U.S.A. 53, 20, 1965. 3. Unanue, E. R., and Askonas, B. A., Zmnzunology 15, 287, 1968. 4. Mitchison, N. A., Immunology 16, 1, 1969. 5. Seeger, R. C., and Oppenheim, J. J., J. Exp. Med. 132, 44, 1970. 6. Hersh, E. M., and Harris, J. E., J. Immamol. 100, 1184, 1968. 7. Mosier, D. E., Fitch, F. W., Rowley, D. A., and Davies, A. J. S., Fed. Proc. Fed. Amer. Sot. Exfi. Biol. 26, 375, 1969. 8. Pierce, C. W., and Benacerraf, B., Science 166, 1002, 1969. 9. Bosman, C., and Feldman, J. D., Amer. J. Pathol. 56, 201, 1970. 10. Farr, R. S., J. Iujec. Dis. 103, 239, 1958. 11. Richerson, H. B., Dvorak, H. F., and Leskowitz, S., J. Exp. Med. 132, 546, 1970. 12. Dvorak, H. F., Dvorak, A. M., Simpson, B. A. Richerson, H. B., Leskowitz, S., and Karnovsky, M. J., J. Exp. Med. 132, 558, 1970. 13. Unanue, E. R., Askonas, B. A., and Allison, A. C., J. Immunol. 103, 71, 1969.
I~~MLYSOLOGY
Role
269-274
2,
of
Macrophages
I. Induction
EMIL Department
(1971)
R.
in
with
Hypersensitivity
Macrophage-Bound
UNANUE
of Experimental 476 Prosfiect
Delayed
’ AND JOSEPH
Pathology, Scrip@ Street, La /olln, Received
Janzcnvy
Antigen
D.
1
FELD&IAN
Clinic and Research California 92037
Foundation,
22, 1971
Macrophage-bound antigen inoculated into footpads of rats induced a state of delayed hypersensitivity (DH) equivalent to that induced by free antigen but less than that induced by antigen incorporated into complete Freund’s adjuvant. Antigen bound to macrophages was more immunogenic than free antigen and elicited antibody responses in all injected rats. Macrophages injected intradermally into rats immunized with antigen in complete Freund’s adjuvant excited an acute inflammatory reaction consisting of numerous polymorphonuclear leucocytes and abscesses. In the same animals free antigen produced typical DH lesions, and antigen bound to macrophages produced a mixture of polymorphonuclear and mononuclear cell infiltrates. Intradermal deposit of macrophages or macrophages with antigen increased the number of basophils in areas of inflammation. Basophils did not vary in number or distribution in DH lesions elicited by free antigen at 14 or 28 days after inoculation.
INTRODUCTION During induction of an immune response macrophages appear to play a role of variable importance ( 1). These phagocytic cells take up a variety of antigens, which, depending upon their physicochemical state, may be retained at the surface membranes of the macrophages and/or endocytosed and catabolized (reviewed in 1). Association of some antigens with macrophages favors immunogenicity (2), i.e., doses of antigens that would ordinarily not elicit antibody in viva will do so when the antigens are bound to macrophages (3, 4). Macrophage-bound antigen may also, augment manyfold the production of antibody (3, 4). In in vitro systems, macrophages appear to be needed for lymphocytes to proliferate (5, 6) or to synthesize antibody in the presence of antigen (7, 8). The participation of macrophages in delayed hypersensitivity (DH) has not been fully explo’red. although it is well known that these cells form a major part of the 1 This is Publication No. 467 from the Department of Experimental Pathology, Clinic and Research Foundation, La Jolla, Calif. This work was supported by a United Public Health Grant -41-07007 and Atomic Energy Commission Contract -4T (04-3) -779. 2 Supported by a Senior Present Address : Department
Scripps States
Fellowship of the iZmerican Cancer Society, California Division. of Pathology, Harvard Medical School, Boston, Mass. 02115.
270
UNANUE
AND
FELDMAN
inflammatory DH skin lesion (9). In this paper we report on the role of macrophages in the induction of DH and in the following paper the effect of anti-macrophage antibodies oa the expression of DH. MATERIALS
AND
METHODS
Inbred Lewis (Le) or (Lewis X Buffalo)F, (LBfF,) rats, (both strains purchased from Simonsen Laboratories, Gilroy, Calif., or Microbiological Associates, Inc., Walkersville, Md.) weighing 250-300 g, were used as donors of macrophages and as test animals for the induction of DH. Macrophages were harvested from the peritoneal cavities of rats injected intraperitoneally 3 days before with 3 ml of peptone (Difco Laboratories, Detroit, Mich.) . They were incubated in suspension for 1 hr at 37” with appropriate quantities of heat-aggregated 1311-labeled human serum albumin ( lSII-HSA) (Reheis Chemical Co., Division of Armour Pharmaceutical Co., Los Angeles, Calif.) and then were washed 3 times with balanced salt solution.3 An aliquot of cells was counted in a gamma ray spectrometer. In different experiments about 1 X lo7 cells bound from 1 to 10 PLg of antigen as calculated from radioactive counts. Groups of Le or LBfF, rats were inoculated in their footpads with sufficient syngeneic cells (generally 4-5 X 107) to deliver 5-50 pg of antigen, and 14 or 28 days later they were skin tested with 10 pg of soluble HSA (nonaggregated) in 0.1 ml of saline. Erythema and induration of skin test sites were measured in millimeters at 6 and 24 hr, and rats were bled at 24 hr for detection of circulating antibody by the Farr technique (10). The percentage of 1311-HSA bound at a 1:2 dilution of sera was determined. No attempt was made to establish the dilution of sera binding 33% of the antigen. For comparison, other groups of rats were simultaneously prepared by injecting into the footpads equivalent amounts of free antigen or antigen in a water and oil emulsion containing 1 mg of dried M. tuberc&sis (H37Ra) (Difco Laboratories, Detroit, Mich.) per ml of emulsion (CFA). Several groups of rats were given 5-50 pg of free or macrophage-bound antigen subcutaneously, intravenously, and intraperitoneally. They were skin tested between 6 and 28 days later. Induction of DH with antigen administered by these routes was infrequently achieved, and when successful, skin reactions were weakly positive. The results of these groups will therefore not be reported. In another series of experiments, the efficacy of antigen bound to viable macrophages in eliciting positive skin tests was compared with the efficacy of free antigen. Groups of rats were inoculated in their footpads with 1 mg of antigen in CFA. Fourteen days later each rat was injected intradermally in three different sites with 5 X lo6 syngeneic macrophages carrying 10 PLg of antigen, 5 X lo6 syngeneic macrophages without antigen, and 10 pg of free antigen. A control group, uninoculated, was skin tested simultaneously with the three different preparations. Skin test sites from all rats were measured grossly at 24 hr and removed. After fixation the biopsy was sliced into four l-mm wide strips and these were sectioned, stained with hematoxylin-eosin and toluidine blue, and examined microscopically. 3 Nonaggregated albumins, both human but were taken up by macrophages in small
and bovine, were amounts and were
tested in preliminary experiments therefore difficult to use.
MACROPHAGES
Each test site was evaluated cellular infiltration in each number of sections exhibiting To determine the number magnifications) were scanned subcutaneous fibrofatty tissue single high power field (HPF)
IN
DELAYED
HYPERSENSITIVITY
271
I
histologically on a scale of l-4 based on the extent of section, the amount of cellular infiltration, and the an inflammatory response. of basophils, 10 contiguous high power fields (250 in the superficial dermis, the deep dermis, and the of each section. The average number of basophils per was calculated. RESULTS
Macrophage-bound antigen, injected into the footpads of rats, was capable of inducing DH (Table 1). Generally, the skin test reactions were macroand microscopically similar in rats given free or bound antigen. Also, there was a poor
TABLE Su~x~ay
OF EXPERIMENTS IX WHICH RATS WERE INOCULATED WITH AGGRBGATITI HSA TO VIABLE MACROPHAGES, FREE, OR EMULSED IN COMPLETE FREUND'SADJUVANT Aggregated
Macrophage No. x
1
cells 106
A. 14 Days 4
HSA
bound amount (Pd after
footpad 10
Free amount bg)
CFA amount
10 50 50 50 B. 28 Dal-5 4
after
footpad
Induration (mm)
Ab in serum (range of values)
‘X3,6,7
1,2,1,2
0,3,3,5
1,1,1,2
Neg.
15,16,18
4.4,4
+
(>60)
2,3,4,7
2.2.2,l
+
(21-26)
2,5,6,7
1,1,2.2
Neg.
8,10,10,12,13
4,4,4.4,4
Not
3,4,4,5,5 6,6,7,10,11
2,3,4,1,1 3,3,1..1.3
+
wmw
1,2.1,2.2 1,1,1.1,1
Neg.
1,2,2,3,1 1,2&r 1
+
(31-40)
4,6,7,11 0,2,2,3,4
1,1,2,1,2
+
(O-0.4)
4,5,6,7
1,2,2,1
4
+
(l-13)
done
injection
10
10
0,3,4,5,5 4
Histology
injection
10
4
BIXJND
‘W,2,3,3
50
50
n Gross measurements are matched to corresponding histologic evaluation. b Percentage of HSA bound at a 1:2 dilution of sera. c Acute polymorphonuclear leucocyte inflammation and abscess.
(8-28)
6
272
UNANUE
AND
FELDMAN
correlation between the gross induration and the histologic evaluation, although the largest lesions showed the most severe microscopic infiltrations (see Table 1, CFA groups). In one group prepared with 10 pg of macrophage-bound antigen and tested 28 days later, 6 of 10 reactions were rated histologically 3 and 4; in the comparable groups of 10 rats inoculated in their footpads with free antigen, none developed a lesion that was rated 3 or 4. Detectable differences between lesions of other groups of rats were not consistently observed. Inoculation of either 10 or 50 pg of HSA incorporated into CFA induced a high degree of DH as manifested by the vigorous skin reactions in test sites 14 days later. Control rats inoculated with 10 and 50 pg of antigen in CFA and tested 28 days later were not included in Table 1 because their skin lesions were apparently mixtures of both Arthus and DH reactions. As can be seen in Table 1, antibody was found in the sera of all groups of animals that were given macrophage-bound antigen or antigen in CFA, either 14 or 28 days after injection. In one group of rats injected with 50 pg of free heat-aggregated antigen five of nine rats had antibody in their sera at low concentrations, while all of nine given macrophage-bound antigen had antibody in their sera. In Table 2 are shown the results of skin testing with macrophage-bound antigen, macrophages alone, and free antigen. In order to communicate more accurately the effect of macrophages on the skin test reactions, lesions were divided into small (3-5 mm of induration) and large (6-12 mm of induration). Macrophage-bound antigen elicited larger macroscopic lesions at 6 and 24 hr than did free antigen or macrophages without antigen, and the histologic lesions were composed of mixtures of numerous polymorphonuclear and mononuclear leucocytes and of abscesses. Free antigen elicited typical DH responses, composed chiefly of mononuclear cells; they were generally smaller than those induced by
TABLE RESULTS
IN RATS (I TESTED MACROPHAGES,
INTKADERMALLY AND WITH LIVE
Induration
2
WITH 10 pg OF HSA, FREE OR BOUND MACKOPHAGES LACKING ANTIGEN
(mm)
6 Hr
Experimental Mac + Ag Mac Ag Controls Mac Mac &
+ Ag
a Unimmunized
TO LIVE
Histology
24 Hr
3-s
6-12
3-5
6-12
2/11
9/11
s/11
6/11
2/9 8/11
7/9 3/11
7/9 8/l 1
2/9 3/11
PMN’s and mononuclear cells, abscesses Chiefly PMNs, absczssx Chiefly memo ~uclxu c-lls
o/4
4/4 4/4 o/4
2/4
2/4 o/4 o/4
PMN’s and abscesses PMN’s and abscesses No reaction
o/4 4/4 controls
and rats
immunized
4/4 4/4 3 weeks
previously
with
1 mg of HSA
in CF4.
MACROPHAGES
IN
DELAYED
HYPERSENSITI\‘ITY
I
273
i7lacropl?age-bo~Ind antigen. Skin reactions to macrophages that were syngeneic in the tested rats were also smaller than those produced by macrophage-bound antigen and were composed of numerous polymorphonuclear leucocytes and abscesses. A study of the number and distribution of hasophils in test sites was prompted by the recent observations on skin reactions to hapten protein conjugates (11, 12). Test sites were examined at 14 days when basophil response was reported to be maximal in guinea pig skin reactions and at 28 days when basophil response was absent or minimal. ?To significant differences were noted among the different groups of rats listed in Table 1. Basophils ranged from 5-lO/HPF in the deep dermis and subcutaneous tissues and from 2-3/HPF in the superficial dermis beneath the epithelium. The number and distribution were similar in lesions elicited at 14 or 25 days in all groups and in skin unaffected by an inflammatory response. The amount of macroscopic induration and the quality and quantity of cellular infiltration did not alter the coutits of basophils. By contrast, in the groups of rats listed in Table 2 basophils were increased to between 30-70/HPF in lesions elicited by macrophages or macrophages with antigens but not by free antigen. DISCUSSION We conclude from the results of this study that macrophage-bound antigen was capable of inducing DH. By implication, the antigen was bound to cells in such a fashion, presumably on their surfaces, as to be available to the host and to he immunogenic, i.e., not all of it was destroyed by intracellular digestion. That macrophages. in this sytem, might enhance the immunogenicity of antigen for the induction of DH was not decisively determined. In one set of experiments the skin test reactions were detectably more severe in rats inoculatd with antigen 11ound to macrophages than in rats inoculated with free antigen. On the other hand, macrophage-bound antigen was consistently more immunogenic than free antigen in the production of antibody, a result that corroborated and confirmed the enhancing effect of macrophages in other species (2, 4). It would be fruitless speculation to discuss how macrophages might augment the immunogenicity of antigen for the induction of DH, especially since the results were not decisive. The presence or absence of antibody in serum was not related to the development, size, and severity of DH reactions. Nor would it be possible to ascertain whether macrophages by binding antigen might help in the induction of DH in living animals that are suitaMy inoculated. It has been shown that macrophages, treated with adjuvants, augmented in vim the immune response to protein antigens ( 13). Macrophages were irritating in the skins of syngeneic hosts. producing severe acute polymorphonuclear leucocytic inflammation. Xntigen bound to macrophages elicited the most severe reactions in properly prepared hosts and the reactions appeared to be a summation of the effects produced by macrophages alone and by antigen alone, i.e., a DH response was added to the acute nonspecific inflammation. In areas of acute inflammation kindled by the intradermal injection of macrophages or macrophages with antigen, there was a striking increase of basophils. l\*hether this increase was due directly to the presence of macrophages or their cel-
274
UNANUE
AND
FELDMAN
Mar contents, or due to the appearance of chemical mediators in response to macrophages, was not determined.
provided
by the host
REFERENCES 1. Unanue, E. R., and Cerottini, J.-C., Semin. Hematol. 7, 225, 1970. 2. Frei, P. C., Benacerraf, B., and Thorbecke, G. J., Proc. Nat. Acad. Sci. U.S.A. 53, 20, 1965. 3. Unanue, E. R., and Askonas, B. A., Zmnzunology 15, 287, 1968. 4. Mitchison, N. A., Immunology 16, 1, 1969. 5. Seeger, R. C., and Oppenheim, J. J., J. Exp. Med. 132, 44, 1970. 6. Hersh, E. M., and Harris, J. E., J. Immamol. 100, 1184, 1968. 7. Mosier, D. E., Fitch, F. W., Rowley, D. A., and Davies, A. J. S., Fed. Proc. Fed. Amer. Sot. Exfi. Biol. 26, 375, 1969. 8. Pierce, C. W., and Benacerraf, B., Science 166, 1002, 1969. 9. Bosman, C., and Feldman, J. D., Amer. J. Pathol. 56, 201, 1970. 10. Farr, R. S., J. Iujec. Dis. 103, 239, 1958. 11. Richerson, H. B., Dvorak, H. F., and Leskowitz, S., J. Exp. Med. 132, 546, 1970. 12. Dvorak, H. F., Dvorak, A. M., Simpson, B. A. Richerson, H. B., Leskowitz, S., and Karnovsky, M. J., J. Exp. Med. 132, 558, 1970. 13. Unanue, E. R., Askonas, B. A., and Allison, A. C., J. Immunol. 103, 71, 1969.