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Mechanisms in the in vivo release of lymphokines
CELLULAR
IMMUNOLOGY
64, 203-219 (1981)
Mechanisms I. Comparative
in the in l&o Release of Lymphokines’ Kinetics in the Release of Six Lymphokines in Inbred Strains of Mice
RUTH NETA,* S. B. SALVIN,? AND MOHAMED SABAAWI~ *Department of Microbiology, University of Notre Dame, Notre Dame, Indiana 64556, and TDepartment of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania I5261 Received March
16. 1981; accepted July 7. 1981
Lymphokines were detected in the sera of 16 strains of inbred mice sensitized intravenously with cell walls of Mycobacterium bovis strain BCG and challenged subsequently intravenously with old tuberculin (OT). Variations occurred between the strains in the types and quantities of six lymphokines studied, namely, chemotactic factor (CF), type II interferon (IF II), lymphotoxin (LT), migration inhibitory factor (MIF), mitogenic factor (MF), and skin reactive factor (SRF). The times for maximum release of the lymphokines in the different strains were similar for MIF, IF II, SRF, and MF, but differed for CF and LT. The degree of activity of MIF and IF II generally paralleled one another in the different strains but such parallelism did not occur for the other four lymphokines. Each of the 16 strains was a high responder for at least one of the lymphokines, indicating that in sensitized mice the release or presence in the circulation of lymphokines in response to a specific antigen is a selective process. Thus, each strain may have an individual combination of lymphokines, interactions of which may determine the types of pathway utilized in an immunological response.
INTRODUCTION Lymphokines are lymphocyte-derived soluble mediators of cellular immunity. They were originally described as products of sensitized lymphoid cells after in vitro stimulation with specific antigen (1, 2). Numerous investigations resulted in (a) description of various effects that these soluble mediators had on the cells of the immune and other body systems (3); (b) recognition that cells other than lymphocytes (stimulated or unstimulated), i.e., macrophages (4), fibroblasts (5), and various cell lines (6) produced soluble substances with properties similar to those of lymphokines; (c) the finding that stimulation of lymphoid cells in vitro with a variety of agents might result in efficient production of lymphokines (7); and (d) the accumulation of evidence that such lymphokines were important in the development of cell-mediated immunity (CMI) (8). Because of difficulties encountered in the separation and purification of various lymphokines, evidence for ’ This work was supported by the Public Health Service, National Institute of Allergy and Infectious Diseases (Grant AI-16064) @B.S.) and by the American Lung Association and National Institutes of Health, IF 32CA 06519 (R.N.). 203 0008-8749/81/160203-17$02.00/O Copyright 0 198 I by Academic Rss, Inc. All rights of reproduction in any form reserved.
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their role in cell-mediated immunity has been limited to the demonstrations that lymphokine-enriched preparations can cause inflammatory responses (9), skin reactions ( lo), and rejection of certain tumors ( 1l- 13). Lymphokine release was detected in vivo at the site of an immune response (14). However, it is still not known whether some or all of the known lymphokines are necessary for the normal functions of the immune system. Consequently, the question arises as to whether certain immunodeficiency diseases develop because one or several of the lymphokines are selectively not produced. Clinical data indicate a possible deficiency in the production of some and not other lymphokines in certain disease states (15, 16). Yet, limited information is available on similar differences in the production of inflammatory lymphokines in experimental animals (17). In a previous report ( 18), evidence was presented that marked differences existed in the amounts of type II interferon (IF II)’ and of migration inhibitory factor (MIF) released in vivo into the circulation of different inbred strains of mice after sensitization with cell walls of Mycobacterium bovis strain BCG and subsequent challenge with old tuberculin (OT). The occurrence of these variations introduced a possible means for (a) separation of the mechanisms of production of individual lymphokines and (b) studies of the role of the individual lymphokines in cell-mediated processes. In this paper, evidence is presented that activities associated with such lymphokines as IF II, MIF, SRF (skin reactive factor), CF (chemotactic factor), MF (mitogenic factor), and LT (lymphotoxin) are not necessarily released in vivo in parallel to one another in time or in quantity. MATERIALS
AND METHODS
Mice. Outbred female Swiss Webster mice were purchased from Taconic Farms, Germantown, New York. Peritoneal exudate cells were harvested from these mice approximately 5 days after intraperitoneal injection of 5 ml light mineral oil. The 16 strains of inbred mice, purchased from Jackson Laboratory, Bar Harbor, Maine, were as follows: A/J, C57B1/6J, C57Bl/lOJ, C57Bl/lOSNJ, C57L/J, Balb/cByJ, C57Bl/KsJ, DBA/2J, Sec/lReJ, CBA/CaJ, C3H/HeJ, AKR/J, RF/J, DBA/ lJ, SWR/J and SJL/J. Antigens. Lyophilized BCG cell walls were prepared by and obtained through the courtesy of Dr. Edgar Ribi, Rocky Mountain Laboratory, Hamilton, Montana. The cell walls were ground in Drakeol, then emulsified in Tween 80-saline (19). Mice were sensitized by intravenous injection of 300 gg BCG cell walls in 0.2 ml, and 3 weeks later challenged intravenously with 50 mg old tuberculin (OT) in 0.2 ml, purchased from Jensen-Salsbury, Kansas City, Missouri. The mice were exsanguinated at varying intervals after challenge, and the sera removed for assay. “Zero” time refers to bleeding of sensitized, nonchallenged mice. Purified protein derivative (PPD), obtained from either Parke, Davis & Company, Detroit, Michigan, or from the National Institutes of Health, Bethesda, 2 Abbreviations used: BCG, Mycobacrerium bovis strain BCG; CF, chemotactic factor; CMI, cellmediated immunity; CW/Dr, cell walls of BCG in Drake&Tween 80 emulsion; IF II, type II interferon; LT. lymphotoxin; MEM, minimum essential medium; MF, mitogenic factor; MIF, migration inhibitory factor; MLR, mixed lymphocyte reaction; OT, old tuberculin; PE, peritoneal exudate; PPD. purified protein derivative of old tuberculin; SRF, skin reactive factor.
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Maryland, was used for footpad testing for delayed hypersensitivity by injection of 30 pg in 0.05 ml in a hind footpad. A similar quantity of normal autologous serum was injected as a control into the other hind footpad. Migration inhibitory factor (MIF). This lymphokine was titered by determining what dilution of experimental mouse serum resulted in an inhibition of 20% or more of the migration of peritoneal exudate cells on agar, in comparison with the effect of control serum from the same strain of inbred mice (20). The serum dilution (0.6 ml) was mixed with 2.4 ml of an agar medium containing medium 199, 0.5% Oxoid ion agar, and 100 units/ml of penicillin and 100 pg/ml of streptomycin at 45°C all dispensed in a volume of 3 ml in a 30-ml Falcon flask. A small drop of packed peritoneal exudate cells was placed on the surface of the agar, and after incubation at 37°C for 48 hr the distance of migration was measured with the aid of a micrometer in a low-power microscope. The percentage of inhibition of migration was equal to l-
migration of PE cells in experimental serum x 100. migration of PE cells in control serum
The reciprocal of the maximum dilution of experimental serum causing at least 20% migration inhibition was regarded as the titer of MIF activity. Interferon. Interferon activity was determined by an assay involving the use of encephalomyocarditis (EMC) virus hemagglutination reduction yield (2 1). Here L-929 cells were cultured overnight in minimum essential medium (MEM) with 10% fetal calf serum in glass tissue culture tubes to form monolayers. The medium was decanted and the cells were washed twice with phosphate-buffered saline. The experimental and control sera were diluted lo-fold to obtain 10m2to 10m6dilutions of each serum in MEM containing 2% fetal calf serum. The tubes with the cell monolayers were inoculated in triplicate with 1 ml of each of the serum dilutions. After a 24-hr incubation period at 37°C the medium was removed, cell monolayers were washed twice with phosphate-buffered saline, and 0.25 ml EMC virus (hemagglutination titer, 1:16,384) in a buffer containing 0.5% gelatin and 0.2% lactoalbumin hydrolysate in Hanks’ solution was added to the monolayers. After 30 min incubation at 37°C the infected cells were washed twice with PBS, fresh MEM with 2% fetal calf serum was added, and the tubes were incubated for an additional 18 hr. The virus was harvested by repeated freezing and thawing, and the titer was determined in a hemagglutination assay using human type “0” erythrocytes. The titer of interferon was expressed as a reciprocal of the serum dilution that produced a reduction of 0.5 log hemagglutination yield of EMC virus. Ten units of interferon in this assay was equivalent to 1 unit of a reference mouse interferon standard (obtained from The Antiviral Substances Program of NIAID, NIH). Skin reactive factor. Serum (0.05 ml) from one of the inbred strains of mice was injected into a rear footpad of a normal Swiss Webster mouse; control serum was injected into one hind footpad, experimental serum, into the other hind footpad. The extent of swelling was then measured at 1/2-h intervals up to 4 hr postchallenge with the aid of a calipers-micrometer (Schnelltlster). An increase greater than 10% was considered to be indicative of a positive response. Histologic sections of appropriate footpads were made in order to determine the type of cellular infiltrate.
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Tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Chemotacticfactor. Chemotaxis was assayed by a technique involving the migration of adherent peritoneal exudate (PE) cells under agar (22). Here, one part of 5% agarose, i.e., 10 ml, was mixed with 5 parts, i.e., 50 ml, of the following medium: five milliliters medium 199 (lox), 0.5 ml 200 mM L-glutamine, and 100 units per 100 kg/ml penicillin-streptomycin, brought to 50 ml at 45°C with distilled water. Hepes buffer was added (1 M, pH 8.0) to adjust the pH to 7.2. Five milliliters of the agarose medium were added to each 60 X 15-mm tissue culture dish (#3002, Falcon Plastics, Oxnard, Calif.) and allowed to harden at room temperature before transfer to 4°C for at least an hour. With the aid of a template, a series of three wells, 2.4 mm in diameter and 2.4 mm apart, was cut into the agar and the plugs were removed with an 18-gauge needle. Adherent peritoneal exudate cells were obtained by suspending 1 X 10’ cells/ml in MEM containing 10% guinea pig serum in a petri plate for an hour at 37°C in 5% CO*, decanting the supernatant, removing the adherent cells with a rubber policeman, then reconstituting the cells to 1 X lO’/ml in a medium (Hanks’ supplemented with L-glutamine) containing 1% human serum albumin. With the aid of an automatic pipet, 5~1 of the cell suspension were placed in the center well, and either experimental serum or normal autologous serum placed in the outside wells. After incubation at 37°C in a COZ incubator for 18 hr. the cells were fixed by the addition of 3 ml absolute methanol for 30 min and stained with Wright’s stain. Distances of migration were determined in a low-power microscope with the aid of an ocular micrometer cell. Results are presented as the difference in units of migration between experimental and normal autologous sera. Each unit represents 1.75 mm. Mitogenic factor. The capacity of lymphokine-containing sera to stimulate cell replication was assessedwith spleen cells from normal Swiss Webster mice. The lymphoid cells were recovered from the interphase of a Ficoll-Hypaque gradient of teased spenic cells (23). Of these cells 4 X 10’ in 0.1 ml were cultured in each well of a microtiter plate containing medium RPM1 1640 with Hepes buffer, and 4% of either control serum or experimental serum. After 48 hr of incubation at 37°C in 5% CO*, 1 &i of tritiated thymidine was added to each well. Twentyfour hours later, the cells were harvested with a MASH II Systems Harvester (Microbiological Associates). The filter disks with the cellular material were placed in scintillation vials with 9 ml Econofluor, and the counts per minute of the samples was measured in a liquid scintillation counter. The data are reported as the stimulation index of triplicate samples, namely, cpm of cells in experimental serum cpm of cells in control serum * Colony inhibition assay. In vitro-propagated PA III tumor cells (24) derived from the spontaneous prostate carcinomas of germ-free Lobund Wistar rats were removed from the monolayers by trypsinization in 0.25% trypsin solution. The cells were washed and counted and then suspended to a final concentration of 6 X lo’/ ml cells in MEM supplemented with 10% fetal calf serum. The suspension (0.45 ml) was distributed in tissue culture petri dishes (Falcon 3008). Normal control mouse sera and the assayed sera were added to make up the final concentration
MECHANISMS
IN THE IN VIVO RELEASE
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207
of 2.5% of the mouse serum. Cells in culture in triplicates were incubated for 5 or 6 days. The medium was removed and the cells were fixed in methanol and stained with 0.1% crystal violet solution. The number of cell colonies consisting of five or more cells was counted with the aid of a stereomicroscope. The data are expressed either as percentages of suppression of target cell growth (Table 1) or as percentages of cell growth in experimental serum in comparison with cell growth in control serum (Fig. 6). RESULTS Release of AUF Sixteen strains of inbred mice belonging to six different H-2 haplotypes were assayed for their capacity to release MIF in vivo, after intravenous sensitization with 300 pg of cell walls of iU. bovis strain BCG in Drakeol-Tween 80 emulsion (CW/Dr), intravenous challenge 3 weeks later with 50 mg old tuberculin (OT), and exsanguination 4 hr later (Table 1). This schedule was used, since maximum titers were obtained in Swiss Webster outbred mice at this time interval in previous experiments (25). The titers of MIF in the mouse sera varied from less than 4 to 256, depending on the strain. Four strains of the H-2’ haplotype and two of the H-2q haplotype were low responders, i.e., had titers ranging from less than 4 to 16. Three strains of the H-2b haplotype were high responders, in that their sera had MIF titers of 256; the sera of a fourth strain (C57L/J) had a titer of 16. Three of four strains of H-2d haplotype had sera with titers of 64 to 256; the fourth strain, Balb/cByJ, varied in different experiments from less than 4 to 256 in its titer. The single strains belonging to the H-2” (A/J) and H-2” (SJL/J) haplotypes were high responders, with titers of 256 and 64, respectively. The possibility was present that these differences were due to variations in the times for the release of maximum activities of MIF. Accordingly, 10 inbred strains of mice were assayed for their capacity to release MIF at times varying from 0 to 24 hr after challenge with OT. Five of the strains were high responders, i.e., A/J, C57B1/6J, C57Bl/lOSNJ, C57Bl/KsJ, and SJL/J, while five other strains were low responders, i.e., AKR, CBA/CaJ, C3H/HeJ, DBA/lJ, and RF/J (Fig. 1). In all cases,maximum titers were obtained at approximately 4 hr post challenge. Release of Type II Interferon (IF II) The sera of 16 strains, obtained 4 hr after challenge, were also assayed for their contents of IF II after the mice had been appropriately sensitized and challenged (Table 1). The titers in the sera of these strains varied from less than 100 to more than 1,000,OOO.For example, the 4 strains of the H-2’ haplotype had titers of less than 100 to 21,000; the 2 strains of the H-2q haplotype had titers of ~100 and 11,500. Three of the four strains of the H-2b haplotype had titers of about l,OOO,OOO, while the fourth strain (C57L/J) had a titer of 14,000. Three of four H-2d strains had titers higher than 100,000, while the fourth strain, Balb/cByJ mice, in one experiment, had a titer higher than 10,000; in another experiment, a titer of 2500, while in five other experiments the IF II titers were lower than 100. In studies on the time intervals after challenge for maximum release of IF II, the highest titers were found in the sera at 4 hr after challenge (Fig. 2).
950,000 500,000 I ,300,OOo I, 100,000 14,000 2,500* I ,300,000 120,000 200,000 19,500 850 1,100
256 256 256 256 I6 32* 256 256 64 16 4 4 14 <4 <4 64
H-2
S
a b b b b d d d d k k k k q 9
IF II titer
MIF titer 24.0 f 13.7 ic 25.9 + 17.0 k 22.3 k 24.3 f 30.6 + 13.8 k 22.0 f 13.0 +
0.5 2.3 2.5 3.2 2.1 2.3 3.2 0.5 2.2 6.5 0 + 1.05 -7.5 + 0.78 N.D. +I.81 + I.17 N.D. -3.12 + 1.6 -4.68 + 0.6 Ok0 N.D. +3.0 + 0.93 +0.68 + 0.68 0 + 1.77 +I.5 + 0.46 +4.17 + 1.1 N.D. -1.25 + 0.72
CF (unit of migration + SE)
of 16 Inbred Strains of BCG-Sensitized
SRF (% increase of swelling f SE
Released in Vitro into the Circulation
Lymphokines
of Six Different
1
3.6 + 0.3 2.0 + 0.4 N.D. 2.3 + 0.64 N.D. 1.4 f 0.2 1.1 + 0.32 1.2 + 0.37 N.D. 3.4 + 0.7 2.3 + 0.5 I.5 + 0.46 N.D. 0.5 + 0.21 3.6 + 0.3 1.3 +- 0.2
MF (stimulation index + SE)
Mice 4 hr after Challenge
84.4 + 4.4 32.4 + 9.1 N.D. N.D. N.D. 15.6 f II.7 -3.5 f 9.2 N.D. N.D. 39.1 ? 6.7 N.D. 48.8 -t 9.2 10.0 f 2.9 N.D. N.D. 78.8 If: 4.9
LT (% cytotoxicity k SE)
with OT
Note. See Materials and Methods for procedures for sensitization and challenge, units of migration and units of measurement. In each experiment, a pool of sera from IO mice was used. Exclusive of strains SWR/J and C57L/J, the figures represent results from two to five experiments. * Indicates variable results, cf text. N.D., not done.
C57Bl/6J C57Bl/lOJ C57Bl/lOSNJ CS’IL/J Balb/cByJ C57BI/KsJ DBA/2J Set/ I ReJ CBA/CaJ C3H/HeJ AKR/J RF/J DBA/IJ SWR/J SJL/J
A/J
Strain
Activities
TABLE
if $
5
s
? 5
3
it
MECHANISMS
IN THE IN VW0 RELEASE OF LYMPHOKINES
512 -
209
. A/J d C57BVlOSnJ 0 C57BIIKsJ
C3WHeJ + RF/J
l
0
2
4
6 6 IO TIME (hours)
12 N
24
FIG. I. Titers of MIF at different time intervals after challenge. Mice were sensitized intravenously with 300-rg cell walls of BCG in Drakeol-Tween 80 emulsion, and 3 weeks later challenged intravenously with 50 mg OT. A minimum of 10 mice was exsanguinated at varying time intervals thereafter. The titers are the reciprocal of the serum dilutions that inhibited the migration of normal peritoneal exudate cells by at least 20%.
. AN 0 C57BVlOSnJ q C57BIfKsJ x C57BI/&l n CBA/CaJ 0 SJLN A AKWJ l C3WHeJ 0 DBA/lJ
-+ a
2
I 4
1 6 TIME
I 1 8 IO (hours)
I 12
I%
FIG. 2. Titers of IF II at different time intervals after challenge. See Fig. 1. The titers are the reciprocals of the serum dilution that produced a reduction of 0.5 log hemagglutination yield of EMC virus growth in L cells. Ten units of interferon in this assay was equivalent to 1 unit of a reference mouse interferon standard.
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0 C57BVlOSnJ n CBA/CaJ l C3H/HeJ 0 DBAllJ v DBAl2J
6 8 IO TIME (hours)
24
A
FIG. 3A. Extent of footpad swelling induced by sera obtained at different time intervals after challenge from five strains of inbred mice. See Fig. 1. The percentage increase in footpad thickness was obtained by comparing one hind footpad injected with experimental serum with the other hind footpad injected with control serum. A minimum of four mice was used for experiment. FIG. 39. Section of a footpad of a mouse inoculated with control serum from C57BI/KsJ mice, and sacrificed 2% hr later. X750.
MECHANISMS
IN THE IN VIVO RELEASE
OF LYMPHOKINES
serum of C57BI/ KsJ FIGi. 3C. Section of a footpad of a mouse inoculated with lymphokine-containing mice and sacrificed 2% hr later. X750. FIGi. 3D. Section of a footpad of a mouse actively sensitized subcutaneously with 300 rg CW, /Dr. tested ! in the footpad 3 weeks later with 30 pg PPD. and sacrificed 24 hr later. X750.
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A striking parallelism was apparent in the different strains of mice in the quantities of IF II and MIF released in vivo into the circulation at varying times of exsanguination after challenge (Table 1; Figs. 1 and 2). The presence of low titers or the absence of detectable titers of IF II and MIF in the sera of AKR/J, C3H/ HeJ, CBA/CaJ, RF/J, and DBA/ 1J strains at all times after challenge indicates the inherent inability of these strains to develop high titers of these two lymphokines under the conditions of experimentation. Presence of Skin Reactive Factor (SRF) Introduction of lymphokine-containing preparations intradermally results in an inflammatory reaction that resembles delayed hypersensitivity (10). What lymphokine, or lymphokines, is necessary for this reaction to develop has not been established. Because marked differences occurred in the amounts of MIF and IF II in the sera of the various strains of mice, experiments were initiated to determine whether a parallelism existed in the capacity of a given strain to release the two lymphokines and the capacity of the serum from that strain to induce an inflammatory skin response, presumably of the mononuclear or delayed type. Accordingly, outbred Swiss Webster mice were inoculated in one hind footpad with experimental serum and in the other hind footpad with control serum. Analysis of the footpad reactions by the sera of each of 15 strains of mice indicated that a correlation did not always exist between the presence of the two lymphokines in a serum and that of SRF (Table 1). Sera obtained from 13 of the 15 strains at 4 hr after OT challenge had the capacity to induce footpad reactions passively of more than 10%. Of sera from 6 strains that were low responders in the release of MIF and IF II and that were examined for skin reactivity, 2 did not induce footpad reactions passively, i.e., C3H/HeJ and AKR, and 4 did, i.e., DBA/lJ, CBA/CaJ, SWR/J, and C57L/J. The maximum swelling developed with sera from bleedings at 2-4 hr post challenge (Fig. 3A). Of 2 strains that were low responders for MIF (4 or ~4) and IF II (
MECHANISMS
-6
-cc!
0 C57BVlOSnJ
-8
0
4
8
12 TIME
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IN THE IN VIVO RELEASE OF LYMPHOKINES
16 (hours)
20
24
. A/J C3HlHeJ . DSAIPJ
I IL
0
l
4
8
12 TIME
16 (hours)
20
24
6
A +4 c C57BVKs.l I C57SV6J
q
+2 z
i I
-
/
-10 \\/’ I 0
C
I 4
I
x I1 8
I 12 TIME
I1 16 (hours)
I1
I 20
I 24
FIG. 4. (A-C) Chemotactic activity of sera obtained at different time intervals after challenge of 12 inbred strains of mice. Oil-induced peritoneal exudate macrophages were obtained from Swiss Webster mice. Numbers represent the average differences in units of cell migration between experimental and normal autologous sera. Each point represents an average of lo-12 readings. I unit = 1.75 mm.
addition, multiple peaks of activity appeared in some of the sera, thus indicating the possible presence of several chemotactic factors. Of sera examined from 12 strains, 7 indicated chemotactic activity at some period up to 24 hr postchallenge, namely, C57Bl/lOSNJ, Balb/cByJ, AKR/J, RF/J, SJL/J, CBA/CaJ, and DBA/ 1J. Of the two inactive sera (AKR/J and C3H/HeJ) for induction of skin reactivity, one, AKR/J, had chemotactic activity. Also, a correlation did not exist between the capacity of a strain to release IF II and MIF into the circulation and the capacity to release CF in viva Of the low responders for MIF and IF II release,
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four strains had chemotactic activity, namely, AKR/J, CBA/CaJ, RF/J, and DBA/ 1J. Of the high responders for MIF and IF II, four strains did not have detectable quantities of CF in their sera, namely, A/J, C57Bl/KsJ, C57B1/6J, and DBA/2J. Thus, it appears that, in a given strain, the capacity to release SRF, MIF, IF II, or CF is under separate genetic control. Release of Mitogenic Factor (MF) One of the characteristics of cell-mediated immunity and delayed hypersensitivity is the enhanced mitotic activity of sensitized lymphocytes in the presence of specific antigen. This increased lymphocyte replication is due in part to a mediator produced in vitro or in vivo by sensitized lymphocytes exposed to specific antigen. The question arises as to how the release of this factor (MF) in vivo is related to the release of other lymphokines (MIF, IF II, SRF, LT, and CF). The degree of mitotic activity varied between the strains (Table 1) and did not correlate with the levels of MIF and IF II within a given strain. For example, C57Bl/KsJ, DBA/2J, and SJL/J, high responders in the release of MIF and IF, did not have mitogenic activity, while SWR/J and CBA/CaJ, low responders in the release of MIF and IF, had pronounced mitogenic activity. When the sera from seven inbred strains were obtained at different times after challenge and examined for MF, the maximum activity was at 4 hr postchallenge (Fig. 5). Release of Lymphotoxin Another lymphokine, or group of lymphokines, associated with cell-mediated immunity and released by sensitized lymphoid cells after exposure to specific antigen, is lymphotoxin (LT), a substance inhibitory in low concentrations to cell growth and capable in high concentrations of lysis of selected cells (26). Experiments were initiated to determine what correlation existed, if any, in the release of LT with reference to other lymphokines in eight inbred strains. An analysis of sera obtained 4 hr after challenge indicated that the cytotoxic activities varied from strain to strain (Table 1, Fig. 6). A correlation did not exist with the high and low responder strains of the previously mentioned five lymphokines with regard to the degree of response and the time of maximum activity. Serum of a low responder mouse strain of MIF and IF II, such as CBA, was highly cytotoxic, in that a concentration of 1:40 caused 100% lysis of target cells (Fig. 6). On the other hand, high responder strains of MIF and IF II, such as C57B1/6J and C57Bl/KsJ, did not show strong cytotoxicity in that a similar serum concentration reached a maximum lytic activity of only 57% at 24 hr after challenge. In addition, maximum cytotoxic activity of the sera from the various inbred strains developed at different times after challenge. For example, the sera from SJL/J mice had maximum cytotoxicity at 4 hr after challenge; sera from CBA/CaJ, A/J, and RF/J were at a maximum at 8 hr; and sera from C57B1/6J and C57Bl/KsJ had maximum cytotoxicity at 24 hr. Therefore, LT, like CF, varies either in the kinetics of its release or in the appearance of its activity in the serum independently of the other lymphokines in the strains studied.
MECHANISMS
IN THE IN VW0 RELEASE OF LYMPHOKINES
215
A C57SVlOSnJ q C57BVKsJ l CBAlCaJ A AKWJ l CIH/HeJ o DBAflJ . DBA/2J
3.2 2.8 t
TIME (hours) FIG. 5. Mitogenic activity of sera obtained at different time intervals after challenge of seven inbred strains of mice. Numbers represent the ratio of cpm from splenic cells cultured in experimental sera vs. the cpm of those cells cultured in normal autologous sera. The data are derived from two experiments.
DISCUSSION Numerous reports have indicated that variations in cell-mediated immunity and in delayed hypersensitivity to microbial organisms or to purified antigens occur in different strains of inbred mice (27-29). Few studies, however, have been reported on similar variations in the production of lymphokines. Variations have been described in the release of MIF in vitro (30) and MIF and IF II in vivo (18) in different strains of inbred mice. In experiments on five strains of mice, splenic cells from two strains, namely, DBA/2 and C57B1/6 challenged in vitro with either antigen or Con A, did not produce MIF (30). However, lymph node cells or peritoneal exudate cells from C57B1/6 mice did produce MIF after challenge with Con A (31). Intravenous sensitization and challenge of the two above strains caused the release of high titers of IF II and MIF into the sera. Therefore, the capacity to release lymphokines in the spleen may be independent of the capacity to release such lymphokines in other lymphoid organs or into the circulation, possibly because of homing and sequestration of suppressor cells in the spleens (32). The system to measure the release of lymphokines into the blood was chosen, since this release not only reflected immunologic events in vivo in the circulation but also occurred in parallel with the capacity of mice to develop resistance to challenge with virulent organsims ( 19). Mice sensitized with components of BCG in a manner that induced the capacity to release lymphokines into the circulation also had increased resistance to challenge with virulent H37Rv. In contrast, sensitization via the subcutaneous route or with components of BCG which did not induce the release of IF II and MIF in vivo did not produce enhanced resistance
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x C57BVW . CBA/CaJ
+ RF/J v Balb/c ByJ
TIME (hours) FIG. 6. Cytotoxic effect of sera from eight inbred strains obtained at different times after challenge. See Fig. 1 for conditions for obtaining the sera. Each point represents the number of colonies (percentage) of rat prostate tumor cells grown in 2.5% experimental serum, as compared with cells grown in control autologous serum. (Standard errors were less than IO%.)
to challenge with a virulent strain of Mycobacterium tuberculosis. Other experiments ((29), Neta and Salvin, unpublished results) also indicated that C57Bl/ 1OSNJ and C57Bl/KsJ strains, which were high responders in the release of serum MIF and IF II, were resistant to infection with BCG or Cundidu albicans, while low responder strains, such as DBA/l or C3H/HeJ, were highly susceptible to such infections. In the present study of sera from 16 strains, 7 (not including Balb/cByJ, which will be discussed separately) had titers of IF II and MIF markedly lower than the titers in the sera of the other strain, i.e., titers of 16 or less for MIF, and less than 20,000 for IF II (Table 1). The maximal titers of IF II and MIF were detected in the sera of 10 strains of mice, 5 of which were low responders, when these mice were bled 4 hr after challenge (Figs. 1 and 2). The differences in the titers of MIF and IF II between mouse strains could not therefore be explained on the basis of different kinetics of their release in the blood. Occasionally, production of some, but not all, lymphokines has been detected in a given human patient (15, 16). Yet, only few attempts have been made to observe a similar dissociation in the release of various lymphokines in experimental animals (33). The assumption has been that an appropriately sensitized animal can produce
MECHANISMS
IN THE IN VIVU RELEASE
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217
all of the known lymphokines after stimulation with specific antigen or with mitogen. The existence of strains of mice that can release different levels of IF II and MIF into their circulations provided a model to test this assumption. Therefore, other activities of lymphokines were examined in the same sera to determine possible correlation or dissociation of these lymphokines with MIF and IF II. The activities of lymphokines in the sera of sensitized and challenged mice were determined by biological assays. However, the two lymphokines, MIF and IF II, released in vivo into the mouse circulation had been determined to have physicochemical properties similar to those of the in vitro-produced lymphokines (( 34) Bout, David and Salvin, unpublished results). Rigorous definitions in terms of such physicochemical characteristics as molecular weight, electrophoretic mobility, density centrifugation, or sensitivity to specific inhibitors for other lymphokines were not included. The consideration of such characterizations would not make it possible to carry out the work in its present scope. In this regard (a) the challenge of normal mice of the same strain with OT did not result in the presence of detectable MIF, IF, SRF, MF or LT activities in the sera; (b) challenge of strains A/J, Balb/cByJ, C57Bl/KsJ, AKR/J, SJL/J, and DBA/l J previously sensitized subcutaneously with complete Freund’s adjuvant did not result in the release of detectable quantities of IF or LT (R. Neta, unpublished data) despite the fact that such mice produced specific circulating antibody (35);3 (c) heating of the sera to 56°C for 1 hr did not alter the activity of CF. Thus, involvement of the chemotactic components of complement becomes unlikely (36, 37). It may, therefore, be assumed that the presence of all the above activities is related to the function of sensitized lymphoid cells. While the absence of any given lymphokine activity in the presence of MIF and IF indicates (a) a possible selective defect in the release of such a lymphokine or (b) existence of different mechanisms regulating its presence in the circulation, the presence of these activities in the sera will require further confirmation as to their precise characteristics. For example, the nature of SRF and CF activities in the sera of DBA/ 1J mice in which MIF and IF II activities were not detected requires further analysis. Similarly, further investigations will be required to determine the exact nature of the factors causing strong cytotoxicity in the sera of CBA/CaJ and Balb/cBy mice, which were low responders in the release of IF II and MIF. However, (a) lack of cytotoxic, chemotactic, or mitogenic activities in the sera of C57Bl/KsJ mice, or (b) lack of mitogenic and chemotactic activities in DBA/2J or SJL/J mice, which were all high responders in the release of MIF and IF II, could not be related to the low sensitivity of the assays, since sera from DBA/IJ, CBA/CaJ, and SWR/ J mice were obtained under the same experimental conditions and responded in one or more of these assays. The activities represented by MIF and IF II parallel each other in degree and in time of appearance after challenge, with the exception of Balb/cByJ (see Results). The variability in the titers of MIF in this strain may be the result of the activity of an inhibitory substance similar to that active against IF II (38). The lack of correlation in the presence of MIF vs IF observed in this strain may be explained by the existence of multiple molecular entities of these activities (39,40), only some of which may be active in the circulation. ’ R. Neta, S. B. Salvin, and D. C. Shreffler,
Manuscript in preparation.
218
NETA, SALVIN, AND SABAAWI
In some cases (e.g., C57Bl/KsJ and C57B1/6J), migration of macrophages toward the experimental serum was less than that toward the autologous control serum. The possibility therefore exists that suppressor factors for chemotaxis may be induced under these conditions. Suppressor factors for chemotaxis have been reported in sera from normal and diseased humans (41, 42). The significance of the capacity to develop high or low titers of lymphokines in the circulation with respect to the development of a variety of immunologic responsesremains to be determined. For example, if lymphokines play an important role in protection, then differences in local vs systemic resistance (43) may be related to the level of responsiveness of individual lymphoid tissues. Such responsiveness would involve the release of particular lymphokines or pattern of lymphokines. The composition of the lymphokines would then determine the type of immune response. ACKNOWLEDGMENTS The authors thank Robert B. Propes and T. C. Schuler for their outstanding and valuable assistance in the laboratory.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
David, J. R., Al Askari, S., Lawrence, H. S., and Thomas, L., J. Immunol. 93, 264, 1964. Bloom, B. R., and Bennett, B., Science 153, 80, 1966. David, J. R., and David, R. R., Prog. Allergy 16, 300, 1972. Gery, I., and Waksman, B. H., J. Exp. Med. 136, 143, 1972. Tubergen, D. C., Feldman, J. D., Pollock, E. M., and Lerner, R. A., J. Exp. Med. 135, 255, 1972. Papageorgiou, P. S., Henley, W. L., and Glade, P. R., J. Immunol. 108, 494, 1972. Pick, E., Cohen, S., and Oppenheim, J. J., In “Biology of the Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, Eds.). Academic Press, New York, 1979. Adelman, N. E., Hammond, M. E., Cohen, S., and Dvorak, H. F., In “Biology of the Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, Eds.). Academic Press, New York, 1973. Andreis, M., Stastny, P., and Ziff, M., Arthritis Rheum. 17, 537, 1974. Bennett, B., and Bloom, B. R., Proc. Nat. Acad. Sci. USA 59, 756, 1968. Bernstein, I. D., Thor, D. E., Zbar, B., and Rapp, H. J., Science 172, 729, 1971. Papermaster, B. W., Holtermann, 0. A., Rosner, D., Klein, E., and Dao, T., Res. Commun. Chem. Pathol. Pharmacol. 8, 4 13, 1974. Salvin, S. B., Youngner, J. S., Nishio, J., and Neta, R., J. Nat. Cancer Inst. 55, 1233, 1975. Neta, R., and Salvin, S. B., Lymphokines 2, 295-308, 1981. Chilgren, R. A., Meuwissen, H. G., Quie, P. G., Good, R. A., and Hong, R., Lancer 1, 1286, 1969. Kirkpatrick, C. H., Rich, R. R., and Bennett, J. E., Ann. Intern, Med. 74, 995, 1971. Boraschi, D., and Meltzer, M. S., J. Immunol. 125, 777, 1980. Neta, R., and Salvin, S. B., Cell, Immunol. 51, 173, 1980. Salvin, S. B., Ribi, E., Granger, D. L., and Youngner, J. S., J. Immunol. 114, 354, 1975. Salvin, S. B., and Nishio, J., J. Immunol. 103, 138, 1969. Jameson, P., Dixon, M. A., and Grossberg, S. E., Proc. Sot. Exp. Biol. Med. 155, 173, 1977. Nelson, R. D., Quie, P. G., and Simmons, R. L., J. Immunof. 115, 1650, 1975. Boyum, A., Stand. J. Clin. Lab. Invest. 21:Suppl. 97, 51, 1968. Pollard, M., J. Nat. Can. Inst. 51, 1235, 1973. Salvin, S. B., Youngner, J. S., and Lederer, W. H., Infect. Immunity 7, 68, 1973. Granger, G. A., Daynes, R. A., Runge, P., Jeffes, E., and Prieur, A. M., Contemp, Top. Mol. Immunol. 4, 205, 1975. Allen, E. M., Moore, V. L., and Stevens, J. O., J. Immunol. 119, 343, 1977. Cheers, C., McKenzie, I. F. C., Pavlov, H., Waid, C., and York, J., Infect. Immunity 19,763, 1978. Nakamura, M. R., and Tokunaga, T., Infect. Immunity 22, 657, 1978. Adelman, N., Cohen, S., and Yoshida, T., J. Immunof. 121, 209, 1978.
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IN THE IN VW0 RELEASE
OF LYMPHOKINES
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31. Kuhner, A. L., Cantor, H., and David, J. R., J. Immunol. 125, I I 17, 1980. 32. LaGrange, P. H., and Mackaness, G. B., J. Exp. Med. 148, 235, 1978. 33. Wolstencraft, R. A., Bray, M. A., Dumonde, D. C., DeFranco, M. F., Hanson, J. M., Morley, J., and Smart, J. V., Cell Immunol. (Abstr.) 21, 332, 1976. 34. Youngner, J. S., and Salvin, S. B., J. Immunol. 111, 1914, 1973. 35. Neta, R., and Salvin, S. B., Infect. Immunity, in press, 1981. 36. Leonard, E. J., and Meltzer, M. S., Cell Immunol. 26, 200, 1976. 37. Ward, P. A., Cochrane, C. G., and Mtiller-Eberhard. H. J., J. Exp. Med. 122, 327, 1965. 38. Neta, R., Cell Immunol. 60, 100, 1981. 39. Weiser, W. Y., Greineder, D. K. Remold, H. G., and David, J. R., J. Immunol. 125, 1958, 1981. 40. Weiser, W. Y. Remold, H. G., and David, J. R., Fed. Proc. (Abstr.) 40, 1001, 1981. 41. Berenberg, J. L., and Ward, P. A., J. C/in. Invest. 52, 1200, 1973. 42. Ward, P. A., and Berenberg, J. L., N. Engl. J. Med. 290, 76, 1974. 43. LaGrange, P. H., and Hurtrel, B., C/in Exp. Immunol. 38, 461, 1979.
IMMUNOLOGY
64, 203-219 (1981)
Mechanisms I. Comparative
in the in l&o Release of Lymphokines’ Kinetics in the Release of Six Lymphokines in Inbred Strains of Mice
RUTH NETA,* S. B. SALVIN,? AND MOHAMED SABAAWI~ *Department of Microbiology, University of Notre Dame, Notre Dame, Indiana 64556, and TDepartment of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania I5261 Received March
16. 1981; accepted July 7. 1981
Lymphokines were detected in the sera of 16 strains of inbred mice sensitized intravenously with cell walls of Mycobacterium bovis strain BCG and challenged subsequently intravenously with old tuberculin (OT). Variations occurred between the strains in the types and quantities of six lymphokines studied, namely, chemotactic factor (CF), type II interferon (IF II), lymphotoxin (LT), migration inhibitory factor (MIF), mitogenic factor (MF), and skin reactive factor (SRF). The times for maximum release of the lymphokines in the different strains were similar for MIF, IF II, SRF, and MF, but differed for CF and LT. The degree of activity of MIF and IF II generally paralleled one another in the different strains but such parallelism did not occur for the other four lymphokines. Each of the 16 strains was a high responder for at least one of the lymphokines, indicating that in sensitized mice the release or presence in the circulation of lymphokines in response to a specific antigen is a selective process. Thus, each strain may have an individual combination of lymphokines, interactions of which may determine the types of pathway utilized in an immunological response.
INTRODUCTION Lymphokines are lymphocyte-derived soluble mediators of cellular immunity. They were originally described as products of sensitized lymphoid cells after in vitro stimulation with specific antigen (1, 2). Numerous investigations resulted in (a) description of various effects that these soluble mediators had on the cells of the immune and other body systems (3); (b) recognition that cells other than lymphocytes (stimulated or unstimulated), i.e., macrophages (4), fibroblasts (5), and various cell lines (6) produced soluble substances with properties similar to those of lymphokines; (c) the finding that stimulation of lymphoid cells in vitro with a variety of agents might result in efficient production of lymphokines (7); and (d) the accumulation of evidence that such lymphokines were important in the development of cell-mediated immunity (CMI) (8). Because of difficulties encountered in the separation and purification of various lymphokines, evidence for ’ This work was supported by the Public Health Service, National Institute of Allergy and Infectious Diseases (Grant AI-16064) @B.S.) and by the American Lung Association and National Institutes of Health, IF 32CA 06519 (R.N.). 203 0008-8749/81/160203-17$02.00/O Copyright 0 198 I by Academic Rss, Inc. All rights of reproduction in any form reserved.
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their role in cell-mediated immunity has been limited to the demonstrations that lymphokine-enriched preparations can cause inflammatory responses (9), skin reactions ( lo), and rejection of certain tumors ( 1l- 13). Lymphokine release was detected in vivo at the site of an immune response (14). However, it is still not known whether some or all of the known lymphokines are necessary for the normal functions of the immune system. Consequently, the question arises as to whether certain immunodeficiency diseases develop because one or several of the lymphokines are selectively not produced. Clinical data indicate a possible deficiency in the production of some and not other lymphokines in certain disease states (15, 16). Yet, limited information is available on similar differences in the production of inflammatory lymphokines in experimental animals (17). In a previous report ( 18), evidence was presented that marked differences existed in the amounts of type II interferon (IF II)’ and of migration inhibitory factor (MIF) released in vivo into the circulation of different inbred strains of mice after sensitization with cell walls of Mycobacterium bovis strain BCG and subsequent challenge with old tuberculin (OT). The occurrence of these variations introduced a possible means for (a) separation of the mechanisms of production of individual lymphokines and (b) studies of the role of the individual lymphokines in cell-mediated processes. In this paper, evidence is presented that activities associated with such lymphokines as IF II, MIF, SRF (skin reactive factor), CF (chemotactic factor), MF (mitogenic factor), and LT (lymphotoxin) are not necessarily released in vivo in parallel to one another in time or in quantity. MATERIALS
AND METHODS
Mice. Outbred female Swiss Webster mice were purchased from Taconic Farms, Germantown, New York. Peritoneal exudate cells were harvested from these mice approximately 5 days after intraperitoneal injection of 5 ml light mineral oil. The 16 strains of inbred mice, purchased from Jackson Laboratory, Bar Harbor, Maine, were as follows: A/J, C57B1/6J, C57Bl/lOJ, C57Bl/lOSNJ, C57L/J, Balb/cByJ, C57Bl/KsJ, DBA/2J, Sec/lReJ, CBA/CaJ, C3H/HeJ, AKR/J, RF/J, DBA/ lJ, SWR/J and SJL/J. Antigens. Lyophilized BCG cell walls were prepared by and obtained through the courtesy of Dr. Edgar Ribi, Rocky Mountain Laboratory, Hamilton, Montana. The cell walls were ground in Drakeol, then emulsified in Tween 80-saline (19). Mice were sensitized by intravenous injection of 300 gg BCG cell walls in 0.2 ml, and 3 weeks later challenged intravenously with 50 mg old tuberculin (OT) in 0.2 ml, purchased from Jensen-Salsbury, Kansas City, Missouri. The mice were exsanguinated at varying intervals after challenge, and the sera removed for assay. “Zero” time refers to bleeding of sensitized, nonchallenged mice. Purified protein derivative (PPD), obtained from either Parke, Davis & Company, Detroit, Michigan, or from the National Institutes of Health, Bethesda, 2 Abbreviations used: BCG, Mycobacrerium bovis strain BCG; CF, chemotactic factor; CMI, cellmediated immunity; CW/Dr, cell walls of BCG in Drake&Tween 80 emulsion; IF II, type II interferon; LT. lymphotoxin; MEM, minimum essential medium; MF, mitogenic factor; MIF, migration inhibitory factor; MLR, mixed lymphocyte reaction; OT, old tuberculin; PE, peritoneal exudate; PPD. purified protein derivative of old tuberculin; SRF, skin reactive factor.
MECHANISMS
IN THE IN VlVO RELEASE
OF LYMPHOKINES
205
Maryland, was used for footpad testing for delayed hypersensitivity by injection of 30 pg in 0.05 ml in a hind footpad. A similar quantity of normal autologous serum was injected as a control into the other hind footpad. Migration inhibitory factor (MIF). This lymphokine was titered by determining what dilution of experimental mouse serum resulted in an inhibition of 20% or more of the migration of peritoneal exudate cells on agar, in comparison with the effect of control serum from the same strain of inbred mice (20). The serum dilution (0.6 ml) was mixed with 2.4 ml of an agar medium containing medium 199, 0.5% Oxoid ion agar, and 100 units/ml of penicillin and 100 pg/ml of streptomycin at 45°C all dispensed in a volume of 3 ml in a 30-ml Falcon flask. A small drop of packed peritoneal exudate cells was placed on the surface of the agar, and after incubation at 37°C for 48 hr the distance of migration was measured with the aid of a micrometer in a low-power microscope. The percentage of inhibition of migration was equal to l-
migration of PE cells in experimental serum x 100. migration of PE cells in control serum
The reciprocal of the maximum dilution of experimental serum causing at least 20% migration inhibition was regarded as the titer of MIF activity. Interferon. Interferon activity was determined by an assay involving the use of encephalomyocarditis (EMC) virus hemagglutination reduction yield (2 1). Here L-929 cells were cultured overnight in minimum essential medium (MEM) with 10% fetal calf serum in glass tissue culture tubes to form monolayers. The medium was decanted and the cells were washed twice with phosphate-buffered saline. The experimental and control sera were diluted lo-fold to obtain 10m2to 10m6dilutions of each serum in MEM containing 2% fetal calf serum. The tubes with the cell monolayers were inoculated in triplicate with 1 ml of each of the serum dilutions. After a 24-hr incubation period at 37°C the medium was removed, cell monolayers were washed twice with phosphate-buffered saline, and 0.25 ml EMC virus (hemagglutination titer, 1:16,384) in a buffer containing 0.5% gelatin and 0.2% lactoalbumin hydrolysate in Hanks’ solution was added to the monolayers. After 30 min incubation at 37°C the infected cells were washed twice with PBS, fresh MEM with 2% fetal calf serum was added, and the tubes were incubated for an additional 18 hr. The virus was harvested by repeated freezing and thawing, and the titer was determined in a hemagglutination assay using human type “0” erythrocytes. The titer of interferon was expressed as a reciprocal of the serum dilution that produced a reduction of 0.5 log hemagglutination yield of EMC virus. Ten units of interferon in this assay was equivalent to 1 unit of a reference mouse interferon standard (obtained from The Antiviral Substances Program of NIAID, NIH). Skin reactive factor. Serum (0.05 ml) from one of the inbred strains of mice was injected into a rear footpad of a normal Swiss Webster mouse; control serum was injected into one hind footpad, experimental serum, into the other hind footpad. The extent of swelling was then measured at 1/2-h intervals up to 4 hr postchallenge with the aid of a calipers-micrometer (Schnelltlster). An increase greater than 10% was considered to be indicative of a positive response. Histologic sections of appropriate footpads were made in order to determine the type of cellular infiltrate.
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Tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Chemotacticfactor. Chemotaxis was assayed by a technique involving the migration of adherent peritoneal exudate (PE) cells under agar (22). Here, one part of 5% agarose, i.e., 10 ml, was mixed with 5 parts, i.e., 50 ml, of the following medium: five milliliters medium 199 (lox), 0.5 ml 200 mM L-glutamine, and 100 units per 100 kg/ml penicillin-streptomycin, brought to 50 ml at 45°C with distilled water. Hepes buffer was added (1 M, pH 8.0) to adjust the pH to 7.2. Five milliliters of the agarose medium were added to each 60 X 15-mm tissue culture dish (#3002, Falcon Plastics, Oxnard, Calif.) and allowed to harden at room temperature before transfer to 4°C for at least an hour. With the aid of a template, a series of three wells, 2.4 mm in diameter and 2.4 mm apart, was cut into the agar and the plugs were removed with an 18-gauge needle. Adherent peritoneal exudate cells were obtained by suspending 1 X 10’ cells/ml in MEM containing 10% guinea pig serum in a petri plate for an hour at 37°C in 5% CO*, decanting the supernatant, removing the adherent cells with a rubber policeman, then reconstituting the cells to 1 X lO’/ml in a medium (Hanks’ supplemented with L-glutamine) containing 1% human serum albumin. With the aid of an automatic pipet, 5~1 of the cell suspension were placed in the center well, and either experimental serum or normal autologous serum placed in the outside wells. After incubation at 37°C in a COZ incubator for 18 hr. the cells were fixed by the addition of 3 ml absolute methanol for 30 min and stained with Wright’s stain. Distances of migration were determined in a low-power microscope with the aid of an ocular micrometer cell. Results are presented as the difference in units of migration between experimental and normal autologous sera. Each unit represents 1.75 mm. Mitogenic factor. The capacity of lymphokine-containing sera to stimulate cell replication was assessedwith spleen cells from normal Swiss Webster mice. The lymphoid cells were recovered from the interphase of a Ficoll-Hypaque gradient of teased spenic cells (23). Of these cells 4 X 10’ in 0.1 ml were cultured in each well of a microtiter plate containing medium RPM1 1640 with Hepes buffer, and 4% of either control serum or experimental serum. After 48 hr of incubation at 37°C in 5% CO*, 1 &i of tritiated thymidine was added to each well. Twentyfour hours later, the cells were harvested with a MASH II Systems Harvester (Microbiological Associates). The filter disks with the cellular material were placed in scintillation vials with 9 ml Econofluor, and the counts per minute of the samples was measured in a liquid scintillation counter. The data are reported as the stimulation index of triplicate samples, namely, cpm of cells in experimental serum cpm of cells in control serum * Colony inhibition assay. In vitro-propagated PA III tumor cells (24) derived from the spontaneous prostate carcinomas of germ-free Lobund Wistar rats were removed from the monolayers by trypsinization in 0.25% trypsin solution. The cells were washed and counted and then suspended to a final concentration of 6 X lo’/ ml cells in MEM supplemented with 10% fetal calf serum. The suspension (0.45 ml) was distributed in tissue culture petri dishes (Falcon 3008). Normal control mouse sera and the assayed sera were added to make up the final concentration
MECHANISMS
IN THE IN VIVO RELEASE
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207
of 2.5% of the mouse serum. Cells in culture in triplicates were incubated for 5 or 6 days. The medium was removed and the cells were fixed in methanol and stained with 0.1% crystal violet solution. The number of cell colonies consisting of five or more cells was counted with the aid of a stereomicroscope. The data are expressed either as percentages of suppression of target cell growth (Table 1) or as percentages of cell growth in experimental serum in comparison with cell growth in control serum (Fig. 6). RESULTS Release of AUF Sixteen strains of inbred mice belonging to six different H-2 haplotypes were assayed for their capacity to release MIF in vivo, after intravenous sensitization with 300 pg of cell walls of iU. bovis strain BCG in Drakeol-Tween 80 emulsion (CW/Dr), intravenous challenge 3 weeks later with 50 mg old tuberculin (OT), and exsanguination 4 hr later (Table 1). This schedule was used, since maximum titers were obtained in Swiss Webster outbred mice at this time interval in previous experiments (25). The titers of MIF in the mouse sera varied from less than 4 to 256, depending on the strain. Four strains of the H-2’ haplotype and two of the H-2q haplotype were low responders, i.e., had titers ranging from less than 4 to 16. Three strains of the H-2b haplotype were high responders, in that their sera had MIF titers of 256; the sera of a fourth strain (C57L/J) had a titer of 16. Three of four strains of H-2d haplotype had sera with titers of 64 to 256; the fourth strain, Balb/cByJ, varied in different experiments from less than 4 to 256 in its titer. The single strains belonging to the H-2” (A/J) and H-2” (SJL/J) haplotypes were high responders, with titers of 256 and 64, respectively. The possibility was present that these differences were due to variations in the times for the release of maximum activities of MIF. Accordingly, 10 inbred strains of mice were assayed for their capacity to release MIF at times varying from 0 to 24 hr after challenge with OT. Five of the strains were high responders, i.e., A/J, C57B1/6J, C57Bl/lOSNJ, C57Bl/KsJ, and SJL/J, while five other strains were low responders, i.e., AKR, CBA/CaJ, C3H/HeJ, DBA/lJ, and RF/J (Fig. 1). In all cases,maximum titers were obtained at approximately 4 hr post challenge. Release of Type II Interferon (IF II) The sera of 16 strains, obtained 4 hr after challenge, were also assayed for their contents of IF II after the mice had been appropriately sensitized and challenged (Table 1). The titers in the sera of these strains varied from less than 100 to more than 1,000,OOO.For example, the 4 strains of the H-2’ haplotype had titers of less than 100 to 21,000; the 2 strains of the H-2q haplotype had titers of ~100 and 11,500. Three of the four strains of the H-2b haplotype had titers of about l,OOO,OOO, while the fourth strain (C57L/J) had a titer of 14,000. Three of four H-2d strains had titers higher than 100,000, while the fourth strain, Balb/cByJ mice, in one experiment, had a titer higher than 10,000; in another experiment, a titer of 2500, while in five other experiments the IF II titers were lower than 100. In studies on the time intervals after challenge for maximum release of IF II, the highest titers were found in the sera at 4 hr after challenge (Fig. 2).
950,000 500,000 I ,300,OOo I, 100,000 14,000 2,500* I ,300,000 120,000 200,000 19,500 850 1,100
256 256 256 256 I6 32* 256 256 64 16 4 4 14 <4 <4 64
H-2
S
a b b b b d d d d k k k k q 9
IF II titer
MIF titer 24.0 f 13.7 ic 25.9 + 17.0 k 22.3 k 24.3 f 30.6 + 13.8 k 22.0 f 13.0 +
0.5 2.3 2.5 3.2 2.1 2.3 3.2 0.5 2.2 6.5 0 + 1.05 -7.5 + 0.78 N.D. +I.81 + I.17 N.D. -3.12 + 1.6 -4.68 + 0.6 Ok0 N.D. +3.0 + 0.93 +0.68 + 0.68 0 + 1.77 +I.5 + 0.46 +4.17 + 1.1 N.D. -1.25 + 0.72
CF (unit of migration + SE)
of 16 Inbred Strains of BCG-Sensitized
SRF (% increase of swelling f SE
Released in Vitro into the Circulation
Lymphokines
of Six Different
1
3.6 + 0.3 2.0 + 0.4 N.D. 2.3 + 0.64 N.D. 1.4 f 0.2 1.1 + 0.32 1.2 + 0.37 N.D. 3.4 + 0.7 2.3 + 0.5 I.5 + 0.46 N.D. 0.5 + 0.21 3.6 + 0.3 1.3 +- 0.2
MF (stimulation index + SE)
Mice 4 hr after Challenge
84.4 + 4.4 32.4 + 9.1 N.D. N.D. N.D. 15.6 f II.7 -3.5 f 9.2 N.D. N.D. 39.1 ? 6.7 N.D. 48.8 -t 9.2 10.0 f 2.9 N.D. N.D. 78.8 If: 4.9
LT (% cytotoxicity k SE)
with OT
Note. See Materials and Methods for procedures for sensitization and challenge, units of migration and units of measurement. In each experiment, a pool of sera from IO mice was used. Exclusive of strains SWR/J and C57L/J, the figures represent results from two to five experiments. * Indicates variable results, cf text. N.D., not done.
C57Bl/6J C57Bl/lOJ C57Bl/lOSNJ CS’IL/J Balb/cByJ C57BI/KsJ DBA/2J Set/ I ReJ CBA/CaJ C3H/HeJ AKR/J RF/J DBA/IJ SWR/J SJL/J
A/J
Strain
Activities
TABLE
if $
5
s
? 5
3
it
MECHANISMS
IN THE IN VW0 RELEASE OF LYMPHOKINES
512 -
209
. A/J d C57BVlOSnJ 0 C57BIIKsJ
C3WHeJ + RF/J
l
0
2
4
6 6 IO TIME (hours)
12 N
24
FIG. I. Titers of MIF at different time intervals after challenge. Mice were sensitized intravenously with 300-rg cell walls of BCG in Drakeol-Tween 80 emulsion, and 3 weeks later challenged intravenously with 50 mg OT. A minimum of 10 mice was exsanguinated at varying time intervals thereafter. The titers are the reciprocal of the serum dilutions that inhibited the migration of normal peritoneal exudate cells by at least 20%.
. AN 0 C57BVlOSnJ q C57BIfKsJ x C57BI/&l n CBA/CaJ 0 SJLN A AKWJ l C3WHeJ 0 DBA/lJ
-+ a
2
I 4
1 6 TIME
I 1 8 IO (hours)
I 12
I%
FIG. 2. Titers of IF II at different time intervals after challenge. See Fig. 1. The titers are the reciprocals of the serum dilution that produced a reduction of 0.5 log hemagglutination yield of EMC virus growth in L cells. Ten units of interferon in this assay was equivalent to 1 unit of a reference mouse interferon standard.
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0 C57BVlOSnJ n CBA/CaJ l C3H/HeJ 0 DBAllJ v DBAl2J
6 8 IO TIME (hours)
24
A
FIG. 3A. Extent of footpad swelling induced by sera obtained at different time intervals after challenge from five strains of inbred mice. See Fig. 1. The percentage increase in footpad thickness was obtained by comparing one hind footpad injected with experimental serum with the other hind footpad injected with control serum. A minimum of four mice was used for experiment. FIG. 39. Section of a footpad of a mouse inoculated with control serum from C57BI/KsJ mice, and sacrificed 2% hr later. X750.
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serum of C57BI/ KsJ FIGi. 3C. Section of a footpad of a mouse inoculated with lymphokine-containing mice and sacrificed 2% hr later. X750. FIGi. 3D. Section of a footpad of a mouse actively sensitized subcutaneously with 300 rg CW, /Dr. tested ! in the footpad 3 weeks later with 30 pg PPD. and sacrificed 24 hr later. X750.
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A striking parallelism was apparent in the different strains of mice in the quantities of IF II and MIF released in vivo into the circulation at varying times of exsanguination after challenge (Table 1; Figs. 1 and 2). The presence of low titers or the absence of detectable titers of IF II and MIF in the sera of AKR/J, C3H/ HeJ, CBA/CaJ, RF/J, and DBA/ 1J strains at all times after challenge indicates the inherent inability of these strains to develop high titers of these two lymphokines under the conditions of experimentation. Presence of Skin Reactive Factor (SRF) Introduction of lymphokine-containing preparations intradermally results in an inflammatory reaction that resembles delayed hypersensitivity (10). What lymphokine, or lymphokines, is necessary for this reaction to develop has not been established. Because marked differences occurred in the amounts of MIF and IF II in the sera of the various strains of mice, experiments were initiated to determine whether a parallelism existed in the capacity of a given strain to release the two lymphokines and the capacity of the serum from that strain to induce an inflammatory skin response, presumably of the mononuclear or delayed type. Accordingly, outbred Swiss Webster mice were inoculated in one hind footpad with experimental serum and in the other hind footpad with control serum. Analysis of the footpad reactions by the sera of each of 15 strains of mice indicated that a correlation did not always exist between the presence of the two lymphokines in a serum and that of SRF (Table 1). Sera obtained from 13 of the 15 strains at 4 hr after OT challenge had the capacity to induce footpad reactions passively of more than 10%. Of sera from 6 strains that were low responders in the release of MIF and IF II and that were examined for skin reactivity, 2 did not induce footpad reactions passively, i.e., C3H/HeJ and AKR, and 4 did, i.e., DBA/lJ, CBA/CaJ, SWR/J, and C57L/J. The maximum swelling developed with sera from bleedings at 2-4 hr post challenge (Fig. 3A). Of 2 strains that were low responders for MIF (4 or ~4) and IF II (
MECHANISMS
-6
-cc!
0 C57BVlOSnJ
-8
0
4
8
12 TIME
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16 (hours)
20
24
. A/J C3HlHeJ . DSAIPJ
I IL
0
l
4
8
12 TIME
16 (hours)
20
24
6
A +4 c C57BVKs.l I C57SV6J
q
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i I
-
/
-10 \\/’ I 0
C
I 4
I
x I1 8
I 12 TIME
I1 16 (hours)
I1
I 20
I 24
FIG. 4. (A-C) Chemotactic activity of sera obtained at different time intervals after challenge of 12 inbred strains of mice. Oil-induced peritoneal exudate macrophages were obtained from Swiss Webster mice. Numbers represent the average differences in units of cell migration between experimental and normal autologous sera. Each point represents an average of lo-12 readings. I unit = 1.75 mm.
addition, multiple peaks of activity appeared in some of the sera, thus indicating the possible presence of several chemotactic factors. Of sera examined from 12 strains, 7 indicated chemotactic activity at some period up to 24 hr postchallenge, namely, C57Bl/lOSNJ, Balb/cByJ, AKR/J, RF/J, SJL/J, CBA/CaJ, and DBA/ 1J. Of the two inactive sera (AKR/J and C3H/HeJ) for induction of skin reactivity, one, AKR/J, had chemotactic activity. Also, a correlation did not exist between the capacity of a strain to release IF II and MIF into the circulation and the capacity to release CF in viva Of the low responders for MIF and IF II release,
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four strains had chemotactic activity, namely, AKR/J, CBA/CaJ, RF/J, and DBA/ 1J. Of the high responders for MIF and IF II, four strains did not have detectable quantities of CF in their sera, namely, A/J, C57Bl/KsJ, C57B1/6J, and DBA/2J. Thus, it appears that, in a given strain, the capacity to release SRF, MIF, IF II, or CF is under separate genetic control. Release of Mitogenic Factor (MF) One of the characteristics of cell-mediated immunity and delayed hypersensitivity is the enhanced mitotic activity of sensitized lymphocytes in the presence of specific antigen. This increased lymphocyte replication is due in part to a mediator produced in vitro or in vivo by sensitized lymphocytes exposed to specific antigen. The question arises as to how the release of this factor (MF) in vivo is related to the release of other lymphokines (MIF, IF II, SRF, LT, and CF). The degree of mitotic activity varied between the strains (Table 1) and did not correlate with the levels of MIF and IF II within a given strain. For example, C57Bl/KsJ, DBA/2J, and SJL/J, high responders in the release of MIF and IF, did not have mitogenic activity, while SWR/J and CBA/CaJ, low responders in the release of MIF and IF, had pronounced mitogenic activity. When the sera from seven inbred strains were obtained at different times after challenge and examined for MF, the maximum activity was at 4 hr postchallenge (Fig. 5). Release of Lymphotoxin Another lymphokine, or group of lymphokines, associated with cell-mediated immunity and released by sensitized lymphoid cells after exposure to specific antigen, is lymphotoxin (LT), a substance inhibitory in low concentrations to cell growth and capable in high concentrations of lysis of selected cells (26). Experiments were initiated to determine what correlation existed, if any, in the release of LT with reference to other lymphokines in eight inbred strains. An analysis of sera obtained 4 hr after challenge indicated that the cytotoxic activities varied from strain to strain (Table 1, Fig. 6). A correlation did not exist with the high and low responder strains of the previously mentioned five lymphokines with regard to the degree of response and the time of maximum activity. Serum of a low responder mouse strain of MIF and IF II, such as CBA, was highly cytotoxic, in that a concentration of 1:40 caused 100% lysis of target cells (Fig. 6). On the other hand, high responder strains of MIF and IF II, such as C57B1/6J and C57Bl/KsJ, did not show strong cytotoxicity in that a similar serum concentration reached a maximum lytic activity of only 57% at 24 hr after challenge. In addition, maximum cytotoxic activity of the sera from the various inbred strains developed at different times after challenge. For example, the sera from SJL/J mice had maximum cytotoxicity at 4 hr after challenge; sera from CBA/CaJ, A/J, and RF/J were at a maximum at 8 hr; and sera from C57B1/6J and C57Bl/KsJ had maximum cytotoxicity at 24 hr. Therefore, LT, like CF, varies either in the kinetics of its release or in the appearance of its activity in the serum independently of the other lymphokines in the strains studied.
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215
A C57SVlOSnJ q C57BVKsJ l CBAlCaJ A AKWJ l CIH/HeJ o DBAflJ . DBA/2J
3.2 2.8 t
TIME (hours) FIG. 5. Mitogenic activity of sera obtained at different time intervals after challenge of seven inbred strains of mice. Numbers represent the ratio of cpm from splenic cells cultured in experimental sera vs. the cpm of those cells cultured in normal autologous sera. The data are derived from two experiments.
DISCUSSION Numerous reports have indicated that variations in cell-mediated immunity and in delayed hypersensitivity to microbial organisms or to purified antigens occur in different strains of inbred mice (27-29). Few studies, however, have been reported on similar variations in the production of lymphokines. Variations have been described in the release of MIF in vitro (30) and MIF and IF II in vivo (18) in different strains of inbred mice. In experiments on five strains of mice, splenic cells from two strains, namely, DBA/2 and C57B1/6 challenged in vitro with either antigen or Con A, did not produce MIF (30). However, lymph node cells or peritoneal exudate cells from C57B1/6 mice did produce MIF after challenge with Con A (31). Intravenous sensitization and challenge of the two above strains caused the release of high titers of IF II and MIF into the sera. Therefore, the capacity to release lymphokines in the spleen may be independent of the capacity to release such lymphokines in other lymphoid organs or into the circulation, possibly because of homing and sequestration of suppressor cells in the spleens (32). The system to measure the release of lymphokines into the blood was chosen, since this release not only reflected immunologic events in vivo in the circulation but also occurred in parallel with the capacity of mice to develop resistance to challenge with virulent organsims ( 19). Mice sensitized with components of BCG in a manner that induced the capacity to release lymphokines into the circulation also had increased resistance to challenge with virulent H37Rv. In contrast, sensitization via the subcutaneous route or with components of BCG which did not induce the release of IF II and MIF in vivo did not produce enhanced resistance
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x C57BVW . CBA/CaJ
+ RF/J v Balb/c ByJ
TIME (hours) FIG. 6. Cytotoxic effect of sera from eight inbred strains obtained at different times after challenge. See Fig. 1 for conditions for obtaining the sera. Each point represents the number of colonies (percentage) of rat prostate tumor cells grown in 2.5% experimental serum, as compared with cells grown in control autologous serum. (Standard errors were less than IO%.)
to challenge with a virulent strain of Mycobacterium tuberculosis. Other experiments ((29), Neta and Salvin, unpublished results) also indicated that C57Bl/ 1OSNJ and C57Bl/KsJ strains, which were high responders in the release of serum MIF and IF II, were resistant to infection with BCG or Cundidu albicans, while low responder strains, such as DBA/l or C3H/HeJ, were highly susceptible to such infections. In the present study of sera from 16 strains, 7 (not including Balb/cByJ, which will be discussed separately) had titers of IF II and MIF markedly lower than the titers in the sera of the other strain, i.e., titers of 16 or less for MIF, and less than 20,000 for IF II (Table 1). The maximal titers of IF II and MIF were detected in the sera of 10 strains of mice, 5 of which were low responders, when these mice were bled 4 hr after challenge (Figs. 1 and 2). The differences in the titers of MIF and IF II between mouse strains could not therefore be explained on the basis of different kinetics of their release in the blood. Occasionally, production of some, but not all, lymphokines has been detected in a given human patient (15, 16). Yet, only few attempts have been made to observe a similar dissociation in the release of various lymphokines in experimental animals (33). The assumption has been that an appropriately sensitized animal can produce
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217
all of the known lymphokines after stimulation with specific antigen or with mitogen. The existence of strains of mice that can release different levels of IF II and MIF into their circulations provided a model to test this assumption. Therefore, other activities of lymphokines were examined in the same sera to determine possible correlation or dissociation of these lymphokines with MIF and IF II. The activities of lymphokines in the sera of sensitized and challenged mice were determined by biological assays. However, the two lymphokines, MIF and IF II, released in vivo into the mouse circulation had been determined to have physicochemical properties similar to those of the in vitro-produced lymphokines (( 34) Bout, David and Salvin, unpublished results). Rigorous definitions in terms of such physicochemical characteristics as molecular weight, electrophoretic mobility, density centrifugation, or sensitivity to specific inhibitors for other lymphokines were not included. The consideration of such characterizations would not make it possible to carry out the work in its present scope. In this regard (a) the challenge of normal mice of the same strain with OT did not result in the presence of detectable MIF, IF, SRF, MF or LT activities in the sera; (b) challenge of strains A/J, Balb/cByJ, C57Bl/KsJ, AKR/J, SJL/J, and DBA/l J previously sensitized subcutaneously with complete Freund’s adjuvant did not result in the release of detectable quantities of IF or LT (R. Neta, unpublished data) despite the fact that such mice produced specific circulating antibody (35);3 (c) heating of the sera to 56°C for 1 hr did not alter the activity of CF. Thus, involvement of the chemotactic components of complement becomes unlikely (36, 37). It may, therefore, be assumed that the presence of all the above activities is related to the function of sensitized lymphoid cells. While the absence of any given lymphokine activity in the presence of MIF and IF indicates (a) a possible selective defect in the release of such a lymphokine or (b) existence of different mechanisms regulating its presence in the circulation, the presence of these activities in the sera will require further confirmation as to their precise characteristics. For example, the nature of SRF and CF activities in the sera of DBA/ 1J mice in which MIF and IF II activities were not detected requires further analysis. Similarly, further investigations will be required to determine the exact nature of the factors causing strong cytotoxicity in the sera of CBA/CaJ and Balb/cBy mice, which were low responders in the release of IF II and MIF. However, (a) lack of cytotoxic, chemotactic, or mitogenic activities in the sera of C57Bl/KsJ mice, or (b) lack of mitogenic and chemotactic activities in DBA/2J or SJL/J mice, which were all high responders in the release of MIF and IF II, could not be related to the low sensitivity of the assays, since sera from DBA/IJ, CBA/CaJ, and SWR/ J mice were obtained under the same experimental conditions and responded in one or more of these assays. The activities represented by MIF and IF II parallel each other in degree and in time of appearance after challenge, with the exception of Balb/cByJ (see Results). The variability in the titers of MIF in this strain may be the result of the activity of an inhibitory substance similar to that active against IF II (38). The lack of correlation in the presence of MIF vs IF observed in this strain may be explained by the existence of multiple molecular entities of these activities (39,40), only some of which may be active in the circulation. ’ R. Neta, S. B. Salvin, and D. C. Shreffler,
Manuscript in preparation.
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NETA, SALVIN, AND SABAAWI
In some cases (e.g., C57Bl/KsJ and C57B1/6J), migration of macrophages toward the experimental serum was less than that toward the autologous control serum. The possibility therefore exists that suppressor factors for chemotaxis may be induced under these conditions. Suppressor factors for chemotaxis have been reported in sera from normal and diseased humans (41, 42). The significance of the capacity to develop high or low titers of lymphokines in the circulation with respect to the development of a variety of immunologic responsesremains to be determined. For example, if lymphokines play an important role in protection, then differences in local vs systemic resistance (43) may be related to the level of responsiveness of individual lymphoid tissues. Such responsiveness would involve the release of particular lymphokines or pattern of lymphokines. The composition of the lymphokines would then determine the type of immune response. ACKNOWLEDGMENTS The authors thank Robert B. Propes and T. C. Schuler for their outstanding and valuable assistance in the laboratory.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
David, J. R., Al Askari, S., Lawrence, H. S., and Thomas, L., J. Immunol. 93, 264, 1964. Bloom, B. R., and Bennett, B., Science 153, 80, 1966. David, J. R., and David, R. R., Prog. Allergy 16, 300, 1972. Gery, I., and Waksman, B. H., J. Exp. Med. 136, 143, 1972. Tubergen, D. C., Feldman, J. D., Pollock, E. M., and Lerner, R. A., J. Exp. Med. 135, 255, 1972. Papageorgiou, P. S., Henley, W. L., and Glade, P. R., J. Immunol. 108, 494, 1972. Pick, E., Cohen, S., and Oppenheim, J. J., In “Biology of the Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, Eds.). Academic Press, New York, 1979. Adelman, N. E., Hammond, M. E., Cohen, S., and Dvorak, H. F., In “Biology of the Lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, Eds.). Academic Press, New York, 1973. Andreis, M., Stastny, P., and Ziff, M., Arthritis Rheum. 17, 537, 1974. Bennett, B., and Bloom, B. R., Proc. Nat. Acad. Sci. USA 59, 756, 1968. Bernstein, I. D., Thor, D. E., Zbar, B., and Rapp, H. J., Science 172, 729, 1971. Papermaster, B. W., Holtermann, 0. A., Rosner, D., Klein, E., and Dao, T., Res. Commun. Chem. Pathol. Pharmacol. 8, 4 13, 1974. Salvin, S. B., Youngner, J. S., Nishio, J., and Neta, R., J. Nat. Cancer Inst. 55, 1233, 1975. Neta, R., and Salvin, S. B., Lymphokines 2, 295-308, 1981. Chilgren, R. A., Meuwissen, H. G., Quie, P. G., Good, R. A., and Hong, R., Lancer 1, 1286, 1969. Kirkpatrick, C. H., Rich, R. R., and Bennett, J. E., Ann. Intern, Med. 74, 995, 1971. Boraschi, D., and Meltzer, M. S., J. Immunol. 125, 777, 1980. Neta, R., and Salvin, S. B., Cell, Immunol. 51, 173, 1980. Salvin, S. B., Ribi, E., Granger, D. L., and Youngner, J. S., J. Immunol. 114, 354, 1975. Salvin, S. B., and Nishio, J., J. Immunol. 103, 138, 1969. Jameson, P., Dixon, M. A., and Grossberg, S. E., Proc. Sot. Exp. Biol. Med. 155, 173, 1977. Nelson, R. D., Quie, P. G., and Simmons, R. L., J. Immunof. 115, 1650, 1975. Boyum, A., Stand. J. Clin. Lab. Invest. 21:Suppl. 97, 51, 1968. Pollard, M., J. Nat. Can. Inst. 51, 1235, 1973. Salvin, S. B., Youngner, J. S., and Lederer, W. H., Infect. Immunity 7, 68, 1973. Granger, G. A., Daynes, R. A., Runge, P., Jeffes, E., and Prieur, A. M., Contemp, Top. Mol. Immunol. 4, 205, 1975. Allen, E. M., Moore, V. L., and Stevens, J. O., J. Immunol. 119, 343, 1977. Cheers, C., McKenzie, I. F. C., Pavlov, H., Waid, C., and York, J., Infect. Immunity 19,763, 1978. Nakamura, M. R., and Tokunaga, T., Infect. Immunity 22, 657, 1978. Adelman, N., Cohen, S., and Yoshida, T., J. Immunof. 121, 209, 1978.
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31. Kuhner, A. L., Cantor, H., and David, J. R., J. Immunol. 125, I I 17, 1980. 32. LaGrange, P. H., and Mackaness, G. B., J. Exp. Med. 148, 235, 1978. 33. Wolstencraft, R. A., Bray, M. A., Dumonde, D. C., DeFranco, M. F., Hanson, J. M., Morley, J., and Smart, J. V., Cell Immunol. (Abstr.) 21, 332, 1976. 34. Youngner, J. S., and Salvin, S. B., J. Immunol. 111, 1914, 1973. 35. Neta, R., and Salvin, S. B., Infect. Immunity, in press, 1981. 36. Leonard, E. J., and Meltzer, M. S., Cell Immunol. 26, 200, 1976. 37. Ward, P. A., Cochrane, C. G., and Mtiller-Eberhard. H. J., J. Exp. Med. 122, 327, 1965. 38. Neta, R., Cell Immunol. 60, 100, 1981. 39. Weiser, W. Y., Greineder, D. K. Remold, H. G., and David, J. R., J. Immunol. 125, 1958, 1981. 40. Weiser, W. Y. Remold, H. G., and David, J. R., Fed. Proc. (Abstr.) 40, 1001, 1981. 41. Berenberg, J. L., and Ward, P. A., J. C/in. Invest. 52, 1200, 1973. 42. Ward, P. A., and Berenberg, J. L., N. Engl. J. Med. 290, 76, 1974. 43. LaGrange, P. H., and Hurtrel, B., C/in Exp. Immunol. 38, 461, 1979.