Analysis of T-cell-specific functions in continuous lymphocytic cell lines

Experimental Cell Research 94 (1975) 23-30

ANALYSIS

OF T-CELL-SPECIFIC

CONTINUOUS

LYMPHOCYTIC

FUNCTIONS CELL

IN

LINES

I. Combined Expression of Thymocyte Antigen and MIF Production

in Rat-Mouse Hybrid Lines Y. NAMBA andB. H. WAKSMAN Department

of Pathology,

Yale University Medical School, New Haven, CT 06510, USA

SUMMARY Three cell lines have been obtained by hybridizing normal rat thymocytes and mouse leukemic lymphoblasts (L5178Y, HGPRT-) using Sendai virus. By the use of mouse antisera against rat thymocytes and against the hybrid cells themselves, it was shown that these hybrid cell lines express a specific surface antigen that is found in 230% of rat thymus cells and in a minor subpopulation of rat spleen and lymph node cells. Surface antigens common to rat red blood cells are not expressed in these cell lines. These cells also produce migration inhibitory factor (MIF) but neither lymphotoxin nor inhibitor of DNA synthesis. By isoelectric focusing analysis, it was shown that hybrid cells appear to produce both rat and mouse type MIF. Most of the cell lines that reverted to resistance to 8-azaguanine simultaneously lost the surface antigen and the ability to produce MIF.

The genetic control of immune responses has become the object of many investigators’ attention. Most of the immune response genes-those that control an animal’s capacity to respond to a given antigen-are located in the same chromosomal region as the genes that specify strong histocompatibility antigens [14]. These are thought to control recognition of antigens by the reacting cells [5-6]; however, structural genes for immunoglobulin are not located in the same chromosomal region [7]. The mechanisms of genetic control of lymphokine production and the expression of surface antigens specific to lymphocytes or lymphocytic subpopulations are unknown. The technique of somatic cell hybridiza-

tion provides a convenient tool for investigating some aspects of the genetic control of functional phenotype in lymphocytes. For this purpose it is appropriate to hybridize lymphocytes of two different species. In such a cross, the chromosomes from one species have the propensity to be lost from the hybrid cells [8]. We have, therefore, the opportunity of obtaining hybrid cell lines in which only certain sets of lymphocytic functions are expressed and in which we can detect one or more clusters of genes which control the expression of these functions. We report here an initial study of cells obtained by hybridizing mouse leukemic cells (L5 178Y, 8-azaguanine resistant) [9] and normal DA rat thymocytes. It has been Exptt

Cell

Res

94

(1975)

24

Namba and Walisman

possible to demonstrate that MIF (migration inhibitory factor) production and a surface antigen specific to rat thymocytes and a minor subpopulation of rat peripheral lymphocytes were co-expressed in the hybrid cells. Rat antigens characteristic of red blood cells were not detected on the surface of the hybrid cells.

MATERIALS

AND METHODS

Cells of inbred DA rats 4-6 months of age, bred in the Yale animal facility, were used in cell fusion and the MIF analysis. Forthe latteranalysis and also to obtain antiserum, BALB/c mice 5-7 months of age were used. For MIF assay, guinea pigs 4-7 months of age served as a source of peritoneal macrophages.

Animals.

of rats and mice. For the purpose of obtaining MIF produced by rat, mouse, and the hybrid cells. rats and mice were iniected with ovalbumin (200 pg/rat, 50 pg/mouse) mixed with Freund’s complete adjuvant (Bay01 F,85: Arlacel A, 15, containing heatkilled tubercle bacilli 6 mglml) in four foot pads. After 9 days pooled lymph nodes were obtained and the cells were cultured to obtain MIF (see below).

Sensitization

treatment of rats. In order to obtain cortisol resistant thymocytes, rats were injected with hydrocortisone acetate intraperitoneally (130 mg/kg body weight). After 48 h thymocytes were obtained and used in the cytotoxicity test. Cell recovery from the hydrocortisone-treated rat thymus was about 7 % that of control thymus.

Hydrocortisone

Normal

rat

thymocytes

and other

lymphoid

cells.

Thymocytes were obtained from normal DA rats (4-6 months old). The cells were dispersed with scissors and, after the fragments settled by gravity, the suspended cells were washed three times with Hanks’ balanced salt solution (HBSS). A similar procedure was used to obtain spleen, lymph node, and bone marrow cells. Mouse leukemia cells. The cell line (GF3) used in cell

fusion exneriments was a mutant strain deficient in hypoxantbine/guanine phosphoribosyl transferase (HGPRT-), derived from a population of L5178Y (sub-clone 43) that had been treated with the mutagen ICR-372 [lo]. The L5178Y lymphocytic parent strain was first isolated from a leukemic DBA/Z mouse by Law, and later grown in vitro by Fisher. GF3 cells were kindly provided by E. A. Adelberg, Department of Human Genetics, Yale University Medical School. The cells are near diploid and retain Thy 1.2 (thetaC3H) antigen on the cell surface. The cells also retain alloantieens of H-2d allele (contain alloantigen 4, 8, and 35 gut does not contain 23 and 33 as checked with H-2 histocompatibility typing sera). They are also phytohemagglutinin (PHA) sensitive and resistant to Exprl Cell Res 94 (1975)

cortisol up to IO-’ M. These are the characteristic\ expressed. in a minor cell population of normal mouse thymocytes. GF3 are routinely grown in Fisher’s medium supplement with 10% horse serum. Fu.sion procedure. The leukemic cells were harvested at 2-3 x 105/ml, centrifuged at 1200 rpm for 10 min, washed twice with cold Earle’s basic salt solution. Normal rat thymocytes and leukemic cells were mixed in a ratio of 1 : 1 in Earle’s basic salt solution and the cell concentration was adjusted to 2x 10’ cells in a final volume of I ml. To the tube, 2000 hemagglutinating units of Sendai virus (kindly provided by E. A. Adelberg) in 0.5 ml HBSS was added and incubated at 4°C for 20 min. Then the mixture was transferred to 1 37°C water bath, shaken gently for 3 min, and incubated without further shaking for an additional 17 min. After the incubation the cells were washed once with cold Fisher medium supplemented with 10% horse serum. The cells were divided to four plastic flasks and were cultured in Fisher’s medium supplemented with 10% horse serum, thymidine (16 PM), hypoxanthine (100 pM), and aminopterine (0.4 PM) (HAT medium). (a) Mouse anti-rat thymocyte serum was oreuared bv immunizine BALB/c mice (H-Zd) with DA rat ‘thymus-cells (lo8 ceils/mouse, i.p., every 10 days, three times, bled 10 davs after the last immunization) In some experiments, the antiserum was absorbed with DA rat red blood cells (5~ lo9 cells/ml antiserum, at 4°C for 1 h, twice). (b) Mouse anti-rat red blood ceil serum was prepared by immunizing BALB/c mice with DA rat red blood cells (5x lOa cells/mouse, i.p., every 10 davs. 4 times, bled 10 davs after the last immunization).-(c) Mouse anti-hybrid cell serum was prepared bv immunizing BALB/c mice with one of the hybrid cell lines (hyb>d-3). The mice were injected i.p. with 2~ lo5 hvbrid cells twice at intervals of one week followed after one week by another two weekly injection of 2 x lo6 cells. On the tenth day after the fourth injection, 1O’cells were injected i.p.-and the mice were bled after 10 days. In some experiments the antiserum was absorbed with the leukemic cells used for cell hybridization (2x lO*/ml antiserum twice for 1 h at 4°C). (dj Histocompatibility typing sem (anti-H-2) were kindly given by D. Kayhoe, Transplantation Immunology Branch, NIAID, NIH.

Antisera.

test. Antigen content of normal rat Ivmphocvtes, leukemic cells, and the hybrid cells was assayed by the two step cytotoxicity test. Approx. 105 cells in 0.1 ml HBSS were put in successive wells of a micro-titration nlate (Linbro Chemical Co., New HPven, Conn., fla’t bottom type) together with 0.1 ml of diluted antisemm, and the plate was incubated at 4°C for 1 h. In the next step, another 0.1 ml of 50% guinea pig serum was added and the plate was incubated 1 h at 37°C. After the incubation. 0.1 ml of 0.25 % trvpan blue in saline was added to each weU and viable cellnumber was counted in a hemocytometer.

Cytotoxicity

test. The hemagglutinin titers of some antisera were assaved in microtitration plates (Linbro Chemical Co., round bottom type). In successive wells 10’ rat red blood cells in 0.1 ml and another 0.1 ml of serially diluted antiserum were mixed and the

Hemagglutination

Analysis of T-cell-specific functions plate was placed in the cold room (4°C) for 4 h. Agglutination was judged visually. With the mouse antiserum against the hybrid cells, the distribution of cells that have the surface antigen which is expressed on hybrid cells was examined. Washed cells obtained from different rat lymphoid tissues and bone marrow were incubated with heat-inactivated mouse anti-hybrid cell antiserum, diluted with HBSS to 10% concentration. After incubation at room temperature for 1 h, the cells were washed twice with cold HBSS. FITC-conjugated IgG fraction of rabbit anti-mouse Ig antiserum (Cappel Laboratories, Downingtown, Pa) was then added and the cells were incubated at 4°C for 1 h. After washing twice with HBSS the cells were observed under a Zeiss fluorescence microscone. The FITC-coniuaate was absorbed with rat Ig fraction prepared by -ammonium sulfate precipitation and Sephadex gel filtration (7s fmction) in liquid system (50 pg/ml FITC-conjugate at 4°C for 10 h, twice) and rat lymph node cells (lOa cells/ml FITC-conjugate at 4°C for 1 h) prior to use.

25

100

ZmmunojZuorescence.

For the purpose of distinguishing mouse tvne MIF from rat tvne MIF, isoelectric focusing analysis of culture supernatants from rat and mouse lymph node cells stimulated with ovalbumin was carried out. Nine days later immunization with ovalbumin, pooled lymph node cells of rat or mouse were cultured in serum-free RPMI-1640 medium supplemented with ovalbumin in a final concentration of 10 &ml. After 48 h the culture suwrnatants were harv&ed and condensed three-foid using polyethylene glycol No. 20000. After extensive dialysis against water, 40 ml was applied to an electrofocusing column (LRB 8100, 110 ml) in 1% ampholine solution with a pH range of 2.54.0. After electrofocusing for 48 h (450 V), 4 ml fractions were collected and their pH level measured. Ovalbumin (1 mg) was added to each fraction as stabilizer, and each fraction was freed of ampholite and sucrose by dialysis against HBSS for 48 h with change of the external solution every 12 h. The culture supernatant of hybrid cells (Hybrid-3, 106 cells/ml for 2 days) was processed in the same way.

SO

60

40

20

Isoelectricfocusing.

The technique of David et al. [l l] was used with slight modification. Normal guinea pigs were injected i.p. with Bayol F. After 4 days peritoneal macrophages were obtained and washed twice with HBSS and capillaries tilled for the MIF test. After centrifugation (200 g for 5 min), they were cut at the cell-medium interface, and the stub was fixed into a Falcon plastic dish (35~ 10 mm, Falcon Plastics, Oxnard, CA) with paraffin. The capillaries were overlaid with 2 ml of RPMI-1640 medium supplemented with 10% fetal calf serum plus 1 ml of test sample dialysed against HBSS. Four capillaries (two dishes) were used for each group. The area of migration was recorded after 24 h as the product of long and short axes of the oval area, measured by microscopic observation. The percentage of migration inhibition was calculated as

Assay of MZF activity.

(

,_

test migration area x100 control migration area 1

Fig. 1. Abscissa:

concentration of antiserum against rat thymocytes; ordinate: viable cell % of control. Cytotoxic activity of the antiserum against hybrid cells. Cytotoxic activity was measured before (-) and after (- - -) absorption with rat red blood cells. Thymocytes (O-O, l - - -O), original leukemic cells (O-O), hybrid-2 (x-x, x - - -x); hybrid-3 (O-0, O- - -0); and hybrid-4 (A-A) were used as target cells. The cytotoxicity curve of hybrid-l cells was almost the same to that of hybrid-3.

Reagents and culture medium. Ovalbumin (5 x crystal-

line) was purchased from Nutritional Biochemical Corp., Ohio. Fetal calf serum and horse serum were purchased from GIBCo, Grand Island, N.Y., and was used without heat inactivation. RPMI-1640 was purchased from Associated Biomedic Systems, Buffalo, N.Y. Guinea pig serum was purchased from Difco Laboratories, Detroit, Mich., and was absorbed with rat red blood ceils (5x IO9ceUs/ml serum at 4°C for 1 h) before use. Fisher’s medium for leukemic cells of mice was purchased from GIBCo. Ampholine carrier ampholites were purchased from LKB-Produkter, Sweden.

RESULTS Establishment of hybrid cell lines Four cell lines have been established by crossing leukemic cells and normal rat thymocytes. After the fusion procedure, Exptl Cell Res 94 (1975)

26

Namba and Waksman

cytotoxicity testing with a mouse antiserum prepared against rat thymus cells (fig. I). Neither the original leukemic cells nor the Viable cells (% of control) thymocytes of DBA/2 mice reacted with Dilution of antiserum this antiserum. Absorption of the antiserum Target cells I : 16 I :32 1:64 with rat red blood cells had almost no effect on its cytotoxic activity against the Rat thymocytes 0.5 0.7 2.8 Hybrid-l 95.0 93.5 93.0 hybrid cells, suggesting that major histoHybrid-3 97.5 95.5 95.5 compatibility antigens were not expressed Hemagglutinin titer of the antiserum was 1 : 1 280. on the hybrid cells. This inference was further strengthened by the fact that mouse the cells were placed in HAT medium and antiserum against rat red blood cells did not the medium changed every 3 days. During kill the hybrid cells in a cytotoxicity test the first 10 days the viable cell number de- (table 1). These results, however, do not excreased precipitously, then entered a steady clude the possibility that surface antigens state which lasted for one week, after common to red blood cells may be exwhich the viable cell number increased pressed on the hybrid cells in low density steadily in some of the culture flasks. Six- and thus fail to participate in a cytotoxic teen flasks were prepared and cultured in- reaction. In order to exclude this possibildependently. From four of these flasks cell ity, mouse antiserum against one of the lines were established and transferred hybrid cell lines (hybrid-3) was prepared. serially in HAT medium. All of these cell Its hemagglutinin titer against rat red blood cell was negligible; yet it showed high cytolines reacted with H-2 histocompatibility toxic activity against hybrid cells as well typing sera (D-4, D-8, and D-35) indicating that they retained H-2 alloantigens of the as against rat thymocytes. The antiserum also showed cytotoxic activity against the original leukemic cells. original leukemic cells due, probably, to tuDetection of rat specific surface antigen mor antigens and the minor histocomon the hybrid cells patibility differences between DBA/2 and In three cell lines out of four, a surface anti- BALB/c mice. Absorption of the antiserum gen common to rat cells was detected by with the original leukemic cells had almost Table I. Cytotoxic activity of mouse antiserum against rat red blood cells

Table 2. Cytotoxic activity of mouse antiserum against hybrid-3 cells Viable cells (% of control) Dilution

Dilution

of antiserum

of absorbed antiseruma

Target cells

I:4

I:8

I:16

I:32

I:64

I:4

I:8

I:16

I:32

I:64

Rat thymocytes Original leukemic cells

5.0 32.4

5.7 77.1

8.6 102.5

16.0 96.4

45.5 98.0

4.8 91.0

5.5 10.8 105.0 94.3

28.0 ND

55.2 ND

2.0 3.8 0.5

6.9 9.0 2.5

11.2 20.6 17.2 38.0 7.2 14.8

32.4 61.2 18.2

5.5 12.7 2.5

8.0 15.9 19.6 31.3 5.4 II.8

31.8 61.2 25.9

54.0 ND ND

Hybrid cells Hybrid-l Hybrid-2 Hybrid-3

a The antiserum was absorbed with original leukemic cells (2x 10s cells/ml antiserum) for I h at 4”c, twice. Exptl Cell Res 94 (1975)

Analysis of T-cell-specific functions

27

Table 3. Distribution of antigen-positive cells in lymphoid tissues Cells containing antigen (%)

Cells tested

Immunofluorescencepositive

Killed by unabsorbed antiserum 1:14

1:8

1: 16

Normal thymocytes Cortisol-resistant thymocytes Mesenteric lymph node cells Cervical lymph node cells Bone marrow cells Spleen

92.0 ND 5.2 5.0 3.4 12.0

95.0 46.0 14.6 14.0 4.5 24.0

94.5 41.1 15.5

87.7 34.6 16.5 4.0 4.0 13.0

E 22.5

Antiserum against hybrid-3 was used. The immunofluorescence-positive cell percentages of spleen and bone marrow cells were adjusted by measuring red cell percentages using a hemocytometer. More than 200 cells were counted in each target tissue except thy&s.

no effect upon its cytotoxic activity against rat thymus cells (table 2). This result further supports the inference that the surface antigen of the hybrid cells which reacted with mouse anti-rat thymocyte antiserum was not a cross-reacting mouse antigen but really rat-specific. Distribution of the rat-specific antigen in lymphoid tissues and bone marrow of rat Using the mouse antiserum against hybrid-3 cells (unabsorbed), in a standard cytotoxicity test, approx. 95% of thymus cells were killed, while less than 20 % of the cells in peripheral lymph nodes, spleen, and bone marrow were killed (table 3). Less than half of cortisol resistant thymocytes were killed under the same conditions. The result of immunofluorescence tests also indicated that the antigen was present on a majority of thymus cells but on less than 15% of the cells in peripheral lymph nodes, spleen, and bone marrow. MIF production by the hybrid cells MIF activity could be detected in the culture supernatants of the hybrid cells (table 4). The three active cell lines were those that expressed the rat surface antigen. In

order to determine whether the MIF was produced under direction of a mouse structural gene or not, it was necessary to seek significant physicochemical differences between mouse and rat MIF. They proved to have slightly different isoelectric points (PI) (fig. 2), that of rat MIF being pH 2.8 and that of mouse MIF pH 3.0-3.1. The MIF activity produced by the hybrid cells (hybrid3), analysed in a same way, was widely distributed between pH 2.73-3.2, suggesting that the activity was due to a mixture of the rat and mouse types of MIF (fig. 3). Neither lymphotoxin nor inhibitor of DNA synthesis [22] was produced by any of the cell lines. Table 4. MIF activity in the culture supernatant of hybrid cells Culture supematants

% migration inhibition

Hybrid-l Hybrid-2 Hybrid-3 Hybrid4 Original leukemic cells Antigen-stimulated lymphocyte

65.8 61.4 55.8 2.2 4.0 48.5

Percent migration inhibition was the average of four capillary tubes (two dishes). Each culture supematant was added in a final concentration of 33 %. Exptl Cell Res 94 (1975)

28

Namba and Waksman

a

1’ 3

b

-2.8

-1

Fin. 2. Abscissa: fraction no. of isoelectric focusing; ordinate: (left) % migration inhibition; (right) pH. Isoelectric focusina analvsis of MIF oroduced bv rat and mouse 1ymphoc;tes. (a) Mouse l&F activity; (b) rat MIF activity. (O-O) MIF activity; (03) pH gradient.

Reversion of the hybrid cells to 8-azaguanine resistance with concurrent loss of the differential marker functions

The hybrid cells were cultured in Fisher’s medium supplemented with 10% horse serum and 20 pg/ml 8-azaguanine. Ten thousand cells in 0.3 ml medium were inoculated in individual wells of microculture plates. Revertants to 8-azaguanine resistance were obtained in high frequency, usually 3-7 out of 96 wells. Most of the revertant cell lines had concurrently lost the marker antigen (table 5). In these same lines, MIF-producing ability was also lost. DISCUSSION

tained in HAT medium must be either hybrids which contains the rat HGPRT gene or revertants to the wild-type of leukemic cell (HGPRT’). Among the four cell lines which were studied, three had a rat-specific surface antigen and produced MIF, while one line lacked both of these functions. It is probable that the latter is a revertant and the former are hybrid lines. In the cross of mouse leukemic cells and normal rat thymocytes, hybrid cell lines were obtained in very low frequency (three such lines from 16 flasks), although after fusion with Sendai virus about 40% of cells were heterokaryons and half of them contained small dense nuclei characteristic of normal thymocytes. Moreover these lines appeared to express very restricted functions of rat cells which, nevertheless, were stable for more than 100 generations in HAT medium. Hybrids of rat and mouse

100 I

15

c 50

1

t

i 5

In HAT medium the original mouse leukemic cells which lack hypoxanthine: guanine phosphoribosyl transferase (HGPRTcell line) can not proliferate. Cell lines obExptl

Cell

Res 94 (1975)

IO

15

20

25

Fig. 3. Abscissa: fraction no. of isoelectric focusina; &&ate: (left) % migration inhibition; (right) pH. -. Isoelectric focusing analysis of MIF produced by hybrid-3 cells. (O-O) MIF activity; (0-O) pH gradient.

Analysis of T-cell-speciJic functions

Table 5. Loss of thymocyte antigen and of MIF-producing

ability in revertant cell lines Viable cells (% of control)

Revertant cell lines From hybrid-2 Revettant-l L

4 From hybrid-3 Revertant-l 2 3 4

1:s

1: 16 1: 32

% migration inhibition”

92.0 90.5 88.7 6.5

95.5 98.4 91.4 18.7

108.0 97.2 102.9 41.3

ND ND ND ND

87.9 94.0 3.8 96.7

98.5 88.0 14.4 84.5

99.0 97.5 35.0 88.7

-11.7 -2.5 40.0 -14.0

For the detection of the surface antigen, the mouse antiserum against rat thymocytes was used after absorption with red cells. In MIF assays, culture supernatant was added in a final concentration of 33 %. a Minus sign signifies increase in migration relative to control.

cells are known to have a propensity to lose rat chromosomes. It seems reasonable to suppose that, by the time we checked the marker functions (at least 20 generations after cell fusion), any hybrid cells had already lost most of their rat chromosomes. A preliminary karyotype analysis of these hybrid cells brought a complex situation to light. The original leukemic cells were near diploid (chromosome number, 39 or 40). The cells of the three hybrid lines each had 5545 chromosomes; however, we could only identify l-3 rat chromosomes. All of these were telocentric or acrocenttic and no metacentric chromosome could be found, although about 30% of rat chromosomes belong to the latter category. The marker functions expressed in hybrid cells which were continuously cultured in HAT medium were stable for more than 100 generations. They were lost in high frequency among revertants to HGPRT-. These results may suggest that the HGPRT enzyme gene and the genes governing the

29

expression of the antigen and MIF production are located in the same chromosome or chromosomal segment. The major rat histocompatibility antigens were not expressed in the hybrid cells. It follows that a single rat chromosome or chromosomal segment has genes governing both the expression of surface antigen in a major population of thymocytes and MIF production but that genes controlling the major histocompatibility complex are not closely linked with these. The surface antigen expressed in the hybrid cells is worthy of further investigation. Approx. 95% of rat thymocytes but only 40 % of thymocytes in hydrocortisonetreated rats have this antigen, suggesting that it is selectively expressed in cortical thymocytes. The appearance of antigenpositive cells in the peripheral lymphoid tissues and bone marrow was infrequent (3-12 % by immunofluorescence tests). These therefore do not correspond to the conventional T-cell population. They may be cells derived directly from cortical thymocytes or, alternatively, precursors of thymocytes. Antisera reactive with rat thymocytes but of widely variable specificity have been obtained by many investigators. Thymocyte-specific antigens have been known for some time [12-141; these are expressed in almost all rat thymocytes but not in (or in less than 10% of) peripheral lymphocytes. More recently, an antigen similar to murine Thy-l (theta) antigen has been described on rat cells [15-191; this antigen is expressed in almost all thymocytes and thymus-derived lymphocytes in the peripheral lymphoid tissues. Some investigators have reported that antiserum against mouse Thy-l. 1 (theta AKR) reacts with thymus-dependent cells from several inbred rat strains [15-161. The antigen reported in the present paper differs Exptl Cell Res 94 (1975)

30

Namba and Waksman

slightly from all these, being found in about 95% of thymocytes and 40% of cortisol-resistant thymocytes, with a distribution pattern in spleen and lymph node like that of the thymus-specific antigen of Colley et al. [14]. In our hands, rabbit antiserum against DA rat brain absorbed with DA rat liver powder and bone marrow cells, which was fairly specific for Tlymphocytes, failed to react with the hybrid cells after absorption with the original leukemic cells (unpublished observation). The immunological function of the antigen-positive cells demonstrated in peripheral lymphoid tissues is open to further study. MIF was produced by the hybrid cells. Isoelectric focusing analysis indicates that the isoelectric point of the product was distributed widely in the pH range of both rat and mouse MIF. Possibly both rat and mouse MIF are produced by the cells. It has been reported, however, that MIF is inactivated by sialidase treatment, indicating that it is glycoprotein [20]. Furthermore, our data show that it is a very acidic macromolecule, and recently Feldman and his colleagues have also reported that the p1 of migration inhibitory activity obtained from fetal calf serum was about 3.1 [21]. These results suggest that the isoelectric point of MIF is not necessarily determined by the charge of polypeptide chain(s) but rather by its content of acidic sugar. Therefore the widely distributed pattern of the MIF produced by hybrid cells in isoelectric focusing is not clearcut evidence for the production of both rat and mouse MIF. It is possible that the polypeptide

Exptl Cell Res 94 (1975)

chains are homogeneous but that their content of sugar is heterogeneous due to a new expression of enzyme(s) concerned with the synthesis of carbohydrate chain(s). Further study is necessary to elucidate this question. This work was supported by USPHS Grants AI-061 12 and AI-06455.

REFERENCES I. McDevitt, H & Chinitz, A, Science 163 (1969) 1207. 2. Green, 1, Inman, J K & Benacerraf, B, Proc natl acad sci US 66 (1970) 1267. 3. Martin, W J, Elliman. L, Green, I & Benacerraf, B, J exp med 132 (1970) 1259. 4. Green, I & Benacerraf. B. .I immunol 107 (1971) 374. 5. Bluestein, H, Green, I & Benacerraf, B, J exp med 134(1971) 458. 6. Grumet, F C, Fed proc 30 (1971) 469. 7. Herzenberg, L A & McDevitt, H 0, Adv gen 2 (1968) 209. 8. Weiss. M C & Green. H. Proc natl acad sci US 58 (1967) 1104. 9. Fisher, G A. Ann NY acad sci 76 (1958) 673. 10. Kao, F T & Puch, T T, J cell physiol74 (1%9) 245. 11. David, J R, Al-Askari, S, Lawrence, H S & Thomas, L, J immunol93 (1964) 264. 12. Potworowski, E F & Naim, R C, Immunology -- 13 (1967) 597. 13. Bachvaroff, R, Galdiero, F & Grabar, P, J immunol 103 (1%9) 953. 14. Colley, D G, Malakian, A & Waksman, B H, J immunol 104 (1970) 585. 15. Iverson, J G, Clin exp immunol6 (1970) 101. 16. Douglas, T C, J exp med 136 (1972) 1054. 17. Peter, H-H, Clagett, J, Feldman, J D & Weigle, W 0, J immunol 110 (1973) 1077. 18. Goldschneider, I & McGregor, D D, J exp med 138 (1973) 1443. 19. Lubaroff, D M, Transplant proc 5 (1973) 115. 20. Remold, H G & David, J R, J immunol 107 (1971) 1090. 21. Fox, R A, Gregory, D S & Feldman, J D, J immunol 112 (1974) 1867. 22. Namba, Y & Waksman, B H, Inflammation 1 (1975). In press. Received December 2, 1974 Revised version received February 17, 1975