A skin reactive factor elaborated from a human lymphoblastoid cell line (RPMI 1788)

CELLULAR

IMMUNOLOGY

A Skin

11,

Reactive

Factor Elaborated from a Human Cell Line (RPM 1788) ‘1 2

DONALD Department

286-303 (1974)

of

L. CALEBAUGH

Microbiology,

3 AND RONALD

University Chicago, Illinois

of Illinois

Received

17, 1973

July

Lymphoblastoid

E. PAQUE cnt the Medical

Center,

60680

The biological characteristics of a soluble factor released from RPM1 1788 human lymphoblastoid cell line are described. The skin reactive factor elicited skin reactions in nonimmunized guinea pigs that were indistinguishable from antigen elicited skin reactions as assessed by macroscopic and microscopic appearance. Histological examination of skin biopsies 18 hr after intradermal injection of the factor indicated substantial numbers of mononuclear cells in the skin lesion. In vitro, the factor was unable to inhibit the migration of normal guinea pig macrophages and was not chemotactic for guinea pig peritoneal exudate cells. Supernatant fluids prepared similarly from other cultures of human lymphoblastoid cells, Raji and 8866, failed to elicit skin reactions either macroscopically or microscopically similar to those observed for the RPM1 1788 lymphoblastoid cell line.

INTRODUCTION Lymphoid cells obtained from animals with delayed hypersensitivity skin reactions to various antigens have been shown to release, in vitro, soluble mediators associated with cell-mediated immunity (1-13). These soluble mediators, released in small quantities and presumably proteins in nature, have been identified by their biological effects in vitro: lymphotoxic activity (14-17), the ability to inhibit the migration of macrophages (Z-13, 18--ZO), the capacity to sltimulate blastogenesis and mitosis of lymphocytes (13, 21-25), and chemotaxis of mononuclear cells through Millipore membranes (26, 27). Other mediators have been described and recently have been designated by Dumonde et al. (28) and Dumonde (29) as lymphokines. Because soluble mediators released iul vitro have been implicated in cell-mediated immune reactions, several investigators have attempted to assesswhether they have biological effects in tivo (10, 13, 2.5, 30, 31). For example, Willoughby et al., and Spector and Willoughby (32-35) reported that the intradermal injection of a factor released in vitro from normal or sensitized lymph node cells elicited a skin lesion indistinguishable histologically from the tuberculin skin reaction in sensi1 Presented in part at the 57th Annual Federation of American Societies for Experimental Biology Meeting, April, 1973. 2 Supported in part by PHS-GRSG 423. 3 In partial fulfillment of the requirements for the Doctor of Philosophy Degree in the Graduate College. Supported by Immunology Training Grant PHS Al 00335. 286 Copyright All rights

1974 by 9 reproduction

Academic Press, in any form

SKIN

REACTI\.E

1JACTOK

2x7

tized animals. Bennett and Bloom (IO) have found primarily mononuclear cells in a skin lesion that was first evident 3-5 hr after the intradermal injection of migration inhibitory factor. Also, Pick and Turk (36-42) and Svejcar ct al. (4.1-45) have reported the release of a factor from guinea pig lymphocytes incubated with specific antigen or mitogen that produced an inflammatory lesion .s hr after intrndermal injection ; histological examination of the injection site revealed tile presence of mononuclear cells after 3 hr, but a granulocytic infiltrate after .!1 hr. Teodorescu ef crl. (46) have reported that the intradermal injection of a factor released from hun~nn lymphocytes incubated in vitro with phytohemagglutinin (I’H .\) caused skin reactions in humans. Since PHA alone caused similar skin reactic~ns and was not removed from the culture fluids. these results are difficult to interpret. Furthermore, the histology of the skin reactions were not recorded. On the othrr hand, Schwartz et al. (47) have reported removal of concanavalin A from supernatant fluids obtained from tissue cultures of guinea pig l~mphoitl cells iiiciil)atf~tl . . . \z,itli the iiltrogen used to elicit skin grafts in guinea pigs. Recently. long term cultures of human lymph& cell lines have been shown to release AlIF, lymphotoxin, and interferon into supernatant fluids during incul):ltion (48-51 ). \1Te have undertaken this investigation with the aim of xssessillg tht ilr Go and in zlifro biological effects of a skin reactive factor that n’as folmtl to 1~ rrleased from a human lymphoblastoid cell line ( Rl’MI 17%) and that tslicits VStensive mononuclear cell infiltration when injected intradermally into uninlmunizetl guinea pigs. The macroscopic and histological characteristics of the skill lesion produced by injection of this factor resemblesan antigen elicited tuberculin skin test. The use of the human lymphoblnstoid cell line for evaluating the biological effects of this skin reactive factor offers scvcral distinct advantages: (‘.I) elimination of the need to add antigens or nonspecific plant lectins t(J trigger release of the nlediator from the cells ; (B) a source of cells readil! grown ill tissue culture that provides substantial quantities of the llledi~lt(Jr which facilimtatesthe biochemical characterization of the soluble mediator in supernatant fluids; ( C:) a grnetically uniform population of lymphoid cells which can readily be propagatrtl in tissue culture ; and (D) a system where the mechanism of biosynthesis of a sc~lubl~~ mediator associated with cell-mediated immunity can 1~ studied irz rsifuo. \\‘ts no\\ rel)ort on sc\.eral biological characteristics of a skin-reactive factor rrleasrtl fr,,nl ;I cell line of human lymphoblastoid cells tlesignatetl as riPnIl 17PS.

Maiutolame

of the Huwmz Lynphoblastoid Cell Lines

The human lymphoblastoid cell lines used in our experiments have been designated RPM1 1788 (Associated Biomedic Systems, Buffalo, KY), Raji, and 8866. The Raji and 8566 human lymphoblastoid cell lines were a gift of Dr. Richard I,erner. Cell line RPM1 1788 was maintained in RPMT 1640 tissue culture medium containing 10% fetal calf serum, 100 U/ml penicillin, 100 pg/ml streptomycin, and 75 pg/ml polymyxin B (Associated Biomedic Systems, Buffalo, NY). The Raji and 8866 cell lines were maintained in Auto-POW BME tissue culture medium (Flow Laboratories, Inc., Rockville, MD) containing 15% fetal calf serum. 100 U/ml penicillin, 100 pg/ml streptomycin, 5 pg/ml amphotericin I’,, and 50 pg/ml glutamine. The lymphoblastoid cells were grown at 37°C in 5% CO-. ‘I’ht~

288

CALEBAUGH

AND

PAQUE

cells were harvested twice weekly and transferred into fresh medium as follows. The cells were collected by centrifugation at 104g for 10 min in sterile plastic 50 ml screw-cap tubes (Falcon Plastics, Cockeysville, MD) and the cell pellets pooled. For subculturing and continued maintenance, the cells were dispersed into 250 ml culture flasks (Falcon Plastics, Cockeysville, MD) and diluted in fresh medium to a cell concentration of 1 X 106/ml. Prepa.ration of Sztpernatant Fluids for Skin Testing The lymphoblastoid cells growing in tissue culture medium were centrifuged at 104g for 10 min in SO-ml sterile plastic screw-cap tubes. The supernatant fluids were removed from the cell pellet by carefully pouring the fluids from the tubes. The cell pellets of each cell line were resuspended in 3-4 ml of cold HBSS and pooled into single 50-ml sterile plastic screw-cap tubes. The cells were washed in 50 ml cold Hank’s balanced salts solution (HBSS) followed by centrifugation at 104g for 10 min and resuspension of the cell pellets in 50 ml HBSS. After three to five washes, the pellets were suspendedin 10 ml of cold HBSS. Small aliquots were removed for counting the cells and for determining their viability by the trypan blue dye exclusion test. Into 250 ml tissue culture flasks containing 50 ml HBSS, 50 X lo6 viable cells of each cell line were dispensed. The flasks were incubated at 37°C in 5% COs for 18 hr. After incubation, the cultures were centrifuged at 140s for 10 min in sterile plastic 50 ml screw-cap tubes. The supernatant fluids were removed and the cell pellets were resuspended in 5 ml HBSS to assesstheir viability after incubation by the trypan blue dye exclusion test. Each supernatant fluid was individually filtered through 0.45 pm disposable Millipore filters (Millipore Corporation, Bedford, MA), and dialyzed 3-4 days at 4°C against three to four changes of 4 liters distilled water. Before use, the dialysis tubing (Scientific Products, Evanston, IL) was boiled for 15 min in distilled water or in 0.001 M ethylenediaminetetraacetic acid (EDTA) . Followin g dialysis, each bag was rinsed in distilled water three to four times and the supernatant fluids were individually filtered through 0.45 p Millipore filters. The volume of each dialyzed supernatant fluid was reduced by ultrafiltration at 4°C to approximately 2.5 ml. As a sham control, HBSS in the absenceof cells was incubated, dialyzed and concentrated in the samefashion. Skin Testing of Guinea Pigs with Supernatant Fluids from Lymphobhtoid Cell Limes Rockefeller strain and inbred Sewall Wright strain-13 guinea pigs were used for skin testing. The sites for skin testing were prepared on the backs of the animals by carefully shaving the hair with an electric razor (Oster size 40, Mode1 ZA, Milwaukee, WI). Areas showing a reddened appearance of irritation after shaving were not used for skin testing. Concentrated supernatant fluids of each lymphoblastoid cell culture (0.1 ml) were injected intradermally and the test sites were inspected at 30 min, 1, 5, and 18 hr. Human serum, fetal calf serum, HBSS, and the dialyzed and concentrated HBSS were injected similarly. At 18-20 hr, perpendicular diameters of induration elicited by the injections were measured with a metric ruler or vernier caliper. The animals were sacrificed by cervical dislocation and biopsy specimenswere excised from each injection site. The biopsies were

SKIN

REACTIVE

FAc’TOR

289

fixed in 10% formalin, dehydrated in dilutions of alcohol and embedded in paraffin blocks. Sections 10 pm in thickness were cut on a model 820 microtome (American Optical CO., Buffalo, NY). Several serial sections from each biopsy were fixed on 3 X 1 in glass slides and stained with hematoxylin (Harris’ mixture) arltl eosin (0.5%) dyes. After preparation of the biopsied skin sections, the slides were examined at 215 X, 500 X, and 1250 x on a Zeiss microscope. Both the measurement of the skin test induration and the examination of the histological sections were done independently by two persons, nne of whom was unfamiliar with the experimental design. Capillav>l Tube Cell Migration Inhibition Assay and Analysis of Data The collection of guinea pig peritoneal exudate cells (GP-PEC) and the nligr:ition inhibition assay were done according to the method described by Jureziz ct nl. (52). In our experiments, the Sykes-Moore tissue culture chambers (1 ml/chamber) were filled with tissue culture medium TCM-199 containing 10% heat inactivated guinea pig serum, 5% fetal calf serum, 100 U/ml penicillin, 100 pg/ml streptomycin, 75 pg/ml polymyxin B, and 0.1 ml dialyzed and concentrated HBSS or supernatant fluid per ml of TCM-199. The chambers containing dialyzed and concentrated HBSS in TCM-199 served as a negative control and as a baseline ior calculating migration indices. After 48 hr incubation, the capillary tubes containing GP-PEC were placed on a Nikon Model GC profile comparator (Nikon. TokJ,tr. Japan) at 20 X magnification and the areas of cell migration were drawn and lne:lsured by planimetry. The raw values of the eight migration areas were used in calculation of a mean migration index (M.I.) similar to that described by Paque et al. (53). X Mathatron Power Log calculator was programmed for calculating the mean migration intles according to the formula: Mean M.T. = (Area of migration xl-it11test supernatant) i x 1()(j - -___.-.-rea of migration with sham supernatant) i :, 2 (A nXm where n represents the number of measured migration areas of cells from the test supernatant capillary tubes and m represents the measured migration areas from the sham control supernatant fluids. Mononuclear ClzewaotaxisAssay ‘, The effect of supernatant fluids on chemotaxis of mononuclear cells was assessed sing the Boyden technique (54) as modified by Demo and Andersen (55). Disposable chemotaxis chambers having Millipore membranes (average pore size, 8.0 pm) Market Engineering Co., Chicago, IL) separating ,the upper and lower compartments were used in our experiments, In the lower compartment, the folowing reagents were introduced : the supernatant fluids of RPM1 1788 cell cultures (0.9 ml dialyzed concentrated supernatant fluid with 0.1 ml of 10 x HBSS), HBSS, 25% normal guinea pig serum, or supernatant fluids from cell cultures of PPD-sensitive lymphoid cells incubated with PPD; IO7 GP-PEC were la!,ered

290

CALEBAUGH

AND

PAQUE

onto the upper side of the Millipore membrane and the chemotaxis chambers (three to five chambers per reagent) were incubated at 37°C in a humidified incubator for 5 hr. The membranes were removed, fixed in 10% formaldehyde, and stained with hematoxylin. The stained membranes were then microscopically examined for the number of mononuclear cells that had passed across the membrane. The mononuclear cells in at least 10 different fields were counted on each membrane at 256 x using a Zeiss microscope. Assessrvlefztof Su,pernatant Fluids for the Presence of Human Iwvvmmoglobulins and Lysozyme Goat anti-human whole serum (Pentex Research Division, Miles Laboratories, Kankakee, IL ; Kalstead Laboratories, Minneapolis, MN) was added to the center well of an Ouchterlony plate. Adjacent wells containing the following reagents: reference human immunoglobulins IgM, IgG, and IgA (Miles-Yeda, Rehovot, Israel; Melpar, Falls Church, VA), whole human serum, and supernatant fluids from the cell cultures. Radial diffusion plates (Kalstead Laboratories, Minneapolis, MN) were also used for the detection of human immunoglobulins. These plates could detect 40 pg IgM, 25Opg IgG, or 70 lug IgA. On other plates goat antihuman lysozyme (Kalstead Laboratories, Minneapolis, MN) was placed in the center well and the adjacent wells contained whole human serum, reference human lysozyme, and supernatant fluids from the cell cultures. A direct biological assay for the presence of lysozyme in the supernatant fluids was done using Micrococcus Zysodeikticus, an organism susceptible to lysis by lysozyme (Kalstead Laboratories, Minneapolis, MN). Supernatant fluids (15 ~1) were deposited in each well eight times. Three separate wells were filled once with 3.5 pg/ml, 14 pg/ml, and 70 pg/ml of lysozyme as positive controls. Clear zones of bacterial lysis around the wells containing reference human lysozyme was evident after 18 hr at 25°C. The diameter of lysis surrounding each well was measured and was proportional to the amount of lysozyme contained in each well. RESULTS Skin Reactivity of Guinea Pigs to Supernatant Human Lynzphobhtoid Cells

Fluids Obtained from Cultwes

of

Supernatant fluids were dialyzed and concentrated 20 X from 10 individual RPM1 1788 cell cultures (5.0 X lo7 cells/50 ml HBSS) incubated 18 hr ; each supernatant fluid was injected intradermally into five unimmunized guinea pigs to assessits ability to elicit a skin reaction (Table 1). Supernatant fluids obtained from each of the 10 cell cultures elicited an indurated skin lesion after 18 hr, which was substantially greater than the sham control of dialyzed and concentrated HBSS. For example, supernatant A5 caused skin induration in guinea pig DC3 of 10 3 11 mm ; however, the control was only 3 X 3 mm on the same animal (Table 1). Supernatant fluids prepared from 11 cell cultures of 8866 and Raji human lymphoblastoid cell lines failed to elicit skin reactions that were similar to those observed with the RPM1 1788 cell line (Table 2). For example, supernatants BS and C3 obtained from the 8866 and Raji human lymphoblastoid cell lines caused skin induration on guinea pig DC7 of 5 X 6 mm and 4 X 7 mm, respectively (Table 2). Although supernatant B6 of the 8866 human cell line elicited a skin

reaction on guinea pig DC6 of 9 X 9 mm, this reaction was not t!yical of the supernatant fluids obtained from the 8866 cell line, and microscopic esnmiix~tioii of the lesion revealed a granulocvtic influx of cells which was histologically clissimilar to the lesions produced by supernatant fluids from the RPM1 cell line. The average area of induration calculated from 145 skin tests site.s pr~xlucctl 1)~ the ilitratlermal injection of supernatant fluids from 12I’M T 1788 cell culture \\YIS suhst:mtially greater (70 mm”) than the ;I\-et-age arz;t of intlurati~~l~ i,~r vithcr the SSff) (25 mm’) or linji (10 mm”‘) h~um:m ccl1 lines ( ‘I‘:~ble 3 1 TIIC illtlxtl~rni;~l injection into unimmunized guinea pigs of th( iollowing rragr~~ts alw failetl to elicit skin reactions that xvere similar to those ol,served with hl I’.\] 1 I/‘ss supernat:mt fluids : A) human serum ; A’) fetal c;di sertun ; C) ICI’~lI 1040 tissue culture nletlium ; or n) HHSS (Table 3). To xsess \vhether the presence of soluble cell surface antigens ill the sul)ern;~tant fluids could elicit the skin reactions, viable KPATI 1788 human I~~~~l~hol~lastoiti cells (IO’-10’) were injected intratlermally into five unimmunized gui,w;l pigs, IGghteen hours later, the injection sites hat1 lesions that resembled an ;wltcs 110~ specific inllanmlatory reaction (56-63 j that is produced h!- the iiljectioll of ~j;Lr-ticulate Iwterials. The lesions were histc~logically ullrelatetl to those rll)ser\.y(l after the injection of supernatant fluids front culturrs of IiF’> IFS 1,.1111~11(,blnstoitl cells.

10 x 8X9 9x9 8X8 11 x 9x9 xx9 8X9 9x9 9 x

10

11

10

9 x 10 9x9 9x9 6X6 9x9 9x9 7X8 10 X 11 9x9 8X8

7X9 9x9 8X9 0 x 10 X 9x9 9 x 9 x 10 X 8 x

10 11 12 9 10 9

10 x 8 x 8X9 9x9 12 X 10 x 10 X 10 x 9 x 7x8

11 9

12 10 11 10 10

10 7 0 8 to 0 9 10 10 8

X x x x x x x X x x

11 h 0 0 II 0 10 IO 10 0

u The RPM 1 1788 human lymphoblastoid cell line was derived from A mmn;tI male indivjdu;~l. b Ten supernatant fluids prepared from ten separate RF’11 I 1788 cell cultures wwy c~~t-ed 01, three unimmu~~ized Rockefeller strain and two Setvall ii’right straill-13 guinea [jigs. C The letter or number in parentheses designate:, the strain oi guinea pig-: (R). Rockef~ll~~r ,tr;lin : (1.31, Sewall \Vright strain 13.

292

CALEBAUGH

AND

TABLE SKIN

TEST INDURATION INTRADERMAL FLUIDS

Supernatant

PAQUE

2

OBSERVED IN NONSENSITIVE GUINEA PIGS 18 HR AFTER IN JECT~ON OF 0.1 ML CONCENTRATED SUPERNATANT FROM CULTURES OF THE 8866 AND RAJI HUMAN LYMPHOBLASTOID CELL LINESQeb

fluids

Skin

test

induration Guinea

DC6(R) HBSS

sham

8866 Cell

control

X mm)

pigsc DCS(13)

DC9 (13)

0x0

2x2

0x0

2x2

4X6

5x4 5X5

6x7 0x0 3x4 9x9 2x2

4X6 5X5 6X6 6X7 5X6 3x3 5X6

5X5 5x5 6X6 3x3 3x3 5x7 6X5

3x4 5x5 2x2 4x5

3x3 3x3 4x7 5x7

3x4 5X6

line

Bl B2 B3 B4 B5 B6 B7 Raji

DC7 (13)

(mm

THE

2x2

6x7 6X6 6X6 5X8 6X6

cell line Cl c2 c3 c4

0 Eleven supernatant fluids prepared from eleven separate cell immunized Rockefeller strain and three Sewall Wright strain-13 b The 8866 human lumphoblastoid cell line was derived from myelogenous leukemia; the Raji human lymphoblastoid cell line Burkett’s Lymphoma. c The number or letter in parentheses designates the strain strain; (13), Sewall Wright strain 13.

3x3 5X6 4x5 5x5

6X6 5x7

cultures were tested on one unguinea pigs. a patient with an acute case of was derived from a patient with of guinea

pig:

Histology of Skin Lesions Elicited After the Intradewnal Inject-ion Fluids Prepared from Cultures of Huwmn Lywphoblastoid Cells

(R),

Rockefeller

of Supematant

Macroscopically, the development of the skin lesions elicited by the RPM1 1788 supernatant fluids paralleled those observed for PPD mediated delayed-type skin tests. Erythema became evident 6 hr after the injection of the RPM1 1788 test supernatant, and induration of the lesion was maxium at 18-20 hr. A central ischemic area was apparent at 18-20 hr, and scarification of this avascular area of the skin test occurred after three days. On the other hand, the intradermal injection of sham controls, human or fetal calf serum, or supernatant fluids prepared from 8866 or Raji cell cultures revealed little erythema at 6 hr, and substantially less induration at 18-20 hr (Table 3). Tissue sections of the skin lesions were prepared and examined microscopically. Typical histological sections of the skin lesions from animal DC-4 injected with supernatant A5 (Table 1) are shown in Fig. 1. The macroscopically indurated skin lesion measured 12x 12 mm. Microscopically, there was 50-70s mononuclear cells consistently seen in the lower part of the dermis especially above the pan-

SKIN

REACTIVE

.x3

FACTOR

niculus C~Y~~OS~LS where many of the cells appeared perivascular (Fig. 2). A diffuse infiltration of mononuclear cells was also seen in the upper portions of the dermis. In the central avascular portion of the skin reaction infiltrating mononuclear cells were observed in the epidermal tissues. Ten to forty per cent of the cells infiltrating the central area of the skin reaction were granulocytes (neutrophils and eosinophils). In less severe reactions of 9 x 9 mm (supernatant A2 on animal DC5 Table I), the incidence of neutrophils was 10-20s and the mononuclear infiltrate was primarily localized above the j~un~~ic&~ ca~~~o.~s. Necrosis was seen only in the central avascular region of severe reactions. Skin sections were also prepared from animals immunized with RCG and skin tested with 10 pg of PPD. Typical histological sections are shown in Fig. 1. The percentages of mononuclear cells present in skin sections from immunized animals approximated those observed in skin sections from animals injected with KPMT 1758 supernatant fluids. Histological skin sections prepared from intradermal injection sites of other reagents did not exhibit extensive mononuclear infiltration after 18 hr (Fig. 1). HBSS, human serum. ant1 the supernatant fluids prepared from the 8866 and Raji human cell lines exhibited little leukocyte infiltration. ;n~d these cells were mostly granulocytes. A typical histological section of a lesion ln-oduced by the 8866 cell line is sholvn in Fig. 1. In addition, concentrated preparations of supernatant fluids from Hep- 2 human epidermoid Gu-CincJllla cells failtstl to elicit skin reactions that were histologically similar to the RPRlI 1788 slopernatant fluids. The intradermal injection of lo”-lo6 viable RPM1 1788 human lymphoblastoitl cells in 0.1 ml HBSS did not produce skin reactions similar to those produced I)\. the RPM1 1785 supernatant fluids. Macroscopically, the injection site containing the RPM1 1788 cells exhibited slight erythema at 18 hr. The erythema was not TABLE SU,MYARY

OF SKIN

AFTER

THE

TEST

INDURATION

INTRADERMAL INJECTION OF HU~IAN LYMPHOBLASTOID SERUM

Reagents

HBSS RPM1

1640~

Human serum

.

Fetal calf serum 8866 cell line Raji cell line 1788 cell line

3

OBSERVED

OR MEDIA

Number of skin tests

40 20 36 6 44 32 115

IN NO~SENWIWE

OF SUPERNATANT CELL LINES ARE

GUINE.~

FLUIDS (CONTROLS

FROM WIW

PIGS

18 HK

CULTURES

IWZLGDED)

Mean area induration (mm2)0 --~ 4fl 15 i 0 f16 It 25 f3 16 f 70 f

3 2 3 3 8

of

--._-

70 ~~ononucle:Ir cells in skin lesion” ___~ _.~~~__ 1O-20 20-30 lo- 20 IO -20 1 o--30 1 w-30

50 -70

(1 The area of induration was calculated by determining an average radius from two perljendicular diameter measurements on each skin test and multiplying the average radius squared 1)) T. The mean area of induration was then determined by summatinq the areas within ;I group .IIK~ dividing by the appropriate number of skin tests performed. * Marginal sections of the skin test lesions. c RPM1 1640 tissue culture medium containing 10% fetal calf serum and antibiotics as described in the 3Iaterials and Methods section.

294

CALEBAIJGH

AND

PAQUE

FIG. 1. Dermal and deep dermal sections of the skin reactions from guinea pigs injected with PPD, RPM1 1788 supernatant fluids, and 8866 supernatant fluids (160 X). (A and B) Sections of dermis and deep dermis respectively from a guinea pig previously sensitized with BCG and skin tested 3 weeks later with 10 fig of PPD. (C and D) Sections of dermis and deep dermis respectively from an unimmunized guinea pig injected with RPM1 1788 supernatant fluids. Note the presence of substantial numbers of mononuclear cells in both antigen-mediated skin reactions (A and B) and in skin reactions excised from an tmimmnnized animal injected with RPM1 1788 supernatant fluids (C and D),

FIG. guinra

1. (E and pig injected

‘F) Sections with 8866

of dermis supernatant

and deep fluids.

dermis

reqectively

from

an unimmmlized

296

C:ALEBA~GH

AND

PAQUE

FIG. 2. Deep dermal section of the skin reaction taken from guinea pig DC-4 (Table 1) corresponding to picture D in Fig. 1. Note the presence of mononuclear cells in the skin section. A=1600X.

SKIN

FIG. 2. B =2500 x.

REACTIVE

FACTOR

20;

298

CALEBAUGH

AND TABLE

PAQUE 4

SUPERNATANTFLUIDSFROM CULTURES OFTHE RPM1 1788 HUMAN LYMPHOBLASTOID CELLLINETESTED INVITROFOR MIGRATION INHIBITORYFACTOR AND MONONUCLEARCHEMOTACTICFACTOR Supernatant

fluids

ControW HBSS MCF NGPS

Skin testsa Mean area of induration (mm2)d

S&4 -

MIF Assayb ye Mean migration inhibition

Chemotaxis assayc Mean number of mononuclear cell9

100 -

7 40 180

1788 cell line Al A2 A3 A4 A5 A6 A7 A8 A9 A10

74 54 61 53 88 66 74 72 71 56

f f &8 f f 3~ f zk f f

10 12 11 12 6 12 12 7 9

92 113 111 113 114 9.5 103 100 130 90

f zt f f f f f f zk *

5 8 9 13 8 6 12 8 15 5

12 3 6 9 15 7 4 5 10 7

a Ten supernatant fluids prepared from ten separate RPM1 1788 cell cultures were assessed for skin reactivity on two unimmunized Rockefeller strain and three Sewall Wright strain-13 guinea pigs. The individual in oivo skin reactions are presented in Table 1. b-0 Migration inhibition and mononuclear chemotaxis of the RPM1 1788 supernatant fluids were tested on peritoneal exudate cells from unimmunized Sewall Wright strain-13 guinea pigs. d The mean area of induration was calculated as described in footnote a of Table 3. e The mean number of mononuclear cells was calculated by observing the number of cells migrating across the membrane from a minimum of ten fields (256X). A differential cell count was performed and only mononuclear cells were recorded. f Abbreviations: HBSS, Hanks’ Balanced Salt Solution; MCF, supernatant fluids prepared from cultures of lymphoid cells from an immunized (Mycobacteria tuberculosis, HST Ra) strain-13 guinea pig incubated in vitro with PPD ; NGPS, normal guinea pig serum (250/c).

associated with induration and microscopic examination of the biopsied skin test site revealed a granulocytic infiltration of cells. Intradermal injection of larger quantities of RPM1 1788 cells (lo?-lo*) in 0.1 ml HBSS also did not produce skin reactions that were similar to those of the supernatant fluids alone. Microscopic examination of the skin biopsies 24 hr after injection of the cells showed extensive necrosis at the injection site. Assessment of Supernatant Fluids flow Cultures of the RPMI 1788 Human Lymphoblastoid Cell Line for Cell Migration-Inhibition and Monowclear Chemotaxis Supernatant fluids obtained from the 10 individual cell cultures of RPM1 1788 lymphoblastoid cells (shown in Table 1) were assessed for mononuclear chemotaxis and their ability to inhibit the migration of guinea pig peritoneal exudate cells (GP-PEC). Supernatant fluids prepared from RPM1 1788 cell cultures failed to

inhibit the migration of normal GP-PEC. The mean migration index ranged irollr 9C)-13Oy (Table 4). The same sulJernatant fluids elicited skin reactions characterized I))- induration and nionocptic infiltraticoll ( Table -l 1. 1’11r esanlple, sulwrnatant Al had a mean area of induration of 71 1lm1” ;~nri a twan tnigr;~tioll imlr\ superiJ:Wllt tluitls ~mlx~rctl irojjl 1.2 ;~~l~litioJi;tI of 9.27; (Table -!) 1Ioreovcr, culturrs also Failed to illhil)it (;I’-L’EC nligr:ltitrJJ ; thb IW;LII nJiq:hou i~r~licw \T’(T:’ sinlilar to thosr ~how~i iii Table 4. ior t~ioi~oi~~lcIcar ch~~~c~t~iri~ oi The 10 sul~ernatant fluids were also assessetl g-uinca l)ig. In the top coinpartiiwilt of (; I’-I’EC from an uniiiiiiiunized straiil 1.3 the chtmotasis chamber, 10’ GZ’-I’EC \vere ln!.ered auto ,\llillipore liltcrb. Iti the bottom hali of the chamber, the follo\ving reagmts \vere atltletl : _ I I ~llljcmwtarlt fluids frcmr cell cultures of RI’YIT 171% humall cell line : I? ) IH HSS : C’ 1 2.i:/iZ nornial guiiiea pig serum ; or I,) supernatant fluids obtained from cultures of guinea pig Iynq)hoid cells sensitized to EKG and inculmted 1S Ill- it1 Iif’ I 1fAO ~lretlia \vith [‘I’ll. The chemotaxis chambers were incubated 5 hr at 37°C’. ‘1‘1~ ~r~illil)c m’ ~nen~l~r:~l~cs were then removed, fixed in form;~ltlehvtle, st;liw(l \\.ith h~t~mto\r\~li~~. :iiitl the iiuinl)er of nmionucle;w cells passing through tht, nwinbraiiv cr,iirttc,tl (‘l’;il~lr 4). Supermtant fluids prep-cd from 121‘.\I I 1SM wll cultl1i-c.2 tlitl li0t tlmionstr;~tc nioiionuclear clieiiiotasis. The inan niinil)er of ii~~~i~o~Jticl~:ii~ ( ; I ‘~ I’I~IC‘ passing through the membrane ranged from 3-I 5 ( Tal~le 4 ). These ~~11 ~II~IJIIJWY (hi the other linntl. tlw ti1:211 :Ire similar to those observed with HHSS do~w. nunil~er of mononuclear cells traiis\wsiil g the iiieiitltranc~ iii the lwsiti\c cr~iitix~l5 \V:LSsignificantly greater. For esarnl~le. a iiwan of IS0 iiioilcillliclwr rrll5 lwi- lit.ltl tramversed the membrane when 25 /(‘G normal guinea pig w-uni \v:is iii the 11,wichamber. Norinal guinea pig seruiii has IKYII ckw~oiisti-atr.cl11)IJC 5trc,)llg:Iy clrt~1110tactic for guiim pig monoiluclear cells (6-+1. Alorec,\ t’r, \\~lwii su~~eri~at~~iit Illlitlb fro1ii cultures of wr> sensitive Iyq~hoitl cvlI> \\.crc illClllJXtd \\.ith 1’1 ‘1 ) X1(1 tested for nlouonuclcar chenlotasis, wt. obser\wl a ~~(YLII(,i -1-OII~~~IIOIII~CI~;II~~.il,% [ler fieltl.

Supernatant fluids obtained from 18 hr cc*11cultures ot’ 331’MI I/‘SS 1111117;111 I~vq~hoblastoid cells incubated in HKSS \\.et-e ass~ssetlfor the presence cli imniunoglobulins in the dialyzed, 20 X concentrate~l fluids IJ~ ()uchterlon)- tlouble diffusion and I))- radial diffusion. Ten supernatant ~ml~aratitm \vere tested fo1 the presence of human Tgl\J. IgG, or lg:\ 1~). the ( )ucht~rlol~!~ double tlitfusion technique using goat anti-human whole w-ml in the celltc.r ~~~11.-litter 2-l Ilr :lt 2.5OC, 1Jrecipitin bands could not lye oljsen~et! i~withe ~~211.~ coiitaimilrg I( I’Al J 17% sul)ernatant fluids even after the I\-ells I\-cre I-ctillrtl vi&t titlres. On tllc ~thhand. reference hm~an imnlunc~globulil~s( IgAl, Ig(; ;1m1 lg.\ ) incubnttyl ill the. adjacent \vells on the same Ouchtcrlon~~ IJlate g;i\-e sillglc liiii~ of preciIJit;ltic,n. 1liheu the RPA’II 17x8 supernatant fluids \vc’re tvstc(l II>. r;l(li;ll difftlsion Ilsillg AJmcini agar plates saturated with anti-human IgAl, Ig( ;, or Ix:,i ( Kalstc:~(l I ,:kl,oratories. 31inneapolis, i\tS), prccipitiii b;mrls wultl iiot IJC oljserved ill the l~el]s containing Ii [‘&II Ii85 superuatant fluids. f
300

CALEBAUGH

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Supernatant fluids obtained from the same RPM1 1788 cell cultures were assessed for the presence of lysozyme in the concentrated fluids. Experiments were performed by placing goat anti-human lysozyme in the center well of an Ouchterlony plate and the RPM1 1788 supernatant fluids and human lysozyme were deposited in adjacent wells. Precipitin bands were not observed for the RPM1 1788 supernatants, even after the wells were refilled eight times; however, 3.5 pg of human lysozyme gave precipitin bands after 18 hr. The RPM1 1788 supernatant fluids were also assessed for the presence of lysozyme by a direct biological assay. The wells in agar gel plates embedded with Micrococcus lysodeikticus were filled with the RPM1 1788 supernatant fluids and lysozyme reference standards. Clear zones of lysis were not observed around the wells containing the RPM1 1788 supernatant fluids even after ‘the wells were refilled eight times. In contrast, a well containing reference lysozyme standard (3.5 pg) gave excellent zone of lysis measuring from 3.1 mm. DISCUSSION Our data indicate that supernatant fluids prepared by incubating RPM1 1788 human lymphoblastoid cells in HBSS for 18 hr can elicit, after intradermal injection, an indurated skin lesion in unimmunized guinea pigs< which reaches a maximum induration at 18-24 hr and which is macroscopically and histologically similar to a specific antigen mediated delayed skin reaction (65-68). Substantial numbers of mononuclear cells (50-70$&o) were observed in the skin lesion 18-24 hr after injection. These lesions were macroscopically and histologically similar to those described by Bennett and Bloom (10) who injected migration inhibtory factor (MIF) into unimmunized guinea pigs and obtained maximum skin reactions 12-18 hr after injection. We were unable to detect the presence of MIF in our supernatant fluids from the RPM1 1788 cell line. We also assessed our RPM1 1788 supernatant fluids for the presence of mononuclear chemotactic factors that have been implicated in delayed hypersensitivity. Mononuclear chemotactic factors, which are released by sensitized guinea pigs (26, 27) and human lymphoid cells (69) have the ability to attract unsensitized mononuclear cells through millipore membranes in vitro. Our supernatant fluids were not chemotactic for guinea pig peritoneal exudate cells since significant numbers of mononuclear cells failed to cross the membrane in the chemotactic assay. Pick and Turk (36-42) have reported the release of a substance from sensitized guinea pig lymphoid cells that elicits an intradermal skin lesion which is characterized by the appearance of substantial numbers of granulocytes 24 hr after injection. However, the skin lesions produced by intradermal injection of supernatant fluids from the RPM1 1788 cell line were characterized by the presence of substanttial numbers of mononuclear cells at 24 hr. Supernatant fluids from other human lymphoblastoid cell lines have been assessed for release of the skin reactive factor. For example, supernatant fluids obtained from cultures of Raji and 8866 human lymphoblastoid cells failed to produce skin reactions that were similar to those observed with supernatant fluids from cultures of RPM1 1788 lymphoblastoid cells. The Raji and 8866 cells were originally isolated from humans with malignant diseases of hemopoietic tissue and their peripheral lymphocytes were established in tissue culture as long term lymphoblastoid cell lines. Whether long term cultures of lymphoblastoid cells estab-

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lished from these individuals may possibly have lost the capacity for synthesizing certain soluble mediators is unknown at present. We have assessed the possibility of whether our supernatant fluids obtained from RPM1 1788 cell cultures could be nonspecific inflammatory materials released from human lymphoblastoid cells and unrelated to mediators associated with delayed hypersensitivity. For example. lysosomal enzymes released from lymphoitl cells could possibly elicit inflammatory skin reactions; yet the testing of our supernatant fluids with a goat antiserum against human lysozyme failed to exhibit lines of precipitation in double gel diffusion experiments. Furthermore, using a direct enzymatic assay for the detection of human lysozyme did not reveal the presence of the enzyme in our supernatant fluids. hloreover, intraclermal injection of the following materials also failed to elicit skin reactions that were similar to those observed with supernatant fluids obtained from RPM1 1788 cell cultures: A) dialyzed and concentrated HBSS alone, B) HBSS, C) human and fetal calf serum, D) 104-lo* viable RPM1 1788 human lymphoblastoid cells, and E> fluids from cell cultures containing human Hep-2 epidermoid carcinoma cells. Finally, we have considered the possibility that human immunoglobulins could be present in our supernatant fluids and could possibly cause Arthus type skin reactions. Antibody mediated skin reactions have sometimes been misinterpreted as delayed skin reactions, especially when hiStIIJlO&il esamination of the lesion was not performed (64, 69). However, repeated testing of our supernatant fluid preparations by immunological methods failed to demonstrate the presence of human IgM, IgG, or IgA. Although we cannot esclude the involvernent of an immunoglohulin eliciting our skin reactions, the injection of lvhole human serum failed to elicit similar skin lesions. Several possibilities must he considered to explain the appearance of the skin lesions following intradermal injection of RPM1 1788 supernatant fluids. We cannot exclude the presence in our supernatant fluids of other mediators that are mobilizing mononuclear cells. It is possible that two or more mediators acting synergistically could mobilize mononuclear cells. or that a single mediator might act in a similar fashion. In preliminary experiments utilizing acrylamide gel electrophoresis, we have observed five to seven bands in the gel that appear to be protein in nature. We have not detected the presence of MTF or mononuclear chemotactic factors in our concentrated supernatant fluid preparations ipl vitro. L$:e cannot exclude the possibility that MIF may be present in our preparations, and lack of detectable MIF could be due to different quantities of mediators that are effective in viva and apparently not ill z&o. Future experiments in concentrating our supernatant fluids may indeed indicate the presence of MIF. Another possibility is that guinea pigs may possess native delayed sensitivity (71-74) to certain human antigens released into the supernatant fluids during in zitro incubation. These antigens could be present in the supernatant fluids 1)) exudation, secretion or release of internal constituents, or shedding of membraneassociated antigens. These possibilities seem unlikely since the intradermal injection of supernatant fluids from two other human lymphohlastoid cell lines, or injection of 104-10’ viable RPM1 1788 cells failed to elicit skin reactions that were similar to those observed with our 1788 supernatant fluids. On the other hand. these experiments do not completely exclude the possibility that the guinea pigs might have natural delayed sensitivity to a human cell material that is presrnt in the supernatant fluids of RPM1 1788 cells.

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While many investigators have attempted to assess the role of soluble mediators in delayed hypersensitivity in vivo, the results have been inconclusive because of the failure to remove specific antigens and nonspecific mitogens from the supernatant fluids used to trigger their release. Indeed, Teodorescu et al. (46) reported that the intradermal injection of phytohemagglutinin caused indurated skin lesions in humans. These difficulties have been eliminated from the present study by using human lymphoblastoid cells which release a skin reactive factor in the absence of antigen or nonspecific mitogen. The ability of our supernatant fluids to recruit mononuclear cells in tivo following intradermal injection into unimmunized animals may have important practical considerations. For example, Bernstein et al. (31) injected MIF intradermally mixed with syngeneic guinea pig tumor cells subsequently observed regression of tumor growth in Z&JO. These authors concluded that nonspecific mobilization of mononuclear cells by MIF without specific recognition of tumor antigens was responsible for tumor regression. The MIF was demonstrated to be noncytotoxic for tumor cells in vitro. The previous results could also be explained by the presence of a factor similar to the material that is present in our supernatant fluids from lymphoblastoid cells, which nonspecifically mobilizes mononuclear cells in vivo. Further investigations are in progess in order to isolate, purify, and characterize the biologically active material in our supernatant fluids. With our system, large quantities of the factor can be obtained in the absence of antigens, nonspecific mitogens, and serum proteins, and therefore facilitates investigations on the role of soluble mediators in delayed hypersensitivity. 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.

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