Immunology 1978 34 231

Detection of cell-mediated immunity to sheep erythrocytes by the capillarymigration inhibition technique in the lizard, Calotes versicolor

S. JAYARAMAN & VR. MUTHUKKARUPPAN Department of Immunobiology, School ofBiological Sciences, Madurai University, Madurai 625 021, India

Received 7 March 1977; acceptedfor publication 25 April 1977

Summary. Utilizing the in vitro capillary migrationinhibition (MI) technique, the cell-mediated immuneresponse to sheep erythrocytes (SRBC) has beenstudied in the lizard, Calotes versicolor. The viabilityof spleen cells in culture was above 85 %, irrespectiveof the presence or absence of antigen. Both plaque-forming cell (PFC) and MI responses were foundto be antigen-specific. Heat-labile serum factorsdid not seem to have a role in the MI pheno-menon. Both cellular and sonicated membranepreparations of SRBC induced a similar pattern anddegree of MI of sensitized spleen cells. Migration ofspleen cells was observed within 1 h of the initiationof cultures. The maximum difference betweencontrol and experimental cultures occurred by 12 hof incubation. There was an insignificant escapefrom inhibition after 24 h of culture. Administrationof 6 x 108 SRBCvia the intramuscular route favouredboth PFC and MI responses. Although the PFCgeneration was favoured, only a low level of MIwas induced by the intraperitoneal and intracardiacinjections. MI and PFC responses are inverselyrelated to the amount of antigen injected. Adminis-tration of 104 SRBC resulted in a high degree of MIwithout the production of PFC. On the other hand,6 x 108 SRBC produced an abundant PFC responsewith a lesser degree of MI. Incorporation of SRBCinto Freund's complete adjuvant resulted in the

Correspondence: Professor VR. Muthukkaruppan, De-partment of Immunobiology, School of Biological Sciences,Madurai University, Madurai 625 021, India.

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production of MI response with a concurrentreduction in the number of PFC. Formalized SRBCgenerated a good MI response without the inductionof PFC. Sonicated SRBC induced both PFC and MIresponses. MI was shown to be mediated by sensi-tized lymphoid cells. As few as 5% sensitized spleencells were enough to bring about significant MIof unsensitized spleen cells. Thus, MI has beenshown to be an in vitro manifestation of CMI toSRBC in the lizard.

INTRODUCTION

The first in vitro model for studying the cell.mediated immune (CMI) response described byRich & Lewis (1932) was based on the observationthat the migration of cells from spleen or lymphnode explants obtained from tuberculous rabbits orguinea-pigs was markedly inhibited in the presenceof tuberculin in the tissue culture media. Improve-ment in the field was brought about by the introduc-tion of the capillary tube migration inhibition (MI)technique which provided a simple, reproducibleand quantitative in vitro system (Bloom, 1971;Morley 1974; Pekarek & Krejci, 1974). This tech-nique has been found useful in assessing CMI to avariety of antigens (cf. Bloom, 1971) including sheeperythrocytes (SRBC) (Pick, Krejci & Turk, 1972;Gordon & Yu, 1973).

S. Jayaraman & VR. Muthukkaruppan

The capillary MI technique has been applied forthe first time in poikilotherms to assess CMI to skinallografts in the lizard, Calotes versicolor (Jayaraman& Muthukkaruppan, 1977a). On the basis of theencouraging results of this study, we have appliedthis technique to detect CMI to SRBC in order tounderstand the mechanism of modulation of im-mune responses in the lizard (Jayaraman, 1976)which has been used in this laboratory as the modelsystem for studying the development of immunity(Muthukkaruppan, Subramonia Pillai & Jayaraman,1976a; Muthukkaruppan et al., 1976b). In thiscommunication, evidence is presented for con-sidering MI of sensitized spleen cells as a manifesta-tion of CMI in vitro to SRBC in the lizard, Calotesversicolor.

MATERIALS AND METHODS

AnimalsLizards obtained from a local commercial dealerwere maintained in the laboratory as detailed else-where (Muthukkaruppan, Kanakambika, Manicka-vel & Veeraraghavan, 1970). Adult lizards weighing35-65 g were housed in wooden cages and fed on livetermites and water ad libitum.

AntigensSheep blood was drawn into Alsever's solution froma single animal, washed three times with phosphate-buffered saline solution (PBS, pH 7 2) and appropri-ate dilutions were made, shortly before use (100 ulof 25% SRBC contained 6 x 108 cells).Rat red blood cells (RRBC) in Alsever's solution,

obtained by a cardiac puncture of white rats, werewashed thrice with PBS before use (100 ,pl of 25%RRBC contained 1 x 108 cells).

Formalized SRBC (F-SRBC) were prepared byadding an 8% suspension of erythrocytes in PBSto an equal volume of 3% formaldehyde followedby incubation for 18 h at 370 with constant and gentleagitation (Dennert & Tucker, 1972). Treatederythrocytes were washed five times with cold PBSand used for immunization. Erythrocytes so treatedshowed no gross changes in antigenicity as judgedby their agglutination pattern with antibody, raisedagainst native SRBC in rabbits.

Sonicated SRBC (S-SRBC) were prepared follow-ing the method of Palmer (cf. Byrd, Feldmann &Palmer, 1974). A suspension of 10% SRBC in PBS

was given 6-8 bursts of energy of 30s duration,separated by a period of 30-60 s using a Vibronics220 Watt Sonicator (Vibronics Pvt. Ltd., Bombay,India). Less than 5% intact erythrocytes were presentafter sonication. To exclude intact erythrocytes, thesuspension was spun at 500 rev/min for 5 min andthe clear supernatant was stored at -5° until used.Haemolysed sonicated SRBC (HS-SRBC) were

prepared in the same manner except that theerythrocytes were first lysed in triple distilled water,but without centrifugation after sonication.

Antigen-adjuvant emulsions were prepared byadmixing 100 pl of 25% SRBC in PBS with an equalamount of Freund's complete adjuvant (FCA)(Difco, U.S.A.; containing killed Mycobacteriumtuberculosis, H37 Ra, 1 mg/ml).

ImmunizationLizards were immunized by a single intracardiac(i.c.), intraperitoneal (i.p.) or intramuscular (i.m.)injection of an appropriate dilution of the antigenin 100 p1 volume of PBS. Intramuscular injectionswere given in the thigh region of hind legs. 200 p1 ofSRBC-FCA emulsions were injected via the i.m. route.

Plaque-forming cell assayDirect haemolysin producing cells were detectedusing the method described previously for Calotes(Kanakambika & Muthukkaruppan, 1972). Theplaques were developed at 370 for 45 min (Jayara-man, 1976). The viability of spleen cells was judgedby the ability to exclude 0-25 % trypan blue dye.

Migration inhibition assayMigration inhibition (MI) assay adapted for lizardsystem (Jayaraman & Muthukkaruppan, 1977a)was followed. Briefly, a single cell suspension ofspleen cells was made in PBS. The cells were washedtwice with diluted L-15 Leibovitz medium (3 partsmedium and 1 part triple distilled water), supple-mented with 20% normal Calotes serum (de-complemented at 560 for 30 min) and antibiotics.The capillaries (0-8 x 90 mm, Laboratory Diagnos-tics Co., Inc., Morganville, N.J. 07751, U.S.A.) werefilled with spleen cells and sealed at one end withmodelling clay. After spinning the capillaries at500 rev/min for 2 min, a nick was made just belowthe cell-fluid interface with a diamond pencil. Byapplying a gentle pressure the cell pellet was sepa-rated and secured to the bottom of the migration

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chambers (Jayaraman & Muthukkaruppan, 1977a)with silicone grease. After sealing the migrationchambers, medium was introduced gently.For most of the experiments 10 p1 of 10% S-SRBC

or HS-SRBC/ml medium was added. Intact erythro-cytes were added directly to the culture medium at aconcentration of 10 p1 of 1 % erythrocytes/mlmedium (Gordon & Yu, 1973; Jayaraman &Muthukkaruppan, 1977a).The migration cultures were incubated at 370 for

24 h and the whole fan area of migration (Jokipii &Jokipii, 1974; Jayaraman & Muthukkaruppan,1977a) was drawn on a paper with camera lucidaand measured by planimetry. The % MI was cal-culated using the formula:

% MI= 100-100 x Migration area with antigen

Migration area without antigen.

StatisticsStatistical assessment of probability was by Student'st-test.

RESULTS

Influence of route of immunization on the immuneresponse

To study the influence of the route of antigen injec-

tion on the immune response, a standard dose ofantigen, 6 x 108 SRBC (Kanakambika & Muthuk-karuppan, 1972) in 100 ul PBS was injected via thei.p. or i.m. route. Since the peak PFC response wasreported to occur on day 14 with i.p. administrationof 6 x 108 SRBC (Kanakambika & Muthuk-karuppan, 1972), MI assay was also performed 14days after immunization. Additionally, 6 x 108SRBC in 100.p1 PBS incorporated into an equalamount of FCA were given i.m. to a group oflizards. Throughout the entire study, the inhibitionof migration of unsensitized lizard spleen cells inmostinstances was negative, i.e. the migration area waslarger in the presence of antigen than in its absence.On the other hand, lizards that had been injectedwith SRBC showed positive inhibition, i.e. themigration area was smaller in the presence ofspecific antigen than in its absence.As shown in Table 1, the i.m. injection of the

standard dose of SRBC in PBS favoured both PFCand MI responses. On the other hand, i.p. and i.c.routes of immunization did not result in appreciabledegree of MI. Interestingly, the lizards whichreceived SRBC-FCA emulsion produced little orno PFC, while exhibiting the same level of MI asshown by the spleen cells taken from lizards whichreceived SRBC only. This observation leads to thepostulation that MI can be obtained with little orno PFC in the test spleen cell population.

Table 1. Selection of route of immunization for the induction of theimmune response to SRBC

Route of PFC/106 WBC % Migration inhibitioninjection (mean ± s.e.) (Mean ± s.e.)

PBS alone 5-2 ±1-6 7 0 ± 3 5(5) (22)

i.c. 137-2 ±48-9 11 *5 ± 8-0t(7) (7)

i.p. 114-7 ±23-8 18-9 ±9-2t(8) (8)

i.M.:SRBC alone 267-7 ±71-3 24-3 ±6-1*

(6) (6)SRBC-FCA 35 0 ±14-1 25-8 ± 5-8*emulsion (9) (9)

The PFC and MI assays were performed with spleen cells 14 daysafter immunization with 6 x 108 SRBC via the intracardiac (i.c.),intra-peritoneal (i.p.) and intramuscular (i.m.) routes.Number of animals is given in parentheses.* Significantly different from controls (P < 0-001).t i.c. vs i.m., P <0 025.t i.p. vs i.m., P < 0 005.

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Table 2. Effect of antigen dose on the immune response to SRBC

% Migration inhibitionDose of SRBC PFC/106 WBC (Mean ± s.e.)

(mean ± s.e.)24 h* 48 h

104 0 7 ±0 5 72-9 ± 3-9 64-6 ± 10-6(4) (4) (4)

106 22*5 ±6-6 52 5 ± 7-3 44-3 ± 18-0(4) (4) (4)

6 x 108 286-2 ±77-3 22-0± 7-2 18-0 ± 6-3(5) (5) (5)

Fourteen days after i.m. immunization with varying doses of SRBC,PFC and MI assays were performed. The area of migration was traced at24 and 48 h of incubation at 37°.Number of animals investigated is given in parentheses.* Statistical significance: 104 vs 106, P <0-010; 106 vs 6 x 108 SRBC,

P>0.10.

Effect of antigen dose on the immune response

To study the influence of antigen dose on the typeof immune responses elicited, different dilutions ofSRBC in 100 Al PBS were given i.m., since this routeof immunization favoured the induction of both HIand CMI responses (Table 1). Fourteen days later,spleen cells were assayed for PFC and MI. The datapresented in Table 2 suggest that these two types ofimmune response were inversely related to theamount of antigen injected. It is noteworthy thatthe injection of 104 SRBC resulted in a high degreeof MI with a low number of PFC. On the otherhand, high dose SRBC injection induced abundantPFC production with a lesser degree of MI. Theinsignificant escape from inhibition of migration at48 h of incubation is seen in spleen cells of lizardsimmunized with varying doses of SRBC.

Migration kinetics

The slight reduction in the degree of MI after 24 h ofculture suggests that the MI is strong and mightoccur quite early in the culture period (Table 2).This led to the study of kinetics of migration ofsensitized spleen cells. Spleen cells from lizards thatreceived 104 SRBC in PBS 14 days earlier, werepacked in capillaries and cultured with and withoutantigen at 37°. The area of cell migration from eachof the capillaries was traced at different timeintervals. Migration of spleen cells was observedwithin 1 h of the initiation of cultures. The initial

feature of migration is a tongue-like process of cellsmoving from the tube, extending to the surface of theglass-coverslip bottom of migration chambers.Thereafter, cells spread radially to form a fan-likestructure. Some cells appear to be attached to thesurface of the glass-coverslip, while the remainingcells migrate over them. The disc of cells becomesthinner as the area of culture increases. At the end ofthe culture period (24 h), two distinguishable zonesare visible: an inner mass of cells adjacent to thetube and a peripheral thin layer which forms the

40r .000.0 Control

00

30F

c

20E

3:a,

*0

1o0 0

0* C

0 o0

0000

0 00Experiment

0 4 8 12 16 20 24ncubation period (h)

Figure 1. Kinetics of migration of sensitized spleen cells inthe presence and absence of antigen. Spleen cells from lizardssensitized by an intramuscular injection of 104 SRBC in PBSwere cultured at 370 and the area of cell migration wastraced at specified time intervals. The figure represents atypical case. (0) control; (0) experiment.

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Cell-mediated immunity to SRBC

Table 3. Influence of nature of antigen on the immune response toSRBC

Treatment PFC/ 106 WBC % Migration inhibition(Mean + s.e.) (Mean ± s.e.)

6x10'F-SRBC 19-9±6-9 21-1±21-1(8) (4)

6X 10'SRBC 443-7±112-9 23-4±7-3(8) (7)

250 Ilof 10 % 203-6±55-6 39-7±10-5S-SRBC (5) (5)

Seven days after i.m. immunization with formalized SRBC(F-SRBC), sonicated (S-SRBC) and native SRBC, PFC and MIassays were performed.Number of animals investigated is given in parentheses.

border of the migrating fan of cells. These dis-tinguishable features are clearly seen in controlcultures, whereas the sensitized spleen cells inpresence of antigen generally do not show such a

pattern of migration. However, the whole fan area

of migration was taken into account for determiningthe extent of MI (Jokipii & Jokipii, 1974; Jayaraman& Muthukkaruppan, 1977a).

Fig. 1 depicts the migration areas in control andexperimental cultures. The increase in the area ofmigration is greatest during the first 12 h of incuba-tion after which it tapers off. Maximum differencesin the area of migration between control and testcultures occurs by 12 h of incubation and this is a

highly reproducible event. The data emphasize thatthe MI is an antigen-induced phenomenon thatbecomes evident between 12 and 24 h of culture.The viability testing with the trypan blue dye exclu-

sion technique revealed the presence of more than

85 %viable cells in cultures incubated with or withoutantigen up to 48 h. In addition, no drop in pH ofthe culture medium was observed in the presence orabsence of the antigen.

Influence of nature of antigen on the immune response

Lizards were immunized with an i.m. injection of6 x 108 formalized SRBC (F-SRBC) and 7 days later,PFC and MI assays were performed with the spleencells. From Table 3, it is clear that F-SRBC failed toimmunize the lizards for antibody synthesis asevidenced by the small number of PFC produced.However, the degree of MI was found similarto that obtained with the same amount of nativeSRBC.

Since sonicated SRBC (S-SRBC) was added to theculture medium as a source of antigen in MI assay,the following experiment was carried out to verify

Table 4. Effect of antigen presentation in vitro on migration inhibition

Immunizing % Migration inhibitionantigen Antigen in vitro (Mean ± s.e.)

Haemolysed-sonicated SRBC 59-0 8-7(7)

104 SRBC Sonicated SRBC 74-4 ± 66(7)

Intact SRBC 69-8 ±7-5(7)

Seven days after i.m. immunization, the MI assay was performed. Theculture medium was added either with intact (x 10,000) erythrocytes orsonicated antigen preparations.Number of animals investigated is given in parentheses.

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S. Jayaraman & VR. Muthukkaruppan

Figure 2. Migration of sensitized spleen cells in the absenceof antigen. The tongue-like central pellet and a large diffuseouter area of the fan are clearly seen (x 10-8).

whether there was any alteration in the immuno-genicity of the antigen preparation as a consequence

of sonication procedure. Lizards were injected with250.u1 of 10% S-SRBC which is equivalent to thestandard dose, 100 ,ul of 25 % SRBC (6 x 108 SRBC).Assays were carried out on day 7, since peak PFCand MI responses were found to occur after an i.m.injection of 6 x 108 SRBC on day 7 (Jayaraman &Muthukkaruppan, 1977b). The data in Table 3indicate that the sonication procedure did not alterthe immunogenicity of SRBC. Further, this experi-ment provides evidence for the fact that the observedMI is not due to a simple toxic or cytopathic effectof the sonicated form of SRBC when added to theculture medium. This view has been furtherstrengthened by the observation that the same levelof MI could be obtained by supplying intact SRBCto the culture medium (Table 4). Worth mentioningis the fact that the degree of MI was not significantlyaltered by the removal of cellular components ofSRBC by haemolysis. Additionally, intact antigendid not interfere with the measurement of migrationareas (Figs 2 and 3). However, S-SRBC is preferablefor ease in preparation, in long storage and in addingto the culture medium for routine practice.

Role of heat-labile serum factorsIn all experiments, the serum used to make up thetissue culture media was heat-inactivated at 560 for

Figure 3. Migration of the sensitized spleen cells as inFig. 2, in the presence of intact SRBC antigen(s). Theperipheral zone (p) of the migration area is clearly seen

x 10-8).

30 min. However, two experiments were conductedusing media supplemented with fresh normalCalotes serum. In this condition, cells from sensitizedanimals were still markedly inhibited in the presenceof antigen. As can be seen from Table 5, this istrue with spleen cells obtained either from 104 or6 x 108 SRBC injected animals. These results suggestthat complement may not be required for inhibitionto occur.

Specificity of migration inhibitionIn view of the reports available in mammals that MIis an antigen-specific event (David, Al-Askari,Lawrence & Thomas, 1964a; David et al., 1964c;Bloom, 1971) and since we have already reported therequirement of specific antigen for inhibition tooccur in the allograft-sensitized lizard spleen cellmigration (Jayaraman & Muthukkaruppan, 1977a),it was imperative to establish the specificity of theinhibition of SRBC-sensitized spleen cell migration.The results are set out in Table 6. The migration ofspleen cells from SRBC injected lizards was in-hibited by the injected antigen only. This effect wasspecific since cells from the same animal were notinhibited by an unrelated antigen, rat red blood cells(RRBC). Additionally, SRBC-sensitized lizard spleencells produced plaques only when tested with SRBCand not with RRBC. Likewise, cells from lizards

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Cell-mediated immunity to SRBC

Table 5. Role of heat-labile serum factors in migration inhibition

Immunizing % Migration inhibitionschedule Serum nature (Mean ± s.e.)

104SRBC Fresh 59 3 ±21-0(7)

104 SRBC Decomplemented 72-0 ± 5-8(7)

6 x 108 SRBC Fresh 21-5 ± 10 0(5)

6 x 108 SRBC Decomplemented 29-5 ± 5-5(5)

MI assay was performed 7 days after i.m. immunization. Theculture medium was supplemented with either fresh or heat inacti-vated (560 for 30 min) normal Calotes serum.Number of animals investigated is given in parentheses.

injected with RRBC were not inhibited by SRBCantigen. This specificity was confirmed by theobservation that the RRBC-sensitized spleen cellsproduced only a trace amount of plaques whentested with SRBC

Mechanism of migration inhibitionDavid et al. (1964b) and Bloom & Bennett (1966)have shown that only a few sensitized lymphocyteswere enough to bring about MI of macrophagestaken from unsensitized animals, and have calledthe factor released from lymphocytes migration in-

hibitory factor (MIF). The evidence presented so

far leads to the postulate that MI of lizard spleencells could be due to the action of MIF. Experimentswere conducted to test this. The results of theexperiments in which SRBC-sensitized lizard spleencells and normal spleen cells were mixed in varyingproportions are presented in Table 7. The migrationof a mixed population in which normal unsensitizedcells from different individuals mixed in equalproportions did not show any sign of inhibition inthe presence of antigen as a consequence of thepossible mixed lymphocyte reaction during theculture period. It was important to compare the

Table 6. Specificity of the immune response to SRBC

Immunizing Test PFC/106 WBC % Migration inhibition*schedule antigen* (Mean + s.e.) (Mean ± s.e.)

PBS alone SRBC 5-2±1-6 70±3-5(5) (22)

6x108SRBC SRBC 477-5±175-5 28-4±8-5(5) (5)

6x108SRBC RRBC 2-8±0-9 8-7±6-6(5) (5)

1 x 108 RRBC RRBC 68-5 ± 15-0 n.d.(6)

lx108RRBC SRBC 5-5±2-3 6-3±2-5(6) (6)

Seven days after i.m. immunization, the animals were killed to perform PFC andMI assays.Number of animals investigated is given in parentheses.* For MI assay intact erythrocytes (x 10,000) were added in the culture medium.n.d., not determined.

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Table 7. Mechanism of migration inhibition

Cell proportion% Migration inhibition

(Mean ± s.e.)

Control*: 50 % normal + 50 % normal 7 9 ±4-6(11)

50% sensitized + 50 % sensitizedt 67-4 ± 13-8(5)

50% normal + 50 % sensitized 42-5 ± 14-0(6)

80% normal +20% sensitized 46-6 ± 15-8(5)

90% normal + IO% sensitized 42-2 +14-5(5)

95 % normal + 5 % sensitized 36-9 ± 18-5(5)

Seven days after i.m. injection of 104 SRBC in PBS, the sensitizedspleen cells were mixed with unsensitized normal spleen cells invarying proportions and packed in the capillaries to perform MI assay.Number of animals investigated is given in parentheses.* Control cultures were prepared by mixing equal amount of

unsensitized normal spleen cells from different individuals.t Sensitized cells from different individuals were mixed in equal

proportions and cultured.

migration of mixed population with the behaviourof the constituent groups of spleen cells as theymigrated alone. To accomplish this, additionalcontrols were obtained by culturing a mixture ofsensitized spleen cells taken from different indi-viduals in equal proportions in the presence ofantigen. As a consequence of this mixed culture, noalteration in the degree of MI was obtained. Themigration of mixed populations in which sensitizedcells made up to 50, 20 or 10% of the total popula-tion was markedly inhibited in the presence ofantigen. Further, significant MI was observed whenas few as 5 % of the population were sensitized cells.

DISCUSSION

In a recent communication, the use of the capillaryMI technique in assessing CMI to skin allograftshas been reported (Jayaraman & Muthukkaruppan,1977a). The present study deals with the applicationof MI assay to detect the nature of CMI directedagainst SRBC antigen under varying experimentalconditions.Under the culture conditions described, migration

of sensitized spleen cells from capillaries is inhibited

in the presence of antigen. This observed inhibitionof migration could be due to the effect of: (1) un-satisfactory culture conditions; (2) humoral anti-bodies; (3) cytophilic antibodies (Amos, Gurner,Olds & Coombs, 1967); (4) antigen-antibodycomplexes (Bloom & Bennett, 1966; Spitler, Huber& Fudenberg, 1969; Pick & Turk, 1972); or (5) theputative mediator, MIF produced by lymphocytes inthe presence of specific antigen (Bloom & Bennett,1966).To exclude the possibility of the unsuitability of

the culture conditions to bring about MI in a non-specific fashion, evidence has already been presented(Jayaraman & Muthukkaruppan, 1977a). However,observations such as the high viability of cells(> 85% viability) in cultures with or without antigenand the slight but insignificant escape from inhibitionafter 24 h of incubation (Table 2) as observed byBloom & Bennett (1966) together with the main-tenance of the physiological pH of the medium inexperimental cultures, rule out the possibility thatMI could be due to poor culture conditions. Thisview is further strengthened by the observation thatthe inhibition is a slow and steady process whichstarts early in the life of the culture as reported byFimmel & Keast (1974). Besides, MI is not

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dependent upon the cytopathic or cytotoxic potentialof the antigen preparation as evidenced by a steadyincrease in the rate of migration during the earlyperiod of culturing in the presence of antigen.Further, the addition of three forms of antigenpreparation resulted in MI to the same level (Table4). Worth noting is the finding of insignificant MIobtained with unsensitized normal spleen cells inthe presence of antigen (Table 1).The possible inhibitory action of antibodies has

been ruled out in so far as MI could be demon-strated with spleen cells that produce little or noPFC (Tables 1 and 2). Additional evidence for theindependence of MI upon the presence of antibodyproducing cells is provided by the F-SRBC experi-ment (Table 3). Formalized erythrocytes have beenshown to induce T-helper cells specifically, withoutthe generation of PFC in mice and lizards (Dennert& Tucker, 1972; Jayaraman, 1976). For the firsttime the present study demonstrates the generationof MI with F-SRBC.

Cytophilic antibodies are not required for CMIreactions, but the possibility that they may partici-pate if present, is not excluded (Bloom, 1971).Further, the majority of the cytophilic antibodieswere shown to be IgG2 (cf. Bloom, 1971). MIcould not be a result of the action of cytophilicantibodies due to the lack of production of mercapto-ethanol resistant antibodies to SRBC in Calotes(Kanakambika & Muthukkaruppan, 1972). How-ever, sera taken from lizards injected with 6 x 1 08SRBC with or without FCA were tested for thepresence of cytophilic antibodies following themethod of Boyden (1964). In such experiments nocytophilic antibodies could be demonstrated (un-published results).Bloom & Bennett (1966) were the first to report

that MI of normal macrophages could be inducedby antigen-antibody complexes. Subsequently it hasbeen shown that antigen-IgG2 but not antigen-IgMcomplexes are able to inhibit normal macrophagemigration (Spitler et al., 1969; Pick & Turk, 1972).The possibility that antigen-antibody complexesinduced MI of lizard spleen cells can be readily ruledout in as much as the lizards do not produce ME-resistant antibodies (Kanakambika & Muthukkarup-pan, 1972) and MI could be demonstrated withspleen cells which contain little or no PFC (Tables1-3).The exclusion of these possibilities leads one to

consider MIF as the putative mediator of the phe-

nomenon of MI, as has been shown earlier (Bloom,1971; Drossler & Ambrosius, 1972). As with thealloantigen system (Jayaraman & Muthukkaruppan,1977a), the SRBC-sensitized lizard spleen cells uponconfrontation with specific antigen (Table 6) couldproduce MIF. The antigen specificity of the reactionis in line with the findings in mammals (David et al.,1964a,c; Bloom, 1971). Complement componentsdo not appear to be necessary for the elaborationof MIF (Table 5) and this finding confirms theobservations made in classical immune lymphocyte-antigen system by David et al. (1964a) and in theantigen-antibody complex system by Pick & Turk(1972).The phenomenon of MI has been shown to be

mediated by sensitized cells in the lizard (Table 7)and this is consistent with the findings in guinea-pigs (David et al., 1964b; Bloom & Bennett, 1966).Cultures of mixed populations of spleen cells fromdifferent individuals do not result in non-specificinhibition as shown in guinea-pigs (Al-Askari &Lawrence, 1973). The presence of a very few sensi-tized cells resulted in MI of normal cells, suggestingthat this specific inhibition is mediated indirectlythrough the elaboration by lymphocytes, of asoluble material, MIF as in guinea-pigs (Davidet al.,1964b; Bloom & Bennett, 1966). Preliminaryevidence has been obtained for the elaboration ofMIF by sensitized cells upon interaction withspecific antigen in culture (unpublished observa-tions). Besides, MI has been shown to be a T-dependent phenomenon and unaffected by treat-ment with cyclophosphamide while antibodytolerance was induced (Jayaraman, 1976). In thelight of these findings, MI can be considered as anin vitro manifestation of CMI function, directedto a specific antigen, SRBC as shown with theallograft system in the lizard, Calotes versicolor(Jayaraman & Muthukkaruppan, 1977a).

ACKNOWLEDGMENTS

One of the authors (S.J.) wishes to express hisgratitude to Madurai University for the award ofstudentship; to Dr P. W. Askenase for the gift ofcapillaries used in this study and to Professor J. L.Turk for the critical reading of the manuscript andthe advice and suggestions given by him in thepreparation of the manuscript.

This research was supported in part by a grant

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240 S. Jayaraman & VR. Muthukkaruppan

from U.S. National Institutes of Health (01-077),under special foreign currency research programme(PL-480).

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