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Alpha globulin changes during the development of cellular immunity
CELLULAR
IMMUNOLOGY
Alpha Globulin DENIS
8, 147-154 (1973)
Changes During the Development
R. BURGER, DAVID
P. LILLEY,
MARILYK
AND R. MARK The
REID,
of Cellular LAWRENCE
Immunity IRISH,
VETTO
Surgical Research Laboratory, Veterans Administration Hospital and Departmeltt of Microbiology, University of Oregon Medical School, Portknd, Oregon 97207 Received
November
15, 1972
Hartley guinea pigs were sensitized to 2,4-dinitrofluorobenzene, Mycobacterium tuberculosis H37RA, and allogenic skin. During the development of these sensitivities, serum alpha globulin changes were detected by polyacrilamide gel disc electrophoresis. Electrophoretic region 4 (alpha gIobulins) increased, then subsided, concomitant with the development of cellular immunity. Skin testing did not seem to influence alpha globulin levels in sensitized animals. However, high alpha globulin levels decreased skin test responsiveness. It is suggested that the increased alpha globulin noted here is immunoregulative alpha globulin (IRA).
INTRODUCTION The association of serum globulin changes with various cellular immune phenomena has been tenuous. Attempts to correlate serum changes with tuberculin hypersensitivity ( 1) or contact dermatitis (2) using cellulose-acetate and immunoelectrophoresis have been unsucessful. However, Riggio et al. (3) have reported alpha globulin changes in human renal allograft recipients during rejection crises. In addition, alpha globulins from human serum were found to be immunosuppressive in viva (4-6) and in vitro (7-9). I mmunosuppression was induced with increasing amounts of alpha globulins from normal sera and, therefore, was due to a quantitative change in a normal serum constituent (5-8). It has been suggested that the active moiety is an immunoregulative alpha globulin (IRA) which participates in the regulation of T lymphocyte function (7, 8). If IRA is important in regulating T cell dependent immune responses, changes in IRA levels may occur in certain anergic conditions (IO, 11) and during antigen sensitization. The purpose of this work was to monitor serum globulin levels by disc gel electrophoresis during the development of cell-mediated immunity in the guinea pig. During sensitization to dinitrofluorobenzene, tuberculin, and allogenic skin, an alpha globulin fraction increased, then subsided, concomitant with the development of cellular immunity. MATERIALS Sensitization
to ,3,4-L)i)zitrofEuorobcllcellc
AND
METHODS
(DNFB)
Hartley strain guinea pigs weighing 350-600 g were used in this study. Animals were sensitized by topical application of 2% (v/v) DNFB in alcohol each day for 147 Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
BURCXR
148
ET
TABLE SERUM
GL~B~~LIN
I,~VSI,S
AL 1
IN C~NTK~L
GKOUP
GUINEA
Prcs
Bleeding numberb
Electrophorctic fraction”
___
1 2 3 4 5 6 7 8 Y
20.7 22.1 2.6 9.8 5.9 11.0 13.1 7.6 7.1
1 f f & i f + f & i
2 3.1" 2.6 0.6 1.1 0.8 2.2 2.8 1.8 1.3
20.5 21.8 3.0 10.0 6.2 11.0 12.5 7.6 7.2
zk zk f f f f zt f xk
3 2.2 1.7 0.3 1.1 0.9 2.1 2.9 1.9 1.0
20.6 21.2 2.8 10.3 5.9 11.2 13.0 7.2 7.5
zk f * i f f rk z!z f
5
4 1.6 1.6 0.4 1.3 0.9 2.3 1.8 2.4 1.0
20.4 22.4 2.7 10.0 5.4 11.5 12.8 7.1 7.4
+ f f i f zt =t zk i
l.Y 1.9 0.8 0.6 0.9 2.3 2.4 1.9 1.3
20.8 21.3 3.2 9.7 5.9 11.4 12.6 7.6 7.5
i f f i i f f f i
6 1.7 1.6 0.3 0.9 1.0 2.0 2.9 1.6 1.3
20.7 19.8 3.1 9.8 6.0 12.0 13.4 7.3 7.0
f f f f i zk * zk f
1.7 3.4 0.4 0.8 0.9 2.1 2.0 1.6 1.3
e May represent more than one electrophoretic species. b Bleedings taken on day -1, 3, 6, 10, 11, and 17 respective to the DNFB treatment group. L Mean globulin percent &SD. 6 days as described (12). Twenty animals were bled by cardiac puncture 1 day prior to the first DNFB treatment and on days 3, 6, 10, 11, and 17 after the initial treatment. Ten control animals were treated with alcohol alone and bled according to the same schedule. All blood samples (3 ml) were allowed to clot and the serum collected and stored at -20°C. All animals were skin tested on day 10 with 0.5% (v/v) DNFB in olive oil. The reactions were graded 45 hr later according to the following scheme : 0, no detectable reaction ; 1, patchy erythema ; 2, homogenous erythema; 3, marked erythema and raised reaction site. Sensitizatiolz
to Tuberculin
Guinea pigs were sensitized to tuberculin by subcutaneous inoculation of 1 mg heat-killed Mycobacteriurn tuberculosis H37RA in 1 ml Freunds’ incomplete adjuvant (Difco) in the nape of the neck. Thirteen animals were bled 1 day before immunization and on days 5, 10, 15, 20, and 25 after inoculation. Ten control animals were injected with incomplete Freunds’ adjuvant alone and bled according to
I
2
3
4
5
6
FIG. 1. Stained polyacrilamide gels after electrophoresis of serum from sequential bleedings (l-6) of a typical guinea pig during sensitization to DNFB. The arrows designate globulin fraction 4 which increases to a maximum by the third bleeding and then subsides.
ALPHA
~LoxmLrN
CHANGES
IN
CELLULAR
149
IMMUNITY
the above schedule. All animals were skin tested intradermally with 10 pg PPD (Parke-Davis) 24 days after treatment. The reactions were graded by measuring the diameter of induration 48 hr after skin testing. Sensitixtion
to Allogenic
Skin
Fifteen animals were sensitized to allogenic skin by grafting full thickness, 1 cmZ, abdominal skin bilaterally on the back. The skin grafts were protected with Blenderm bandage (331). The grafts were examined after 5 days and animals rebandaged. Ten control animals received autologous skin grafts. All animals were bled 1 day before grafting and on days 3, 6, 10, and 14 after grafting. Allogenic skin grafts were considered rejected when the graft became thick, hard and necrotic. Autografts were uniformly accepted. Disc Gel Glectrophoresis Polyacrilamide disc gel electrophoresis was carried out in 7% acrilamide gels at 1.5 m amps/gel for 45 min according to Davis (13) and Ornstein (14). To facilitate comparison, all serum samples from a single animal were electrophoresed together. The gels were stained with amido Schwartz, destained in 10% acetic acid, and scanned in a densitometer (Photovolt). The percent of each globulin fraction was calculated by automatic integration of the gel tracing. RESULTS Serum
Globulin
Densitometry were sufficiently
Changes
During
Sensitization
to 2,4-Dinitrol4uorobenzcne
of the stained disc gels indicated that nine serum globulin regions distinct to be quantitated for these experiments. Some of these TABLE
2
SERUM GLOBULIN CHANGES IN GUINEA PIGS DUKING SENSITIZATION TO 2, 6DINTIROFLUOKOBENZENE Electrophoretic fraction” 1 2 3 4 5 6 7 8 9
Bleeding 1 19.9 23.6 3.0 10.2 5.3 11.9 12.5 7.7 5.8
f 6.1C i 4.4 i 0.7 zk 1.6 * 1.1 ZJZ2.9 f 5.9 f 1.5 f 1.2
2 18.8 20.4 3.2 17.0 4.7 12.2 10.6 7.2 5.7
f 3.6 f 3.1 f 0.7 f 3.4d f 1.1 f 2.3 f 4.7 f 1.9 f 1.3
number* 4
3 17.9 20.5 3.5 17.1 4.6 12.6 10.4 7.0 6.3
f f i f f f i f f
3.0 2.8 0.5 2.6” 1.1 2.6 4.6 2.0 1.6
20.0 19.8 3.4 13.1 5.3 13.4 10.9 7.8 6.3
f i f f f f i f f
6
5 3.0 2.3 1.1 2.1 1.7 2.7 3.7 2.0 1.4
22.1 21.0 3.0 11.9 5.0 12.7 11.0 7.2 6.0
f 3.0 f 2.8 Ik 1.1 ziz 1.6 f 1.1 f 2.7 f 4.1 f 2.0 zt 0.9
20.8 22.1 3.4 9.6 5.3 12.5 12.0 8.1 6.2
f f f f f f * + f
D May represent more than one electrophoretic species. b Bleedings taken on day -1, 3, 6, 10, 11, and 17 respective to DNFB treatment. c Mean globulin percent &SD. d P < 0.01; compared to fraction 4, DNFB-group, bleeding 1 and control group, bleeding (Table 1). a P < 0.01; compared to fraction 4, DNFB-group, bleeding 1 and control group, bleeding (Table 1).
3.7 3.1 0.7 1.0 1.5 2.0 3.9 2.3 2.0
2 3
150
BURGER
ET
AL.
2. A densitometer scan of a stained polyacrilamide gel after electrophoresis of serum FIG. from a control group animal. The broken line (- - -) represents the change in the scan observed when serum from an animal undergoing sensitization to DNFB was electrophoresed (third bleeding). The scan is divided into nine fractions. The percent globulin of each fraction was calculated by integration and is shown for a control group and sensitized animal (third bleeding). A highly significant increase (P < 0.01) in fraction 4 was observed. regions contained more than one electrophoretic species, but quantitation by planimetry could not distinguish between closely migrating globulins. The variability of these nine regions in control animals is presented in Table 1. No significant differences were noted in any of the globulin regions during the 3-wk study period.
In ten additional animals treated topically with the nonsensitizing
irritants
carbon-
tetrachloride
or petroleum ether, no significant changes were noted. When the nine regions from DNFB treated animals were compared to controls, significant
globulin changes in region 4 were noted on the second and third bleeding (3 and 6 days
Globulin
Fraction
FIG. 3. A comparison of serum globulin changes in animals sensitized to allogenic skin. The shaded areas represent the mean globulin percent 2 SD of nine electrophoretic fractions of serum from autografted animals (third bleeding). The broken line (- - -) represents the significant increase (P < 0.01) in globulin fraction 4 in allografted animals at this time.
ALPHA
GLOBULIN
CHANGES
IN
CELLULAR
IMMUNITY
1.51
during sensitization) (Table 2). Globulin region 4 (alpha globulins) increased from a mean percent of 10.2 * 1.6 prior to treatment to 17.0 * 3.4 and 17.1 f 2.6, 3 and 6 days during treatment respectively. By day 17, this region had returned to normal levels (3 = 9.6 r+ 1.0). These changes are highly significant (P < 0.01) and easily observed in the gels prior to densitometry (Fig. 1) . Densitometer tracings of control and test group sera are shown in Fig. 2 and further demonstrated the significant increase in alpha globulins. There was not a significant change in the total globulin levels during these procedures. It seems, therefore, that the increase in alpha globulin represents a change in absolute amount of this electrophoretic species. Serum
Changes
During
Sensitization
to Mycobacterium
tuberculosis
H37RA
Animals sensitized to Mycobacteriuwt tuberculosis also show a significant rise in alpha globulins (region 4). This increase in alpha globulins was evident at different times after immunization (Table 3). The mean alpha globulin percent from any one bleeding after immunization did not significantly differ from controls or preimmunization values. However, the mean of the highest values obtained after immunization (14.4 * 2.7) was significantly different (P < 0.01) from controls (a = 9.6 * 1.0) and preimmunization values (5 = 9.2 f 1.7). Serum
Globulin
In allografted when compared
Changes
During
TABLE IN SERCM
GLOBULIN
TO Mycobacterium
3
4 DURING SENSITIZATION Tuberculosis H37RA
FRACTION
Immunized
Adjuvant control group Guinea pig Highest values 1 2 3 4 5 6 7 8 9 10 11 12 13 Mean fSD
10.1” 9.2 8.8 11.1 9.1 9.6 9.8 10.3 10.2 12.2 9.1 8.6 a.7 9.8 3~ 1.0
Skin
increase in globulin region 4 was observed The change in alpha globulins was restricted
animals, a significant to autograft recipients.
CHANWS
to Allogenic
Sensitization
group
Postimmunization Preimmunization
9.5 9.6 9.3 6.5 8.9 10.2 7.1 9.2 9.3 12.5 11.8 8.0 8.1 9.2 f
Highest value
Bleeding 6 4 2 4 3 1 6 2 3 4 3 2 3
17.8 14.0 18.7 17.8 13.1 10.2 13.5 12.5 13.3 15.5 17.0 12.7 11.1 1.7
o Percent fraction 4 of total globulin. b P < 0.01; compared to adjuvant alone or preimmunization
14.4 f
values.
2.7b
Percent increase
87 46 101 174 47 0 90 36 43 24 44 59 37
BURGER
152
ET
AL.
DNFB-DERMITITIS
2or
2 2Or
OL
T
7
TUBERCULIN
-7
HYPERSENSITIVITY 1
i I
1 mI--II.. 2
-Li 3
Bleeding
4
5
6
Number
FIG. 4. A comparison of globulin region 4 (alpha) values from control animals ( 0 0 0 ) and animals undergoing sensitization (000) to DNFB (top), allogenic skin (middle), and Mycobacterium tuberculosis (bottom). A significant increase (P < 0.01) in this region in animals sensitized to DNFB and allogenic skin is noted on bleedings two and three. The significant change in the tuberculin group only noted when the highest region 4 values were compared (Table 3), was not evident on any single bleeding.
to 3 and 6 days after grafting as was noted in DNFB sensitization. The maximum increase (9.5 f 1.6 to 15.3 + 2.6, P < 0.01) occurred 6 days after allografting and is plotted in Fig. 3. A comparison of the changes in region 4 for each of the sensitization procedures is presented in Fig. 4. It should be noted that highly significant changes (P < 0.01) are observed on the second and third bleedings in both the DNFB and allografted groups but at no single time in the tuberculin group. Egects
of High
Alpha
Globulin
Levels
on Skin Tests
Animals sensitized to DNFB were usually skin test positive (2-3 + reactions) 11 days after initial treatment. This coincided with a return of region 4 values into in additional experiments 3 of 46 normal ranges (a = 10.6 t 1.3). However, animals at this time showed very weak or negative skin tests (0, I+, l+). These three weak responding guinea pigs had elevated globulins in region 4 (Z = 16.2 -C 1.6, P < 0.01). Conversely, the remaining 43 animals were all skin test positive (2-3 + reactions) and none demonstrated elevated region 4 globulins (x’ = 10.2 +1.2). Eleven of thirteen animals immunized with Mycobacterium tuberculosis demon-
ALPHA
GLOBULIN
CHANGES
IN
CELLULAR
IMMUNITY
153
strated positive skin tests (> 10 mm induration) 25 days later. The 11 skin test positive animals had normal globulin levels in region 4 (5 = 10.1 * 1.6) at the time of skin testing. On the other hand, both of the animals that were skin test negative (Table 2, No. 1 and 7, < 5 mm induration) had significantly elevated region 4 globulins (17.8 and 13.5, respectively) at the time of skin testing. EfJects of Skin
Tests on Alpha
Globulin
Levels
In additional experiments, 20 animals were sensitized to DNFB, rested for 10 days (to allow region 4 globulins to return to pre-DNFB levels), and skin tested. The effect of the skin test on alpha globulin levels was observed at 24 and 48 hr. Although all animals in this group displayed strong (3+) skin reactions, changes in alpha globulins at 24 or 48 hr could not be demonstrated. The mean percent of globulin region 4 was 10.2 2 1.1 prior to skin testing, 10.6 * 1.3 24 hr after testing, and 9.8 -t 1.3 48 hr after testing. DISCUSSION In this study, serum alpha globulin changes were associated with immunizations which lead to the development of cell-mediated immunity. In DNFB-induced contact dermatitis, tuberculin hypersensitivity, and allograft immunity electrophoretic region 4 (alpha) globulins increased, then subsided, concomitant with the development of cellular immunity. In DNFB and allograft sensitization, the rise in alpha globulins occurred predictably 3-6 days after antigen exposure and usually subsided by the tenth day (Fig. 4). The increase in alpha globulins after mycobacterium exposure was observed over a broader period of time (Table 3). This difference may be related to the time required to develop cellular immunity and may, in part, reflect differences in antigen, combination with adjuvant, or route. An alpha globulin change was not detected in one animal in the Mycobacterium group. This represented the only such observation in 91 animals sensitized in one of these three protocols. Since this was observed in the Mycobacterium group, it seems likely that the alpha globulin change was missed between bleedings. In our hands, skin tests applied to sensitized animals did not seem to influence alpha globulin levels. However, this result may be influenced by the inability of these procedures to accurately detect small changes in closely migrating electrophoretic species. On the other hand, high alpha globulin levels seemed to influence skin testing. Animals that had high alpha globulin levels at the time of skin testing responded weakly or not at all. Conversely, all sensitized animals with normal alpha globulin levels had a strong skin test response. This observation may have implications in transient anergic conditions that occur in some human diseases (10) including cancer (11). In the latter case, changes in alpha globulins associated with tumor development have been noted (15, 16). The alpha globulin changes described here may well be changes in IRA levels (8). Others have shown that IRA from man has the capacity to suppress PHA and antigen-induced lymphocyte transformation in vitro (7, 9) and inhibit humoral and cellular responses to specific antigen in viva (17, 6). The mechanism of immune regulation by IRA remains obscure. It does not seem to involve antigenic competition ( 18) or disruption of RNA mediated events by RNase activity (8) as previously proposed (19, 20). The mechanism of regulation may involve modulation
1.54
BURGER
ET
AL.
of T lymphocyte function. Experiments of Cooperband et al. (8) and Glasgow et al. (21) suggest that IRA may function as a competitive inhibitor of early T cell recognition events without directly effecting antigen binding. Physiochemically IRA behaves as a small molecular weight polypeptide (22) which is casually associated with alpha globulins. These characteristics bear similarity to at least two other substances reputed to influence T lymphocyte function : thymosin and transfer factor. The thymic hormone, thymosin, appears to be a polypeptide of 12,600 MW (23) and transfer factor is described as a dialyzable polypeptide-polynucleotide (24, 25). Since T lymphocyte regulation may involve the combined influence of IRA, thymosin, and transfer factor, caution should be exercised in interpreting the influences of small molecular weight dialysates on T cell function in vi770 or in vitro. ACKNOWLEDGMENTS This work was supported in part by the American Cancer Society, Oregon Division. We are grateful to Thomas A. Vetto for skillful technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Dardas, T. J., and Mallmann, V. H., 1. Bacterial. 92, 76, 1966. Burger, D. R., Thesis, The University of Arizona, Tucson, Arizona, June, 1967. Riggio, R. R., Schwartz, G. I-I., Stenzel, K. H., and Rubin, A. L., Lancet 1, 1218, 1968. Kamrin, B. B., Proc. Sot. Exp. Biol. Med. 100, 58, 1959. Mowbray, J. F., Trms~luntation 1, 15, 1963. Mannick, J. A., and Schmid, K., Trux.r~lanfation 5, 1231, 1967. Cooperband, S. R., Bondevik, H., Schmid, K., and Mannick, J. A., Science 159, 1243, 1968. Cooperband, S. R., Davis, R. C., Schmid, K., and Mannick, J. A., Trmsphnt. Proc. 1, 516, 1969. 9. Riggio, R. R., Schwartz, G. H., Bull, F. Geoffrey, Stenzel, K. H., and Rubin, A. L., Transplantation 8, 689, 1969. 10. McFarlin, D. E., and Oppenheim, J. J., 1. Zmmunol. 103, 1212, 1969. 11. Eilber, F., and Morton, D. L., Cancer 23, 362, 1970. 12. Burger, D. R., and Jeter, W. S., Infect. Immun. 4, 575, 1971. 13. Davis, D. B., Ann. N. Y. Acad. Sci. 125, 403, 1964. 14. Omstein, L., Ann. N. Y. Acad. Sci. 121, 321, 1964. 15. Ashikawa, K., Inoue, K., Shimizu, T., and Ishibashi, Y., Jap. J. Enp. Med. 41, 339, 1971. 16. Hsu, C. C. S., and LoGerfo, P., Proc. Sot. Exp. Biol. Med. 139, 575, 1972. 17. Glasgow, A. H., Cooperband, S. R., Occhino, J. C., S&mid, K., and Mannick, J. A., Proc. Sot. Exp. Biol. Med. 138, 753, 1971. 18. Glasgow, A. H., Cooperband, S. R., Schmid, K., Parker, J. T., Occhino, J. C., and Mannick, J. A., Trarzsplavat. Proc. 3, 835, 1971. 19. Mowbray, J. F., Fed. Proc. 22, 441, 1963. 20. Mowbray, J. F., and Scholand, S., Immunology 11, 421, 1966. 21. Glasgow, A. H., Cooperband<, S. R., and Mannick, J. A., Fed. Proc. 31, 3314, 1972. 22. Glasgow, A. H., Occhino, J. C., Badger, A. M., Schmid, K., and Mannick, J. A., Szlrg. Forum 22, 273, 1971. 23. Hardy, M. A., Goldstein, A. L., and White, A., Surg. Forum 23, 305, 1972. 24. Lawrence, H. S., Al-Askari, S., David, J. R., Franklin, E. C., and Zweiman, B., Trans. Ass. Amer. Physicians 76, 84, 1963. 25. Burger, D. R., Vetto, R. M., and Malley, A., Science 175, 1473, 1972.
IMMUNOLOGY
Alpha Globulin DENIS
8, 147-154 (1973)
Changes During the Development
R. BURGER, DAVID
P. LILLEY,
MARILYK
AND R. MARK The
REID,
of Cellular LAWRENCE
Immunity IRISH,
VETTO
Surgical Research Laboratory, Veterans Administration Hospital and Departmeltt of Microbiology, University of Oregon Medical School, Portknd, Oregon 97207 Received
November
15, 1972
Hartley guinea pigs were sensitized to 2,4-dinitrofluorobenzene, Mycobacterium tuberculosis H37RA, and allogenic skin. During the development of these sensitivities, serum alpha globulin changes were detected by polyacrilamide gel disc electrophoresis. Electrophoretic region 4 (alpha gIobulins) increased, then subsided, concomitant with the development of cellular immunity. Skin testing did not seem to influence alpha globulin levels in sensitized animals. However, high alpha globulin levels decreased skin test responsiveness. It is suggested that the increased alpha globulin noted here is immunoregulative alpha globulin (IRA).
INTRODUCTION The association of serum globulin changes with various cellular immune phenomena has been tenuous. Attempts to correlate serum changes with tuberculin hypersensitivity ( 1) or contact dermatitis (2) using cellulose-acetate and immunoelectrophoresis have been unsucessful. However, Riggio et al. (3) have reported alpha globulin changes in human renal allograft recipients during rejection crises. In addition, alpha globulins from human serum were found to be immunosuppressive in viva (4-6) and in vitro (7-9). I mmunosuppression was induced with increasing amounts of alpha globulins from normal sera and, therefore, was due to a quantitative change in a normal serum constituent (5-8). It has been suggested that the active moiety is an immunoregulative alpha globulin (IRA) which participates in the regulation of T lymphocyte function (7, 8). If IRA is important in regulating T cell dependent immune responses, changes in IRA levels may occur in certain anergic conditions (IO, 11) and during antigen sensitization. The purpose of this work was to monitor serum globulin levels by disc gel electrophoresis during the development of cell-mediated immunity in the guinea pig. During sensitization to dinitrofluorobenzene, tuberculin, and allogenic skin, an alpha globulin fraction increased, then subsided, concomitant with the development of cellular immunity. MATERIALS Sensitization
to ,3,4-L)i)zitrofEuorobcllcellc
AND
METHODS
(DNFB)
Hartley strain guinea pigs weighing 350-600 g were used in this study. Animals were sensitized by topical application of 2% (v/v) DNFB in alcohol each day for 147 Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
BURCXR
148
ET
TABLE SERUM
GL~B~~LIN
I,~VSI,S
AL 1
IN C~NTK~L
GKOUP
GUINEA
Prcs
Bleeding numberb
Electrophorctic fraction”
___
1 2 3 4 5 6 7 8 Y
20.7 22.1 2.6 9.8 5.9 11.0 13.1 7.6 7.1
1 f f & i f + f & i
2 3.1" 2.6 0.6 1.1 0.8 2.2 2.8 1.8 1.3
20.5 21.8 3.0 10.0 6.2 11.0 12.5 7.6 7.2
zk zk f f f f zt f xk
3 2.2 1.7 0.3 1.1 0.9 2.1 2.9 1.9 1.0
20.6 21.2 2.8 10.3 5.9 11.2 13.0 7.2 7.5
zk f * i f f rk z!z f
5
4 1.6 1.6 0.4 1.3 0.9 2.3 1.8 2.4 1.0
20.4 22.4 2.7 10.0 5.4 11.5 12.8 7.1 7.4
+ f f i f zt =t zk i
l.Y 1.9 0.8 0.6 0.9 2.3 2.4 1.9 1.3
20.8 21.3 3.2 9.7 5.9 11.4 12.6 7.6 7.5
i f f i i f f f i
6 1.7 1.6 0.3 0.9 1.0 2.0 2.9 1.6 1.3
20.7 19.8 3.1 9.8 6.0 12.0 13.4 7.3 7.0
f f f f i zk * zk f
1.7 3.4 0.4 0.8 0.9 2.1 2.0 1.6 1.3
e May represent more than one electrophoretic species. b Bleedings taken on day -1, 3, 6, 10, 11, and 17 respective to the DNFB treatment group. L Mean globulin percent &SD. 6 days as described (12). Twenty animals were bled by cardiac puncture 1 day prior to the first DNFB treatment and on days 3, 6, 10, 11, and 17 after the initial treatment. Ten control animals were treated with alcohol alone and bled according to the same schedule. All blood samples (3 ml) were allowed to clot and the serum collected and stored at -20°C. All animals were skin tested on day 10 with 0.5% (v/v) DNFB in olive oil. The reactions were graded 45 hr later according to the following scheme : 0, no detectable reaction ; 1, patchy erythema ; 2, homogenous erythema; 3, marked erythema and raised reaction site. Sensitizatiolz
to Tuberculin
Guinea pigs were sensitized to tuberculin by subcutaneous inoculation of 1 mg heat-killed Mycobacteriurn tuberculosis H37RA in 1 ml Freunds’ incomplete adjuvant (Difco) in the nape of the neck. Thirteen animals were bled 1 day before immunization and on days 5, 10, 15, 20, and 25 after inoculation. Ten control animals were injected with incomplete Freunds’ adjuvant alone and bled according to
I
2
3
4
5
6
FIG. 1. Stained polyacrilamide gels after electrophoresis of serum from sequential bleedings (l-6) of a typical guinea pig during sensitization to DNFB. The arrows designate globulin fraction 4 which increases to a maximum by the third bleeding and then subsides.
ALPHA
~LoxmLrN
CHANGES
IN
CELLULAR
149
IMMUNITY
the above schedule. All animals were skin tested intradermally with 10 pg PPD (Parke-Davis) 24 days after treatment. The reactions were graded by measuring the diameter of induration 48 hr after skin testing. Sensitixtion
to Allogenic
Skin
Fifteen animals were sensitized to allogenic skin by grafting full thickness, 1 cmZ, abdominal skin bilaterally on the back. The skin grafts were protected with Blenderm bandage (331). The grafts were examined after 5 days and animals rebandaged. Ten control animals received autologous skin grafts. All animals were bled 1 day before grafting and on days 3, 6, 10, and 14 after grafting. Allogenic skin grafts were considered rejected when the graft became thick, hard and necrotic. Autografts were uniformly accepted. Disc Gel Glectrophoresis Polyacrilamide disc gel electrophoresis was carried out in 7% acrilamide gels at 1.5 m amps/gel for 45 min according to Davis (13) and Ornstein (14). To facilitate comparison, all serum samples from a single animal were electrophoresed together. The gels were stained with amido Schwartz, destained in 10% acetic acid, and scanned in a densitometer (Photovolt). The percent of each globulin fraction was calculated by automatic integration of the gel tracing. RESULTS Serum
Globulin
Densitometry were sufficiently
Changes
During
Sensitization
to 2,4-Dinitrol4uorobenzcne
of the stained disc gels indicated that nine serum globulin regions distinct to be quantitated for these experiments. Some of these TABLE
2
SERUM GLOBULIN CHANGES IN GUINEA PIGS DUKING SENSITIZATION TO 2, 6DINTIROFLUOKOBENZENE Electrophoretic fraction” 1 2 3 4 5 6 7 8 9
Bleeding 1 19.9 23.6 3.0 10.2 5.3 11.9 12.5 7.7 5.8
f 6.1C i 4.4 i 0.7 zk 1.6 * 1.1 ZJZ2.9 f 5.9 f 1.5 f 1.2
2 18.8 20.4 3.2 17.0 4.7 12.2 10.6 7.2 5.7
f 3.6 f 3.1 f 0.7 f 3.4d f 1.1 f 2.3 f 4.7 f 1.9 f 1.3
number* 4
3 17.9 20.5 3.5 17.1 4.6 12.6 10.4 7.0 6.3
f f i f f f i f f
3.0 2.8 0.5 2.6” 1.1 2.6 4.6 2.0 1.6
20.0 19.8 3.4 13.1 5.3 13.4 10.9 7.8 6.3
f i f f f f i f f
6
5 3.0 2.3 1.1 2.1 1.7 2.7 3.7 2.0 1.4
22.1 21.0 3.0 11.9 5.0 12.7 11.0 7.2 6.0
f 3.0 f 2.8 Ik 1.1 ziz 1.6 f 1.1 f 2.7 f 4.1 f 2.0 zt 0.9
20.8 22.1 3.4 9.6 5.3 12.5 12.0 8.1 6.2
f f f f f f * + f
D May represent more than one electrophoretic species. b Bleedings taken on day -1, 3, 6, 10, 11, and 17 respective to DNFB treatment. c Mean globulin percent &SD. d P < 0.01; compared to fraction 4, DNFB-group, bleeding 1 and control group, bleeding (Table 1). a P < 0.01; compared to fraction 4, DNFB-group, bleeding 1 and control group, bleeding (Table 1).
3.7 3.1 0.7 1.0 1.5 2.0 3.9 2.3 2.0
2 3
150
BURGER
ET
AL.
2. A densitometer scan of a stained polyacrilamide gel after electrophoresis of serum FIG. from a control group animal. The broken line (- - -) represents the change in the scan observed when serum from an animal undergoing sensitization to DNFB was electrophoresed (third bleeding). The scan is divided into nine fractions. The percent globulin of each fraction was calculated by integration and is shown for a control group and sensitized animal (third bleeding). A highly significant increase (P < 0.01) in fraction 4 was observed. regions contained more than one electrophoretic species, but quantitation by planimetry could not distinguish between closely migrating globulins. The variability of these nine regions in control animals is presented in Table 1. No significant differences were noted in any of the globulin regions during the 3-wk study period.
In ten additional animals treated topically with the nonsensitizing
irritants
carbon-
tetrachloride
or petroleum ether, no significant changes were noted. When the nine regions from DNFB treated animals were compared to controls, significant
globulin changes in region 4 were noted on the second and third bleeding (3 and 6 days
Globulin
Fraction
FIG. 3. A comparison of serum globulin changes in animals sensitized to allogenic skin. The shaded areas represent the mean globulin percent 2 SD of nine electrophoretic fractions of serum from autografted animals (third bleeding). The broken line (- - -) represents the significant increase (P < 0.01) in globulin fraction 4 in allografted animals at this time.
ALPHA
GLOBULIN
CHANGES
IN
CELLULAR
IMMUNITY
1.51
during sensitization) (Table 2). Globulin region 4 (alpha globulins) increased from a mean percent of 10.2 * 1.6 prior to treatment to 17.0 * 3.4 and 17.1 f 2.6, 3 and 6 days during treatment respectively. By day 17, this region had returned to normal levels (3 = 9.6 r+ 1.0). These changes are highly significant (P < 0.01) and easily observed in the gels prior to densitometry (Fig. 1) . Densitometer tracings of control and test group sera are shown in Fig. 2 and further demonstrated the significant increase in alpha globulins. There was not a significant change in the total globulin levels during these procedures. It seems, therefore, that the increase in alpha globulin represents a change in absolute amount of this electrophoretic species. Serum
Changes
During
Sensitization
to Mycobacterium
tuberculosis
H37RA
Animals sensitized to Mycobacteriuwt tuberculosis also show a significant rise in alpha globulins (region 4). This increase in alpha globulins was evident at different times after immunization (Table 3). The mean alpha globulin percent from any one bleeding after immunization did not significantly differ from controls or preimmunization values. However, the mean of the highest values obtained after immunization (14.4 * 2.7) was significantly different (P < 0.01) from controls (a = 9.6 * 1.0) and preimmunization values (5 = 9.2 f 1.7). Serum
Globulin
In allografted when compared
Changes
During
TABLE IN SERCM
GLOBULIN
TO Mycobacterium
3
4 DURING SENSITIZATION Tuberculosis H37RA
FRACTION
Immunized
Adjuvant control group Guinea pig Highest values 1 2 3 4 5 6 7 8 9 10 11 12 13 Mean fSD
10.1” 9.2 8.8 11.1 9.1 9.6 9.8 10.3 10.2 12.2 9.1 8.6 a.7 9.8 3~ 1.0
Skin
increase in globulin region 4 was observed The change in alpha globulins was restricted
animals, a significant to autograft recipients.
CHANWS
to Allogenic
Sensitization
group
Postimmunization Preimmunization
9.5 9.6 9.3 6.5 8.9 10.2 7.1 9.2 9.3 12.5 11.8 8.0 8.1 9.2 f
Highest value
Bleeding 6 4 2 4 3 1 6 2 3 4 3 2 3
17.8 14.0 18.7 17.8 13.1 10.2 13.5 12.5 13.3 15.5 17.0 12.7 11.1 1.7
o Percent fraction 4 of total globulin. b P < 0.01; compared to adjuvant alone or preimmunization
14.4 f
values.
2.7b
Percent increase
87 46 101 174 47 0 90 36 43 24 44 59 37
BURGER
152
ET
AL.
DNFB-DERMITITIS
2or
2 2Or
OL
T
7
TUBERCULIN
-7
HYPERSENSITIVITY 1
i I
1 mI--II.. 2
-Li 3
Bleeding
4
5
6
Number
FIG. 4. A comparison of globulin region 4 (alpha) values from control animals ( 0 0 0 ) and animals undergoing sensitization (000) to DNFB (top), allogenic skin (middle), and Mycobacterium tuberculosis (bottom). A significant increase (P < 0.01) in this region in animals sensitized to DNFB and allogenic skin is noted on bleedings two and three. The significant change in the tuberculin group only noted when the highest region 4 values were compared (Table 3), was not evident on any single bleeding.
to 3 and 6 days after grafting as was noted in DNFB sensitization. The maximum increase (9.5 f 1.6 to 15.3 + 2.6, P < 0.01) occurred 6 days after allografting and is plotted in Fig. 3. A comparison of the changes in region 4 for each of the sensitization procedures is presented in Fig. 4. It should be noted that highly significant changes (P < 0.01) are observed on the second and third bleedings in both the DNFB and allografted groups but at no single time in the tuberculin group. Egects
of High
Alpha
Globulin
Levels
on Skin Tests
Animals sensitized to DNFB were usually skin test positive (2-3 + reactions) 11 days after initial treatment. This coincided with a return of region 4 values into in additional experiments 3 of 46 normal ranges (a = 10.6 t 1.3). However, animals at this time showed very weak or negative skin tests (0, I+, l+). These three weak responding guinea pigs had elevated globulins in region 4 (Z = 16.2 -C 1.6, P < 0.01). Conversely, the remaining 43 animals were all skin test positive (2-3 + reactions) and none demonstrated elevated region 4 globulins (x’ = 10.2 +1.2). Eleven of thirteen animals immunized with Mycobacterium tuberculosis demon-
ALPHA
GLOBULIN
CHANGES
IN
CELLULAR
IMMUNITY
153
strated positive skin tests (> 10 mm induration) 25 days later. The 11 skin test positive animals had normal globulin levels in region 4 (5 = 10.1 * 1.6) at the time of skin testing. On the other hand, both of the animals that were skin test negative (Table 2, No. 1 and 7, < 5 mm induration) had significantly elevated region 4 globulins (17.8 and 13.5, respectively) at the time of skin testing. EfJects of Skin
Tests on Alpha
Globulin
Levels
In additional experiments, 20 animals were sensitized to DNFB, rested for 10 days (to allow region 4 globulins to return to pre-DNFB levels), and skin tested. The effect of the skin test on alpha globulin levels was observed at 24 and 48 hr. Although all animals in this group displayed strong (3+) skin reactions, changes in alpha globulins at 24 or 48 hr could not be demonstrated. The mean percent of globulin region 4 was 10.2 2 1.1 prior to skin testing, 10.6 * 1.3 24 hr after testing, and 9.8 -t 1.3 48 hr after testing. DISCUSSION In this study, serum alpha globulin changes were associated with immunizations which lead to the development of cell-mediated immunity. In DNFB-induced contact dermatitis, tuberculin hypersensitivity, and allograft immunity electrophoretic region 4 (alpha) globulins increased, then subsided, concomitant with the development of cellular immunity. In DNFB and allograft sensitization, the rise in alpha globulins occurred predictably 3-6 days after antigen exposure and usually subsided by the tenth day (Fig. 4). The increase in alpha globulins after mycobacterium exposure was observed over a broader period of time (Table 3). This difference may be related to the time required to develop cellular immunity and may, in part, reflect differences in antigen, combination with adjuvant, or route. An alpha globulin change was not detected in one animal in the Mycobacterium group. This represented the only such observation in 91 animals sensitized in one of these three protocols. Since this was observed in the Mycobacterium group, it seems likely that the alpha globulin change was missed between bleedings. In our hands, skin tests applied to sensitized animals did not seem to influence alpha globulin levels. However, this result may be influenced by the inability of these procedures to accurately detect small changes in closely migrating electrophoretic species. On the other hand, high alpha globulin levels seemed to influence skin testing. Animals that had high alpha globulin levels at the time of skin testing responded weakly or not at all. Conversely, all sensitized animals with normal alpha globulin levels had a strong skin test response. This observation may have implications in transient anergic conditions that occur in some human diseases (10) including cancer (11). In the latter case, changes in alpha globulins associated with tumor development have been noted (15, 16). The alpha globulin changes described here may well be changes in IRA levels (8). Others have shown that IRA from man has the capacity to suppress PHA and antigen-induced lymphocyte transformation in vitro (7, 9) and inhibit humoral and cellular responses to specific antigen in viva (17, 6). The mechanism of immune regulation by IRA remains obscure. It does not seem to involve antigenic competition ( 18) or disruption of RNA mediated events by RNase activity (8) as previously proposed (19, 20). The mechanism of regulation may involve modulation
1.54
BURGER
ET
AL.
of T lymphocyte function. Experiments of Cooperband et al. (8) and Glasgow et al. (21) suggest that IRA may function as a competitive inhibitor of early T cell recognition events without directly effecting antigen binding. Physiochemically IRA behaves as a small molecular weight polypeptide (22) which is casually associated with alpha globulins. These characteristics bear similarity to at least two other substances reputed to influence T lymphocyte function : thymosin and transfer factor. The thymic hormone, thymosin, appears to be a polypeptide of 12,600 MW (23) and transfer factor is described as a dialyzable polypeptide-polynucleotide (24, 25). Since T lymphocyte regulation may involve the combined influence of IRA, thymosin, and transfer factor, caution should be exercised in interpreting the influences of small molecular weight dialysates on T cell function in vi770 or in vitro. ACKNOWLEDGMENTS This work was supported in part by the American Cancer Society, Oregon Division. We are grateful to Thomas A. Vetto for skillful technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Dardas, T. J., and Mallmann, V. H., 1. Bacterial. 92, 76, 1966. Burger, D. R., Thesis, The University of Arizona, Tucson, Arizona, June, 1967. Riggio, R. R., Schwartz, G. I-I., Stenzel, K. H., and Rubin, A. L., Lancet 1, 1218, 1968. Kamrin, B. B., Proc. Sot. Exp. Biol. Med. 100, 58, 1959. Mowbray, J. F., Trms~luntation 1, 15, 1963. Mannick, J. A., and Schmid, K., Trux.r~lanfation 5, 1231, 1967. Cooperband, S. R., Bondevik, H., Schmid, K., and Mannick, J. A., Science 159, 1243, 1968. Cooperband, S. R., Davis, R. C., Schmid, K., and Mannick, J. A., Trmsphnt. Proc. 1, 516, 1969. 9. Riggio, R. R., Schwartz, G. H., Bull, F. Geoffrey, Stenzel, K. H., and Rubin, A. L., Transplantation 8, 689, 1969. 10. McFarlin, D. E., and Oppenheim, J. J., 1. Zmmunol. 103, 1212, 1969. 11. Eilber, F., and Morton, D. L., Cancer 23, 362, 1970. 12. Burger, D. R., and Jeter, W. S., Infect. Immun. 4, 575, 1971. 13. Davis, D. B., Ann. N. Y. Acad. Sci. 125, 403, 1964. 14. Omstein, L., Ann. N. Y. Acad. Sci. 121, 321, 1964. 15. Ashikawa, K., Inoue, K., Shimizu, T., and Ishibashi, Y., Jap. J. Enp. Med. 41, 339, 1971. 16. Hsu, C. C. S., and LoGerfo, P., Proc. Sot. Exp. Biol. Med. 139, 575, 1972. 17. Glasgow, A. H., Cooperband, S. R., Occhino, J. C., S&mid, K., and Mannick, J. A., Proc. Sot. Exp. Biol. Med. 138, 753, 1971. 18. Glasgow, A. H., Cooperband, S. R., Schmid, K., Parker, J. T., Occhino, J. C., and Mannick, J. A., Trarzsplavat. Proc. 3, 835, 1971. 19. Mowbray, J. F., Fed. Proc. 22, 441, 1963. 20. Mowbray, J. F., and Scholand, S., Immunology 11, 421, 1966. 21. Glasgow, A. H., Cooperband<, S. R., and Mannick, J. A., Fed. Proc. 31, 3314, 1972. 22. Glasgow, A. H., Occhino, J. C., Badger, A. M., Schmid, K., and Mannick, J. A., Szlrg. Forum 22, 273, 1971. 23. Hardy, M. A., Goldstein, A. L., and White, A., Surg. Forum 23, 305, 1972. 24. Lawrence, H. S., Al-Askari, S., David, J. R., Franklin, E. C., and Zweiman, B., Trans. Ass. Amer. Physicians 76, 84, 1963. 25. Burger, D. R., Vetto, R. M., and Malley, A., Science 175, 1473, 1972.