CELLULAR

IMMUNOLOGY

37, 188-198 (1978)

Rosette Formation Erythrocytes I.

between Requires

Characteristics

Rat Thymocytes and Guinea “Active” Fetal Calf Serum

of “Active” Thymocytes

GERALD J. ELFENBEIN Omology

Confer

Pig

Serum Factors and Receptors on and Erythrocytes 1

2 AND JERRY A.

WINKELSTEIN

and Department of Pediatrics, The Johns Hopkins Medicine, Baltimore, Maryland 21205 Received

October

z

University

School

of

21,1977

Rosette formation between rat thymocytes and guinea pig erythrocytes is dependent on at least two factors present in non-heat treated fetal calf serum. One factor is a high molecular weight, heat stable substance and the other factor is a heat labile substance(s) . The rosette formation process is divalent cation dependent and seems to involve the sequential binding to thymocytes of the high molecular weight, heat stable substance, the heat labile substance(s), and then guinea pig erythrocytes. Thymocytes appear to bear a receptor which is dependent on hexose monophosphate shunt metabolism, is not removed by many digestive enzymes, but is blocked with phytohemagglutinin and pokeweed mitogen. Erythrocytes appear to bear a receptor which is dependent on hexose monophosphate shunt metabolism, removed by pronase treatment, and blocked by phytohemagglutinin and concanavalin A.

INTRODUCTION Receptors cytes

for heterologous

have

been

identified

(3,4),

(421,dogs

by rosette

fortnation

of rosettes

between

(12). between

cats (5)) and man (RF) guinea

between

pig RBC

characteristics report

1) . These and

of the factors

of the receptors

of the receptor

(13).

rat THY

(THY)

and guinea

detected

the formation

has been described

(FCS)

which

(T) lymph+ guinea pigs

are usually

Recently,

is required

In this communication

in FCS

on rat THY

bearing

receptors

T cells.

that fetal calf serum

and rat THY

some of the characteristics the characteristics

(6-l

the RBC

pig RBC and rat thymocytes

It has been demonstrated guinea

(RBC) on thymus-derived of animal species including

erythrocytes

in a number

permit

RF

for RF

we present and

SOme of

pig RBC. The biological

are described

in the accompanying

(14).

1 This investigation was supported by Grant NO. CA-15396, awarded by the National Cancer Institute, and Grant No. AI-11637, awarded by the National Institutes of Allergy and Infections Disease, DHEW. 2 Investigators, Howard Hughes Medical Institute. 188 0008~8749/78/0371-0188$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

FETAL

CALF

SERUM

REQUIRED

MATERIALS

FOR

AND

RAT

THYMOCYTE

ROSETTES

189

METHODS

Thynzocytes. Adult (9 to 12 week old) Lewis rats were purchased from Microbiological Associates, Bethesda, Maryland. Animals were sacrificed using ether anesthesia, and thymus glands were removed in their entirety from the anterior mediastinum while avoiding adjacent lymph nodes. Single cell suspensions of THY were made by gently pressing the thymus through a fine mesh screen. THY were wrashed three times in Hanks Balanced Salt Solution (HBSS) free of supplemental serum at 22°C. After washing, THY were diluted to 4 x 10F nucleated cells/ml in HBSS at 22°C. Erythrocytes. Hartley guinea pig blood in modified Alsevers solution at a 1 : 1 ratio (v/v) was obtained from Colorado Serum Co., Denver, Colorado, or by cardiac puncture performed on Hartlky guinea pigs purchased from Buckberg Lab *\nimals, Inc., Thomkins Cove, New York. Stored or fresh RBC were washed free of Alsevers solution and plasma by three washes in HBSS free of supplemental serum at 4°C. After washing, RBC were diluted to 1 X lo* cells/ml (approximately 1% v/v suspension) in HBSS at 22°C. Fetal calf seruT?z.Frozen FCS purchased from a number of suppliers (including Colorado Serum Co. ; Pel Freeze, Rogers, Arkansas ; Reheis, Phoenix, Arizona; GIBCO, Grand Island, New York ; and Microbiological Associates), was shown to be active in the studies outlined below. Frozen FCS was thawed carefully, aliquoted, and refrozen (-20°C). Activity was lost when aliquots were repeatedly frozen-thawed or when thawing was performed too rapidly in hot water baths. Except where indicated below undiluted, non-heat treated FCS was used in all experimental procedures. Rosetfe fonmtion. Optimal conditions in our hands for RF between rat THY and guinea pig RBC in media containing FCS are as follows. Equal volumes of the suspensions of THY and RBC (vide supra) are mixed and undiluted FCS is added in sufficient volume to make a final concentration of 10% (v/v) in the RF mixture. The mixture is incubated for 30 min at 22”C, then centrifuged at 1OOg for 5 min at 4”C, and further incubated for 60 min at 4°C. The pellet is gently aspirated with a Pasteur pipette and the suspension mounted in a hemocytometer. Routinely 200 nucleated cells are counted and those bearing three or more firmly attached erythrocytes are defined as being RF positive. For ease of identification of mononuclear cells by light microscopy one drop of 1% (w/v) toluidine blue is often added to the rosette mixture. Absorption studies. Different volumes of packed rat THY (1 ml N l-2 X 10” cells) or guinea pig RBC were suspended in FCS and incubated at 4°C for 60 min with agitation. Cells were removed from suspension by centrifugation and the absorbed FCS was placed in an RF assay. Precoativzg studies. Different volumes of packed rat THY or guinea pig RBC were mixed with FCS and incubated at 22°C for 30 min with agitation after which the cells were washed three times with HBSS at 22°C before being placed in an RF assay. DiZzbtio+zstudies. Aliquots of FCS were serially diluted twofold in either HBSS without supplemental serum or in heat treated FCS (56°C for 30 min) . An aliquot of each dilution of FCS in either HBSS or heat treated FCS was placed in an RF assay to determine the dilution’s ability to support RF.

190

ELFENBEIN

AND

WINKELSTEIN

Treatment of fetd calf seruwl. Different aliquots of FCS were either heat treated at 22, 37, 45, or 56°C for 30 min, or dialyzed against Dulbecco’s phosphate buffered saline (PBS) containing Ca*+ and Mg?‘, or treated with ammonium sulfate at a final concentration of 40% saturated solution. Other aliquots of FCS were treated for different periods of time (0 to 30 min) at 56°C. Aliquots of treated FCS were placed in RF assays to determine their ability to support RF. Treatment of rosette fortimtion mixture. Either ethylene diamine tetraacetic acid (EDTA) at a final concentration of 0.01 M, sodium azide at a final concentration of 0.01% (w/v), or potassium cyanide at a final concentration of 0.1 mM were added to different RF mixtures prior to the incubation steps of the RF assay. Restoration studies. In an attempt to restore the activity of aliquots of heat treated FCS (56°C for 30 min), a number of non-heat treated normal rabbit sera, guinea pig sera or rat sera were added to the RF mixture at a variety of final concentrations ranging from 1 : 10 to 1: 10,000. Normal rabbit serum, guinea pig serum and rat serum at all dilutions tested did not alone support RF between rat THY and guinea pig RBC. Fractionation of fetal calf seyz&?pz. Aliquots of FCS were passed through 50,000, 100,000, and 300,000 Dalton molecular sieve filters (Amicon, Lexington, Mass.) using compressed nitrogen at 40 PSI. From each filtration run aliquots of the filtrate and retentate were placed in an RF assay. Pretreatment of thymocytes and erythrocytes. Aliquots of rat THY (4 X lo6 cells/ml in HBSS) were exposed to ionizing radiation in z&o [ 1500 to 5000 rads from a dual 137cesium source at 136 rads/min (Atomic Energy of Canada, Ltd.) 1. Aliquots of rat THY were also treated with different enzymes in vitro. Aliquots were exposed to either Vibrio clzolera neuraminidase (VCN) at 50 units/ml for 30 min at 37°C or pronase (4 x lo6 THY suspended in 5 ml of a 2.5% (w/v) solution) for 30 min at 37”C, or DNase (0.0004% (w/v) solution added to 5 ml of THY suspension, mixed, and cells used at once), or RNase (4 X lo6 THY suspended in 5 ml of a 20 pg/ml solution) for 30 min at 37°C or trypsin (4 X 10’ THY suspended in 5 ml of a 0.25% (w/v) solution) for 30 min at 37°C. After all treatments (irradiation or enzymatic) except DNase, treated THY were washed in HBSS and placed in an RF assay. Aliquots of guinea pig RBC at 1% (v/v) concentration were treated in vitro with either VCN at 50 units/ml for 30 min at 37°C or pronase [O.l ml packed RBC suspended in 5 ml of 0.25% (w/v) solution] for 45 min at 37°C. After enzyme exposure, treated guinea pig RBC were washed and placed in an RF assay. Aliquots of rat THY (4 x lo6 cells/ml) and guinea pig RBC (1% v/v) were treated with the metabolic inhibitor 2-deoxyglucose which primarily inhibits the hexose monophosphate shunt but also inhibits anaerobic glycolysis. Aliquots of rat THY or guinea pig RBC were exposed to different concentrations of 2-deoxyglucose (10 to 40 mJJ> for different times (30 to 60 min) at 22°C. Without being washed, treated rat THY were then placed in an RF mixture with fresh guinea pig RBC and FCS and treated guinea pig RBC were then placed in an RF mixture with fresh rat THY and FCS. Rat THY and guinea pig RBC were also treated with lectins as follows. Aliquots of rat THY (5 x 106 cells/ml) were incubated with either phytohemagBeckenham, England) at 100 pg/ml. glutinin (PHA ; Burroughs-Wellcome, concanavalin A (Con A; Sigma, St. Louis, Missouri) at 250 pg/ml, or pokeweed mitogen (PWM ; GIBCO) at a 1: 6 dilution for 30 min at 37°C. Aliquots of

FETAL

CALF

SERUM

REQUIRED

FOR RAT TIIYMOCYTE

TABLE Effect

of Different

Absorptions

Absorptiona

on the Ability

of FCS with

191

1 of FCS to Support

Rosette Formation

Percent rosettes supported by absorbed FCS .___ Expt.

Rat Thymocytes (Guinea pig erythrocytes Roth (in either order) Neither

ROSETTES

1.5 74.5 1.5 80.0

1

Expt.

2

0 86.0 0 90.0

n One vol: 1 vol at 4°C for 60 min.

guinea pig RBC (1% v/v) nere 7 incubated with either PHA at 10 pg/ml, Con A at 0.1 to 0.3 pg/ml, or PWM at a 1 : 60 dilution for 30 min at 37°C. All treated cell aliquots were then washed three times in HBSS and placed in an RF mixture with the appropriate fresh cell type and FCS. RESULTS Dependence of rosette formation between rat thyYnocytes and guinea pig erytlzon non-he& treated fetal calf serum. Initially, RBC from a wide variety of species were screened for their ability to rosette with rat THY. Only guinea pig RBC rosetted with rat THY as has been shown elsewhere (12). Initial assays were performed using non-heat treated FCS. In 102 consecutive Lewis rat thymuses, the percentage of THY participating in RF was 73.1 2 8.4% (one standard deviation) when non-heat treated FCS was present at a final cocentration of 10% (v/v) in the RF mixture. When heat treated FCS (56°C for 30 min) was used at 10% (v/v) with the same rat THY there were no rosettes found. This suggests that non-heat treated (“active”) FCS contains at least one factor that is heat labile and necessary to support RF. Absorption of “active” factor(s) in fetal calf semm by rat thynzocytes. W’hen aliquots of FCS were absorbed with packed rat THY or guinea pig RBC at a 1: 1 ratio (v/v) for 60 min at 4”C, it was noted that all ability to support RF was removed by absorption with rat THY but none by guinea pig RBC (Table 1). When different aliquots of FCS were absorbed with successively smaller volumes of rat THY it was noted that at ratios of THY to FCS of 1 : 1000 to 1 : 10,000 no RF supporting activity was removed and at ratios of 1: 100 to 1: 300 approximately 50% of RF supporting activity was removed (data not shown). Thus, it appears that at least one critical factor for RF in FCS is quantitatively absorbable by rat THY. Precoating of rat thymocytes z&h “active” factor(s) of fetal calf semm supports rosette formation. When aliquots of rat THY or guinea pig RBC were incubated with FCS at 22°C for 30 min at ratios of cells to FCS of 1 to 2 10, it was observed that “precoated” THY were able to rosette with untreated RBC in the absence of additional FCS but precoated RBC were unable to rosette with untreated THY unless in the presence of additional FCS (Table 2). These data suggest the sequential binding of the critical factor(s) for RF in FCS to rat THY after which guinea pig RBC bind to the THY-FCS complex. rocytes

192

ELFENBEIN

AND

WINKELSTEIN

TABLE

2

Demonstration That the Factor(s) in FCS Which Support(s) Rosette Formation Thymocytes First After Which Guinea Pig Erythrocytes Bind to the Thymocyte-FCS Rosette

formation

mixture

Additional FCS

Thymocytes

Guinea pig erythrocytes

Normal Normal FCS precoated” FCS precoated Normal Normal

Normal Normal Normal Normal FCS precoated” FCS precoated

Bind(s) to Complex

Percent rosettes Expt.

+ 0 + 0 + 0

1

Expt.

79.5 0 73.5 70.5 80.0 0

2

72.0 0 85.0 68.0 71.0 0

u One vol cells to 10 vols FCS at 22°C for 30 min.

Fetal calf serum contains at least two “actiz~e” factors. Aliquots of FCS were serially diluted twofold in either HBSS or heat treated FCS (56°C for 30 min) and these dilutions were then assayed for their ability to support RF between rat THY and guinea pig RBC. It was found that higher dilutions of FCS in heat treated FCS than in HBSS were able to support RF (Table 3). In a second experiment aliquots of FCS were heat treated at 56°C for 30 min or carefully absorbed twice with equal volumes of packed rat THY at 4°C for 60 min. Then these aliquots were tested separately and in combination for their ability to support RF. As can be seen in Table 4 the recombination of heat treated FCS and absorbed FCS could support RF whereas neither alone was able to support RF. These data taken together demonstrate that “active” FCS contains at least two factors. Furthermore, one factor is heat stable and another factor is heat labile. TABLE Limiting

Dilution

Final dilution of FCS in mixture

Studies Suggesting That FCS Contains Factors Involved in Rosette Formation Percent

rosettes supported

HBSS Heat treated Expt. lb 1: 10d 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:.5120

3

FCSa

by FCS diluted

in :

HBSS Heat treated Expt. 2=

71.0 53.0 37.0 2.5 1.0 0 0 0 0 0

At Least Two

FCSa

73.0 73.0 62.0 37.0 22.0 5.0 1.0 0 0 0

a 56’C for 30 min. b FCS purchased from GIBCO, Grand Island, New York. c FCS purchased from Pel-Freeze, Rogers, Arkansas. d Undiluted FCS placed in rosette formation assay.

64.0 67.5 63.0 41.0 6.0 0 0 0 0

61.5 61.0 69.0 54.5 36.0 16.5 7.0 1.5 0

FETAL

CALF

SERUM

REQUIRED

FOR

RAT

THYMOCYTE

4

TABLE Recombination

Studies Suggesting That FCS Contains Factors Involved in Rosette Formation

Non-heated FCS

Absorbed FCSe

Percent

Heat treated FCS*

Expt. + 0 0 0 + +

0 + 0 + + 0

..~~ -__~

__-~

a Non-heated FCS absorbed 60 min. * 56’C for 30 min.

103

ROSETTES

at Least Two

Rosettes

1

Expt.

2

72.0 11.0 0 0 0 0 43.0 22.0 55 0 31.0 61 .o 35.0 ~ ~~_~.. ~~ __-.-~-

0 0 + + 0 + ~.._____~~.~~

twice with equal volumes

of packed Lewis thymocytes

at 4’c‘ for

Characteristics of the “active”’ factors in fetal calf serum Numerous experiments were performed to determine the characteristics of the “active” factors in FCS which support RF. These experiments were of two varieties. In the first type of experiment aliquots of FCS were pretreated before being added to rosette mixtures to evaluate their ability to support RF. In the second type of experiment various additives were placed in the rosette mixtures to determine their effects on RF. The results of these studies are presented in Table 5. RF activity was lost when aliquots of FCS were pretreated at 56°C for 30 min. M7hen other aliquots of FCS were pretreated at 56°C for shorter times, it was observed that approximately 65% of RF activity was lost after 5 min at 56°C and 100% was lost after 10 min at 56°C (data not shown). RF activity was retained in aliquots of FCS despite either heat treatment at < 45°C for 30 min, dialysis against Dulbecco’s PBS (with Ca”+/Mg’k), or precipitation with ammonium sulfate at a final concentration of 4070 saturation. No RF was noted in rosette mixtures to which EDTX was added at a final concentration of 0.01 M but RF was normal when sodium azide (final concentration 0.01% w/v) or potassium cyanide (final concentration 0.1 mM) were added to the mixture. Thus, it appears that RF between rat THY and guinea pig RBC is dependent on clivalent cations hut independent of anaerobic glycolysis and oxidative metabolism. At least one critical factor in FCS is “inactivated” during the first 10 min at 56°C. However, none of the critical factors appear to be sensiti\-e to TAHLE Effects

Mode

5

of a Variety of Pretreatments of FCS or Additions Mixture on the Ability to Support Rosette --___-

of manipulation

Rosette -.

to the Rosette Formation

Formation

formation activity --_--.-___.__

Lost

Retained -.--

Pretreatment

Treatment formation

of FCS

of rosette mixture

with:

with:

Heat

EDTh

treatment

(0.01

(56°C

.lI)

for 30 min)

Heat treatment (