[HESIS

 

This is to certify that the

thesis entitled

FRACTIONATION 0F BOVINE LYMPHOCYTES WITH
AN IMMUNOABSORBENT COLUMN AND ANALYSIS
OF CELL SURFACE CHARACTERISTICS

presented by

Daniel R. PenneII

has been accepted towards fulfillment
of the requirements for

M.S. . Microbiology and
degree in

 

 

Public Health

 

Major professor

Date 1.] OL/l/g d

0-7639

 

 

 

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Place In book return to remve
charge from circulation records

 

 

FRACTIONATION OF BOVINE LYMPHOCYTES WITH AN
IMMUNOABSORBENT COLUMN AND ANALYSIS OF

CELL SURFACE CHARACTERISTICS

BY

Daniel R. Pennell

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Microbiology and Public Health

1979

ABSTRACT
FRACTIONATION OF BOVINE LYMPHOCYTES WITH

AN IMMUNOABSORBENT COLUMN AND ANALYSIS
OF CELL SURFACE CHARACTERISTICS

BY

Daniel R. Pennell

Bovine lymphocytes were fractionated into
suprpulations enriched in surface immunOglobulin (519)
positive and 51g negative cells and analyzed for the pres—
ence of immunoglobulin (Ig) and complement (C) receptors
using EA and EAC rosette assays. Incubation of peripheral
blood with zymosan allowed for the development of a leuko-
cyte isolation procedure enabling differentiation between
phagocytes and lymphocytes. Lymphocyte fractionations were
performed using a column with matrix-bound anti-bovine IgG.
Upon passage of lymphocytes through a column and elution
with medium, cell isolates were obtained enriched in 519
negative lymphocytes. Elution with medium containing
bovine gamma globulin allowed for the isolation of cells
enriched in 51g positive lymphocytes. EA and EAC rosetting
frequencies increased with increasing proportions of 519
positive lymphocytes suggesting that 19 and C receptors

are largely B cell markers.

DEDICATION

To my family:

Parents, Patrick R. and Dolores R. Pennell

and sister, Tamera M. Schneider.

ii

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to
my research advisor, Dr. Norman B. McCullough, for his
guidance and encouragement throughout this project.

I would also like to thank Dr. Herbert W. Cox and
Dr. Walter J. Esselman for their assistance during

this study.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . Vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . vii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 2
Lymphocyte Heterogeneity . . . . . . . . . . . . 2
Isolation of Peripheral Blood Lymphocytes . . . . 4
Lymphocyte Receptors for Heterologous
Erythrocytes . . . . . . . . . . . . . . . . 6
Lymphocyte Surface Immunoglobulin . . . . . . . . 8
Lymphocyte Receptors for Immunoglobulin . . . . . 9
Lymphocyte Receptors for Complement . . . . . . . 12
Isolation of Lymphocyte Suprpulations . . . . . 13
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 19
Animals . . . . . . . . . . . . . . . . . . . . . 19
Immunization . . . . . . . . . . . . . . . . . 19
Collection and Treatment of Sera . . . . . . . . 20
Isolation of Bovine Leukocytes . . . . . 20
Enrichment of Rabbit Anti-Bovine IgG Antibody . . 22
Analysis of Enriched Rabbit Anti-Bovine IgG . . . 23
Preparation of Cell Fractionation
Immunoabsorbent Columns . . . . . . . . . . . . 24
Cell Preparation and Fractionation . . . . . . . 24
Rosetting Assays . . . . . . . . . . . . . . . . 25
Detection of Surface Immunoglobulin . . . . . . . 26
Enumeration of Phagocytic Cells in
Fractionation Studies . . . . . . . . . . . . . 27
Viability Assay . . . . . . . . . . . . . . . . . 27
Photography . . . . . . . . . . . . . . . . . . . 27
Statistics . . . . . . . . . . . . . . . . . . . 28
Preparation of Reagents . . . . . . . . . . . .'. 28

iv

Page

RESULTS 0 O O O O O O O O O O O O O O O O O I O O O O 31
Detection of Phagocytic Leukocytes with
Zymosan . . . . . . . . . . . . . . . . . . . . 31
Antibody and Complement Titration for
Rosette Assays . . . . . . . . . . . . . . . . 34
Analysis of Enriched Rabbit Anti-Bovine IgG . . . 36
Fractionation of Bovine Leukocytes . . . . . . . 39
DISCUSSION 0 O O 0 O O O O O O O O O I O O O O O O O 44
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 52

Table

LIST OF TABLES

Wright's stain examination of bovine
leukocytes in zymosen treated and untreated
Wh01e blOOd O I O O O O O O O O O I O O I O O

Turk's stain examination of bovine leukocytes
isolated from Ficoll-Hypaque centrifugation
of zymosan treated and untreated whole blood

Rosette formation by bovine peripheral blood
lymphocytes and sheep erythrocytes sensitized
with variable dilutions of rabbit anti-sheep
erythrocyte serum . . . . . . . . . . . . . .

Rosette formation by bovine peripheral blood
lymphocytes and sheep erythrocytes sensitized
with variable dilutions of mouse serum . . .

Fractionation of 9.04 x 107 bovine leukocytes
at 25°C and flow rate of 0.5 ml per min . . .

Fractionation of 6.27 x 107 bovine leukocytes
at 37°C and flow rate of 0.3 ml per min . . .

vi

Page

32

33

35

37

40

42

Figure

1.

LIST OF FIGURES

Page

Electrophoretic patterns of rabbit

anti—bovine IgG serum (A) showing a high

albumin peak and a relatively low gamma

globulin peak and enriched anti-IgG (B)

showing considerable reduction in non-

gamma globulin protein . . . . . . . . . . . 38

Photomicrograph of two phagocytes with

intracellular zymosan and a rosetting
lymphocyte at 400x . . . . . . . . . . . . . 46

vii

INTRODUCTION

Mammalian peripheral blood lymphocytes appear quite
homogeneous morphologically under light microscopy. However
these cells have been divided into subpopulations based on
the presence of surface immunoglobulin (519) (74), and
surface receptors for immunoglobulin (Ig) (25, 61), and
complement (C) (6, 65). The manner in which these surface
marker defined lymphocyte suprpulations interrelate has
been established quite well for some Species, e.g., human
(29), but not for lymphocytes of other species. One
approach in elucidating these interrelationships involves
the isolation of lymphocyte fractions or suprpulations in
relative purity with respect to one surface marker and sub-
sequent analysis for the presence of the other markers (19).

Recently, assays for the detection of bovine
lymphocyte sIg and receptors for 19 and C (43) have been
developed. The study described herein was undertaken to
develop a lymphocyte immunoabsorbent fractionation column
so that bovine lymphocyte subp0pulations with and without
519 could be obtained. Analysis of these cell isolates for
the presence of receptors for Ig and C would then lend some
insight into how these receptors are distributed amongst

Ig-bearing and non-Ig-bearing lymphocytes.

LITERATURE REVIEW

Lymphocyte Heterogeneity

 

Lymphocytes were first divided into two separate
and distinct groups based upon their development in giyg.
They are the thymus-derived T lymphocytes and bursa- (avian)
or bursa equivalent- (mammalian) derived B lymphocytes (29,
81). Both T and B cells are found in the circulating blood
of birds and mammals. Both also populate specific regions
of the lymph nodes and spleen of man and other mammalian
species (29). B cells reside in the germinal centers of
the lymph nodes and in areas around the splenic vein. T
cells occupy the paracortical areas of the lymph nodes
surrounding the germinal centers as well as the white pulp
of the Spleen and periarterial sheath around the splenic
artery.

B lymphocytes are the precursors of memory and
antibody secreting plasma cells (29, 81). Antibody
production, the central ingredient of humoral immunity,
is essential for normal protection against bacterial
infection, especially encapsulated bacteria, as well
as some immunity to infection of non-bacterial etiology
(29). T lymphocytes are believed to mediate cell-mediated

immune responses such as allograft rejection, graft-versus-

host reactions, and delayed type hypersensitivity (29, 81).
Cellular immunity normally develops in response to viral,
fungal, parasitic, and intracellular bacterial infection
(29). T lymphocytes also play a major role in the early
recognition and processing stages for some antigens

(T dependent antigens) necessary for subsequent B cell
activation and humoral response (29). In addition, T
lymphocyte suprpulations (T helper and T suppressor cells)
are thought to play regulatory roles in both cellular and
humoral immune reactions (29, 61).

Other functional properties attributed to
lymphocytes are thought to be mediated by lymphokines
(23). Most of these lymphocyte produced and secreted
soluble products are well characterized £3 33339 while
their 33 3339 functions remain in question. Lymphokine
production and secretion is generally attributed to T
lymphocyte subpopulations although the well studied
migration inhibition factor (MIF) has been found to
be a product of both T and B lymphocytes (19).

B lymphocytes are identified in peripheral blood
and other tissues as cells with readily detectable 519
(see Lymphocyte Surface Immunoglobulin section) and T
lymphocytes by their ability to bind to heterologous
erythrocytes (see Lymphocyte Receptors for Heterologous

Erythrocytes section). This surface marker defining of

lymphocytes has led to identification of the so-called
undefined lymphocytes (UL's) (25) or null cells (19)

which lack either marker although these cells are probably
either thymic or bursa equivalent in origin (19) or
monocyte-macrophage related (25). Some caution is
warranted in equating marker defined and ontogenetically
defined T and B lymphocytes since some evidence has been
collected suggesting that not all B cells have readily
detectable sIg (l9) and some sIg positive lymphocytes

may bind to heterologous erythrocytes (26).

The subject of the lymphocyte surface has received
considerable attention in recent years. This dynamic and
complex membrane contains a number of cell markers in
addition to 519 and receptors for heterologous erythrocytes,
including receptors for lg (see Lymphocyte Receptors for
Immunoglobulin section) and C (see Lymphocyte Receptors
for Complement section). For a discussion and references
regarding the subject of other cell receptors, including
those for mitogens and hormones, and antigens, including
those of the histocompatibility complex, see Laudoulis

et a1. (52).

Isolation of Peripheral Blood
Lymphocytes

 

By far the most commonly employed method for the

isolation of blood lymphocytes entails centrifugation of

anticoagulated whole blood or buffy coat cells layered
over a mixture of Ficoll and sodium metrizoate (ISOpaque)
(9, 10) or sodium diatrizoate (Hypaque) (32). Originally
described by Boyum (9) in the late 19603 for human blood
samples, the technique has since been adOpted for use with
lymphocytes from other animal species (10). Upon centri-
fugation at 800 to 1200 x g the white cell layer at the
plasma-Ficoll-ISOpaque (Hypaque) interface is removed.
Although the presence of a small percentage of poly-
morphonuclear cells and erythrocytes is not infrequent,
this cell layer consists principally of mononuclear cells
(lymphocytes and monocytes) and platelets.

Removal of contaminating phagocytes has been
accomplished by incubating whole blood or buffy coat
cells with iron carbonyl prior to centrifugation over
gradient (38). Phagocytic ingestion of the iron particles
increases the density of the cells such that they will
sediment to the bottom of the tube upon centrifugation.
Removal of these iron laden leukocytes with a magnet is
also possible. Alternatively, phagocytes may be labeled
with latex (16) so that they may be differentiated from
lymphocytes upon microscopic examination.

A useful modification of the Ficoll-Hypaque
centrifugation procedure is outlined by English and

Anderson (32). These authors have developed a discontinuous

gradient centrifugation technique using two Ficoll-Hypaque
solutions of different concentrations of Hypaque. Solution
1 of specific gravity 1.077 gm/ml is layered over solution 2
with a specific gravity of 1.119 gm/ml. Diluted heparinized
human blood was layered over the gradient. After centri-
fugation three distinct cell layers were observed; layer 1
(plasma-solution 1 interface) consisted of mononuclear cells
and platelets, layer 2 (solution l-solution 2 interface) of
polymorphonuclear cells, and layer 3 of erythrocytes.

Lymphocyte Receptors for Heterologous
Erythrocytes

 

 

Although Thy-1 and Ly antigens allow for rapid and
specific immunological identification of mouse T lymphocytes
(29), no such antigens have been identified for most other
mammalian T cells. The observation that sheep erythrocytes
(SRBC's) (11) as well as other heterologous RBC's (54, 82)
attach to the surface of some human lymphocytes, i.e., form
E rosettes, has led to the development of reliable methods
of T cell detection and enumeration. Evidence that the E
rosette assay detects T lymphocytes is based on a number
of observations. It has been shown that lymphocytes with
detectable sIg do not bind SRBC's to their surfaces (13,
48) with the exception of one study finding that 2-4% of
peripheral blood lymphocytes with sIg bound neuraminidase-

treated SRBC's (26). Mouse studies have shown that the

organ distribution of SRBC binding lymphocytes is inversely
correlated to the presence of cells carrying Ig with the
frequencies of rosette formation occurring at the expected
values (77). Finally, the tissue distribution of E rosette
forming lymphocytes were found to correspond to the
thymus-dependent areas (80).

The E rosette assay is accomplished by incubating
lymphocytes with erythrocytes and subsequent microscopic
examination looking for lymphocytes with attached erythro—
cytes, i.e., rosettes. Procedures for detecting E rosetting
lymphocytes vary considerably making it difficult to compare
the results from one laboratory to another. A number of
erythrocyte treatments have been introduced in order to
minimize the variability and/or enhance the frequency of
rosette formation including treatment with neuraminidase
(26, 33, 54, 83), papain (33), and 2—aminoethylisothio-
uronium bromide (AET) (68, 69). Formation of rosettes
in the presence of fetal calf serum (88) and dextran (8,
13) has also proved to enhance rosetting frequencies.

A modified rapid E rosette test has been described
to identify "active" E rosette forming cells which are
thought to represent a subpopulation of T lymphocytes
(88, 89). The significance of this cell population has
not been established. E rosette assays have been developed
for a number of non-human lymphocytes (8, 33, 83) including

lymphocytes of bovine origin (42, 68, 85).

Lymphocyte Surface Immunoglobulin

 

Although the concept of surface receptors for
antigen had been prOposed as early as 1900 by Ehrlich
(31), it was not until 1961 when Moller found that anti-
mouse Ig serum reacted with some mouse lymphocytes that
the presence of surface-bound Ig was suggested (60). It
is now generally accepted that lymphocytes with readily
detectable sIg are B lymphocytes, a conclusion suggested
by the distribution of sIg—bearing cells in lymphoid tissues
(39). Receptors on T lymphocytes have been demonstrated
(3, 59, 76) but the chemical nature of these receptors
remains unresolved.

Routine procedures in both clinical and research
laboratories for the detection and enumeration of B
lymphocytes usually entails the use of fluorescenated
antisera against polyvalent Ig of the species under inves-
tigation. Antisera labeled with peroxidase (39) can also
demonstrate sIg. Enumeration of B cell suprpulations is
possible with labeled antisera to heavy (class specific)
or lambda and kappa light chains (1, 51, 70, 72, 73). A
recent publication by Winchester et al. (87) recommends
the use of labeled F(ab')2 to avoid staining of lympho-
cytes bearing Fc receptors but lacking 519. Some perform
the staining procedure in the presence of sodium azide to

prevent capping of sIg (51, 70).

Lymphocyte Receptors for
Immunoglobulin

 

 

The subject of lymphocyte receptors for Ig became
an extensive area of research interest in the late 19605
and continues to receive attention today. Receptors for
IgG were the first to be demonstrated and characterized
[see Dickler (25) for a recent review]. Since the obser-
vation of human lymphocyte receptors for IgM in 1975 (62)
the attention has shifted considerably to this subject
[see Moretta et a1. (61) for recent review]. Recent
investigations have demonstrated receptors for IgE on
human B lymphocytes (40) and human and rat T lymphocytes
(90) as well as IgA receptors on human T cells (56).

Early investigations into the nature of lymphocyte
receptors for lg demonstrated that binding is stable only
when Ig is complexed to antigen (5) or in aggregated form
(aggIg) (27). Early studies also showed the binding to be
specific for the Fc fragment of IgG but not IgM (21, 36).
Hence, lymphocytes with receptors for the Fc fragment of
IgG (FcRL's for IgG) are most frequently detected with
labeled aggIgG or erythrocytes sensitized with either
heat inactivated anti-erythrocyte serum or purified IgG
(EA or EAIgG).

The question as to whether the two assays detect
the same receptor or two different receptors has been

raised, since the EA rosette assay generally identifies

10

a lower percentage of cells as FcRL's (29). Depletion of
rosetting cells from a population of lymphocytes incubated
with EAIgG results in a suprpulation of lymphocytes with
very few cells that can bind aggIgG (20) showing that the
EAIgG assay detects a subset of those lymphocytes capable
of binding aggIgG. This observation suggests that the
receptors are the same and that the EA assay is simply
less sensitive. Inhibition experiments also provide
evidence that the same receptor binds both aggIg and
antibody-antigen complexes (24).

Early studies with both mouse and human lymphocytes
suggested that the Fc receptor for IgG was largey if not
exclusively a B cell marker (5, 27). Later reports, how-
ever, show that at least some human T cells also carry
this receptor (12, 21, 36).

FcRL's for IgG have been shown to mediate antibody-
dependent cell-mediated cytotoxicity (ADCC). Incubation
of lymphocytes with aggIgG inhibits this 33 33333 cytolytic
phenomenon (24), whereby antibody coated target cells are
lysed by non-sensitized lymphocytes. These effector cells
are frequently referred to as killer (K) cells (29). ADCC
is in fact another assay for lymphocytes with Fc receptors
for IgG (29).

Receptors for IgM went undetected in all early

studies due to the necessity for overnight incubation of

ll

lymphocytes at 37°C in IgM free medium before EAIgM
rosetting could occur (57, 62). One explanation that
accounts for this incubation requirement is that IgM
receptors are saturated with serum IgM £2_3i3g and that
during 13.33339 incubation new receptors are synthesized
(34).

IgM receptors were first demonstrated on human T
lymphocytes (57, 62) and later on B cells as well (34, 71).
IgM receptors bind to the Fc portion of either monomeric
(14, 75) or pentameric (14, 35) IgM. Moretta has found
that human T FcRL's for IgG (T.G) and T FcRL's for IgM
(T.M) to be separate and distinct cell pOpulations (63).
Moreover, T suppressor activity seems to reside within the
T.G population and T helper activity within the T.M pOpu-
lation suggesting a regulatory role for these receptors.

Considerable evidence has shown that 319 and Fc
receptors for IgG and IgM are structurally independent of
each other. As mentioned above no T cells carry both IgM
and IgG receptors. In addition, IgM receptors on T lympho-
cytes are trypsin sensitive whereas receptors for IgG on T
lymphocytes are trypsin resistant (58). On B lymphocytes,
binding of EAIgM requires the presence of divalent cations
and is not inhibited by preincubation of cells with aggIgG
whereas binding of EAIgG does not require the presence of

divalent cations and is inhibited by preincubation of cells

12

with aggIgG (14, 71). Furthermore, capping of sIg with
anti-Ig serum does not effect the distribution of Fc
receptors for IgG (24) or IgM (71).

Receptors for lg of one class or another are found
on mast cells (22), macrophages (22), monocytes (45, 53),
neutrophils (53), and baSOphilS (46). An EA rosette assay

has been used to study bovine lymphocytes (43).

Lymphocyte Receptors for Complement

 

In 1965 Uhr observed that antibody-antigen-
complement complexes bind to some lymphocytes (84). Since
that observation a considerable amount of research effort
has been directed toward the so-called complement receptor
lymphocyte (CRL). [See Nussenzweig (65) and Bianco and
Nussenzweig (6) for recent reviews.]

Detection of CRL's is usually accomplished by
incubating lymphocytes with erythrocytes sensitized with
antibody and a non-lytic complement source. The complement
source may be non-lytic by nature, e.g., mouse serum, or
diluted to a sublysing titer. Antibody sensitization of
erythrocytes with IgM to avoid antibody binding to Fc
receptors is suggested for human lymphocyte studies (2).
Alternatively, erythrocytes are sensitized with anti-
erythrocyte serum and the data is interpreted with the
understanding that FcRL's may also be detected. Erythro-

cytes that bind poorly to T lymphocytes are preferred to

13

decrease the possibility of E rosette formation with
EAC's (2).

The C receptor is generally considered a B cell
marker although it may be that not all B lymphocytes are
CRL's. Many report that there are fewer CRL's than lympho-
cytes with 519 in peripheral blood (29, 67, 77), although
the EAC rosette assay may not be sensitive enough to detect
all CRL's. CRL's correspond well with B cell ontogeny in
that lymphocytes with C receptors are rare in the thymus
(7, 53, 67) and show the characteristic distribution in
thymus-independent tissue regions (28, 80).

Human lymphocyte EAC studies have shown that CRL's
can bind both C3b and C3d (30). Unlike Fc receptors for
IgG, C receptors are destroyed by trypsin (66). Capping
of sIg has no effect on the distribution of C receptors
(66). C receptors have been demonstrated on red blood
cells (64), macrophages (53), monocytes (53) and gran-
ulocytes (30, 53). An EAC rosette assay has been used
to study bovine lymphocytes (43, 50).

Isolation of Lymphocyte
Subpopulations

 

 

Obtaining lymphocyte subpopulations in relative
purity has contributed considerably to the understanding
of lymphocyte marker interrelationships. There are a

number of approaches used in obtaining such cell isolates.

14

Patients of immunodeficient status have been used as
lymphocyte sources for this purpose. Thus, patients
with X-linked Agammaglobulinemia have no CRL's (37).
A number of £3 33352 manipulations have been
developed for isolating lymphocyte subpopulations.
These procedures can be divided into two types.

1. There are methods which deplete or exclude a
defined set of lymphocytes from the original
lymphocyte population. The remaining cells are
generally defined in the negative context, e.g.,
Fc receptor negative lymphocytes.

2. Lymphocyte fractionation results in two or more
subpopulations, defining one from another according

to some lymphocyte characteristic.

Selection of a lymphocyte separation technique
depends to some degree upon the nature of the investigation.
Factors to consider include: the time, cost and assessabile
ity of supplies necessary to perform the separation; the
purity, viability, and cell recovery that can be expected;
and the number of cells which can be processed.

Julius et a1. (49) have described a now rather
popular method for T lymphocyte isolation involving the
incubation of lymphocytes in nylon wool. Subsequent elution
of non-adhering cells results in a cell isolate with only

5% cells with sIg. Recovery of T lymphocytes ranged from

15

50-90%. The basis for retention of Ig-bearing lymphocytes
with nylon wool is not known and selective loss of T cell
subpopulations is possible.

Rosette depletion has been accomplished with E
(18, 44, 78), BA (20, 67), and EAC (7, 18, 67) rosette
forming cells. After lymphocytes are allowed to form
rosettes, they are centrifuged over a gradient, e.g.,
Ficoll-Hypaque, allowing non-rosetting lymphocytes to
be recovered. The success of this technique depends to
a major extent upon the stability of the rosettes formed.
When rosettes are stable, however, purity is generally
very good and large numbers of cells can be processed.
Saxon et a1. (78) have recently described an E rosette
depletion technique allowing for the isolation of the
rosetting lymphocytes as well as the non-rosetting lympho—
cytes by lysing the RBC's of the rosetting cells after
centrifugation.

Gutierrez et al. (41) have described a discontinuous
density gradient centrifugation procedure which allows for
very good recovery of highly purified sIg negative lympho-
cytes, however the B cell subpopulations are of relatively
poor purity (72.8% with detectable sIg). This one step
method is very simple and rapid and allows for large

numbers of cells to be processed.

16

Methods offering the greatest success for obtaining
highly pure lymphocyte subpopulations involve the separation
of cells with an immunoabsorbent column. Such columns
involve the binding of antigen specific antibody to column
matrix. Upon application of cells to the column, antigen
positive cells are retained while antigen negative cells
can be eluted.

Wigzell et a1. (86) first introduced such a
column for the isolation of sIg negative mouse lymphocytes
by utilizing what they refer to as the "double layer
principle." Plastic beads are first coated with mouse
immunoglobulin. The antigen coated beads are then labeled
with excess anti-Ig so that free antigen binding sites on
the antibody molecules are available to interact with Ig—
bearing lymphocytes. Although some non-specific retention
of cells was noted, only 0.8% of those lymphocytes passing
through the column were sIg positive. No attempt was made
to recover bound cells.

Campbell and Grey (15) introduced a similar column
also for mouse lymphocytes. This column, however, was made
by binding anti-gamma globulin to plastic beads directly.
Upon serial passage of lymphocytes through four columns
only 0.2% were 519 positive although cell recovery was

poor. Again, there was no attempt to recover bound cells.

17

Schlossman and Hudson introduced the first column
allowing for the recovery of retained cells (79). This
time, however, anti-serum was made to F(ab')2 and the
antibody fraction of the serum purified. The purified
anti-F(ab')2 was bound to dextran gel beads (Sephadesz-ZOO).
Retained mouse lymphocytes were eluted by digesting the
beads with dextranase. Non-specific retention of cells
was reduced considerably presumably due to the use of
purified antibody. Dextranase eluted cells were difficult
to analyze since they were covered with anti-F(ab')2 but
unbound lymphocytes had only 3-6% cells with 319. A
similar column for guinea pig lymphocytes allowed for the
demonstration of transfer of delayed type hypersensitivity
(DTH) from immune to non-immune animals with sIg negative
lymphocytes (47). AAttempts to determine whether the
dextranase eluted cells could also transfer DTH failed
due to an inflammatory reaction presumably caused by
undigested dextran residue.

With only minor modification a similar immuno-
absorbent column was developed for human lymphocytes (17).
Again, purified anti-F(ab')2 was bound to Sephadex G-200.
Elution of retained cells, however, was accomplished by
competitive inhibition with human gamma globulin. Data
from ten lymphocyte fractionations resulted in unretained

cells with an average of less than 2% of the cells positive

18

for sIg. Gamma globulin eluted cells averaged 95.2%
cells positive for sIg. Viabilities were greater than
95% for all populations.

A lymphocyte immunoabsorbent fractionation column
is somewhat time consuming to construct and operate but
requires no specialized equipment and allows for the

processing of a wide range of cell numbers.

MATERIALS AND METHODS

Animals

Adult New Zealand female rabbits (Spartan Research
Animals, Inc., Haslett, Michigan) were immunized for the
production of anti-bovine IgG.

Adult ICR female mice (Harlan Industries, Inc.,
Cumberland, Indiana) were used as a source of complement
for EAC rosette studies.

Holstein heifers, used as a source of blood for
leukocyte studies, were housed at the Large Animal Clinic
at Michigan State University. All bovine data appearing
in this report were collected using leukocytes from animal

#223629, age 2, in good health according to clinic records.

Immunization

 

Rabbits were immunized by injecting 2 mg of bovine

IgG (Miles Laboratories, Inc., Elkhart, Indiana) intra-
muscularly in Freund's complete adjuvant (Difco Labora-
tories, Detroit, Michigan) and one week later the same
dosage was injected subcutaneously in sterile saline.
Rabbits were bled 8 weeks after the last injection.
Sufficient humoral response to the immunization was
determined when a visible precipitin band was observed

using Ouchterlony gel diffusion.

l9

20

Collection and Treatment of Sera

 

Rabbit blood, obtained from the marginal ear vein,
was allowed to clot for 2 to 3 hr and spun at 1000 x g for
30 min at 4°C. The anti-bovine IgG serum was harvested,
pooled and stored at -20°C.

Mouse blood was obtained from the subclavian artery,
allowed to clot for 30 min in an ice bath and centrifuged
at 3000 x g for 15 min at 4°C. The serum was harvested,
pooled and absorbed against an equal volume of washed
SRBC's for 45 min at 0°C. Absorbed serum was harvested
by centrifugation at 3000 x g for 15 min at 4°C and stored
in 0.2 ml aliquots at -70°C.

Rabbit anti-SRBC serum, used in all EA and EAC
rosette studies, was obtained from Dr. E. Sanders, Michigan
State University. The antiserum was heat inactivated at

56°C for 30 min and stored in 0.5 ml aliquots at -20°C.

Isolation of Bovine Leukocytes

 

Heifers were bled from the caudal vein using 20 ml
vacutainer tubes (Becton-Dickinson, Rutherford, New Jersey)
containing sodium heparin. Blood leukocytes were isolated
using the Ficoll—Hypaque centrifugation method described by
Boyum (10) with slight modification. Warm blood, diluted
1:2 with EDTA-phosphate buffered saline (EPS) was layered
over 3 ml Ficoll-Hypaque and spun at 1100 x g for 30 min

at room temperature. The white cell layer at the

21

Ficoll-Hypaque—supernatant interface was removed with

a Pasteur pipette and washed three times with Dulbecco's
phosphate saline (PBS) (Grand Island Biological Co., Grand
Island, New York) recovering cells by centrifugation at
500 x g.

An alternate leukocyte isolation procedure enabling
rapid differentiation between phagocytic and non-phagocytic
cells was established. Twenty-five milligrams of washed
(PBS) zymosan A (Sigma Chemical Co., St. Louis, Missouri)
in 2 ml Hank's balanced salt solution (HBSS) (Grand Island
Biological Co.) were added to 10 ml whole blood and incu-
bated for 30 min with end-over-end mixing at 37°C. The
zymosan-blood mixture was diluted 1:2 with EPS and leuko-
cytes isolated as described above. Cells with intracellular
zymosan were scored as phagocytes.

A comparison study involving the two leukocyte
isolation procedures was conducted in order to establish
zymosan treatment as an adequate method for the identifi-
cation of phagocytic cells. Differentials were performed
on leukocytes in whole blood with Wright's stain and
Ficoll-Hypaque isolated white cells with Turk's stain
from zymosan treated and untreated blood. Each luekocyte
isolation procedure was performed in triplicate and cell
differentials were determined by counting 200 cells

microscopically. The zymosan-Ficoll-Hypaque isolation

22

procedure was adopted for use in subsequent bovine

leukocyte fractionation studies.

Enrichment of Rabbit Anti-Bovine
IgG Antibody

 

 

Antibody from anti-bovine IgG serum was enriched
with an insoluble immunoabsorbent of bovine gamma globulin
(Miles Laboratories) covalently bound to cyanogen bromide
activated Sepharose 4B(Pharmacia Fine Chemicals, Piscataway,
New Jersey). The procedure used is a modification of that
outlined by Chess and Schlossman (18). Thirty milligrams

of CNBr activated sepharose 4B, swollen in 10-3

M HCl, were
mixed with 200 mg bovine gamma globulin, dissolved in borate
buffered saline (pH 8.3) (coupling buffer) and gently mixed
for 2 hr at room temperature. The resulting protein-bead
complex was washed with coupling buffer to remove unbound
protein and resuspended in 0.5 M Tris-HCl (blocking reagent)
for 2 hr at room temperature to block remaining active
protein binding sites. Excess blocking reagent was washed
out with three rounds of 0.1 M acetate buffer followed by
coupling buffer before a final wash with several volumes

of PBS. Antiserum (100 ml) was thawed to room temperature
and slowly passed through 10 ml of immunoabsorbent packed
in a l x 20 cm glass column. Unbound serum components

were eluted with PBS, and bound antibody with 0.1 M

glycine-HCl (pH 2.5) collected in 2.0 M phosphate buffer

23

(pH 8.0). The latter eluate was repeatedly 1y0philized
and dialyzed against coupling buffer to achieve a final
concentration of 10 mg protein per ml and stored at -70°C.
Protein determinations were performed as described by
Lowry (55).

Analysis of Enriched Rabbit
Anti-Bovine IgG

 

 

Rabbit anti-bovine IgG serum, enriched antibody,
and normal human control serum were examined by electro-
phoresis to assess the effectiveness of the purification
procedure. Fractionation of samples was performed on
60 x 75 mm Titan III cellulose acetate plates (Helena
Laboratories, Beaumont, Texas) containing eight strips
per plate. Plates were soaked in Electra HR buffer (Helena
Laboratories) of pH 8.6 and ionic strength 0.05 and 5.5 ul
of sample applied to each strip. A constant current of
6 ma was applied to each plate (0.78 ma per strip) in
Electra HR buffer for 15 min. Samples were stained with
Ponceau S (Gelman Instrument Co., Ann Arbor, Michigan),
dried, and read with a Gelman ACD-l8 automatic computing
densitometer (Gelman Instrument Co.) at a wavelength of
500 nm. All samples were run in duplicate.

Precipitin activity of enriched antibody, diluted
to the original serum volume, was compared with the activity
of undiluted immune rabbit serum and a normal rabbit serum

control using Ouchterlony gel diffusion.

24

Preparation of Cell Fractionation
Immunoabsorbent Columns

 

 

The procedure for preparation of cell fractionation
columns outlined by Chess and Schlossman (18) will be
described briefly. Swelled Sephadex G-200 (60 m1)
(Pharmacia Fine Chemicals) was activated with 100 mg
of cyanogen bromide (Eastman Kodak Company, Rochester,
New York), maintaining a constant pH of 10.2 with l N
NaOH, and a constant temperature of 20°C with ice, with
gentle mixing for 1 hr. The activated Sephadex, washed
in coupling buffer was mixed with 20 mg of enriched anti-
IgG antibody and incubated for 4 hr at room temperature.
The Sephadex G-200—antibody conjugate was transferred to
a sintered glass funnel and washed with cold coupling
buffer. The resulting immunoabsorbent was stored in PBS
containing 0.1% sodium azide (Eastman Kodak Company) at
4°C. Disposable 10 ml syringes (Becton-Dickinson) fitted
with 35 mm pore polyethylene discs (Bell Art Products,
Benawalk, New Jersey) were packed with 9 ml of immuno-
absorbent and washed with medium 199 containing 5% fetal
calf serum and 2.5 mM EDTA (cell medium) before use in

cell fractionation.

Cell Preparation and Fractionation

 

The procedure used is a modification of the methods

described by Chess and Schlossman (18). Briefly, bovine

25

leukocytes isolated by the zymosan-Ficoll-Hypaque procedure
were washed three times with cell medium and concentrated
to l x 107 cells per ml in cell medium before application
to fractionation columns. Lymphocyte suspension (5-10 ml)
was applied to columns, run at a flow rate of 0.3 to 0.5 ml
per min. Unbound cells were eluted with 45-90 m1 of cell
medium and retained cells with 45 m1 of cell medium con-
taining 10 mg bovine gamma globulin per ml. During the
elution of bound cells the column material was gently mixed
with a Pasteur pipette. The recovered cell populations were
then washed with cell medium before analysis. A retained
portion of unfractionated cells was also analyzed. All

cell populations were analyzed at the same time.

Rosetting Assays

 

EA and EAC rosette procedures were performed
according to the methods of Higgins and Stack (43) with
slight modification. Washed SRBC's from whole blood in
Alsever's solution (Colorado Serum Company, Laboratories,
Denver, Colorado) were suspended to 5% in complement fixa-
tion buffer (CFB) and incubated with an equal volume of
anti-SRBC serum for 30 min at 37°C. The antibody sensitized
erythrocytes (EA's) were washed three times with CFB and
resuspended to 5% in CFB with 1% gelatin (CFBG). A portion
of the EA suspension was incubated with an equal volume of

mouse serum for 30 min at 37°C for complement sensitization.

26

The resulting antibody, complement sensitized erythrocytes
(EAC's) and the retained portion of the EA's were washed
three times in CFBG and resuspended to l x 108 cells per ml.

6 cells

A 0.15 ml portion of leukocyte suspension at 2 x 10
per ml was mixed with an equal volume of sensitized erythro-
cytes and incubated for 15 min at 37°C. The cell mixture
was then spun at 200 x g for 5 min and reincubated for 16
to 18 hr in an ice bath. One drOp of 1:5 diluted crystal
violet was added to each tube and the cells gently resus-
pended. Lymphocytes, examined under phase microscopy, with
three or more erythrocytes attached were scored as rosettes.
At least 200 cells were counted from each tube. Each test
was run in triplicate.

Optimal antibody and complement titers were deter-
mined by testing several dilutions of each and finding
those dilutions resulting in the highest frequency of
rosetting cells. E, EC, and EA controls were included

in all titration studies. E controls were included with

all leukocyte fractionation studies.

Detection of Surface Immunoglobulin

 

Surface immunoglobulin was detected with FITC
conjugated IgG fraction of rabbit anti-bovine gamma
globulin (Cappel Laboratories, Inc., Cochranville,
Pennsylvania). A 0.1 ml portion of leukocyte suspension

in PBS with 0.1% sodium azide at a concentration of 2 x 106

27

cells per ml was incubated with an equal volume of
fluorescein-antibody conjugate for 30 min at 37°C and
washed three times with PBS with 0.1% sodium azide.
Fluorescent activity was studied with a Zeiss fluorosc0pe
equipped with a dark field condenser and an Osram HBO 200
mercury lamp using exicter filter BGS and barrier filters
47 and -65. For each test 200 cells were counted and
scored.

Enumeration of Phagocytic Cells
in Fractionation Studies

 

 

Enumeration of phagocytes in fractionation studies
was determined by scoring 200 cells for intracellular

zymosan using leukocytes in the E controls.

Viabilit33Assay

 

Viabilities were determined with trypan blue (Grand
Island Biological Company). A 0.1 ml portion of leukocyte
suspension at a concentration of 2 x 106 cells per ml in
HBSS were incubated with 0.05 ml of 1:5 diluted trypan blue
for 15 min at 37°C. Two hundred cells were scored in each
test. Viabilities of greater than 90% were achieved in

all studies unless otherwise specified.

Photography

 

The photomicrograph appearing in this report was
taken with Tri-X-pan Kodak film at an exposure time of

1/8 sec using a Zeiss microscope.

28

Statistics

 

All tests run in triplicate are expressed in terms
of mean percent 1 standard deviation (E i S.D.). The
student's t-test was used to evaluate results when

indicated.

Preparation of Reagents

 

- Ficoll-Hypaque density gradient
72.8 gm Ficoll 400 (Pharmacia Fine Chemicals)
480 ml Hypaque (sodium diatrizoate)
25% aqueous solution (Winthrop
Laboratories, New York, New York)
720 ml Distilled water.
The specific gravity was determined with a hydro-
meter (Arthur Thomas Company, Philadelphia,
Pennsylvania) and always found to be between
1.076 and 1.079. The solution was filter sterilized

with a 0.2 pm Nalgene filter unit (Sybron Corpora-

tion, Rochester, New York) and stored at 4°C.

. EDTA-phosphate buffered saline (EPS)

3.58 gm EDTA-trisodium salt (Sigma Chemical
Company)

9.00 gm NaCl
1.08 gm KH2P04
1.00 K Distilled water.
The solution was filter sterilized (0.2 um) and

stored at 4°C. The pH was adjusted to 7.2 to 7.4

before use.

29

- Complement fixation buffer (with 0.1% gelatin)
(CFB and CFBG)
83.00 gm NaCl
10.19 gm Sodium 5,5-diethyl barbituric acid
34.60 ml 1N HCl

5.00 ml Stock solution containing 1 M MgCl

and 0.3 M CaClz.

The above reagents were dissolved in distilled water

2

and adjusted to 2 Z. The solution was filter
sterilized (0.2 pm) and stored at 4°C. CFB, made
by diluting a portion of the above stock CFB 1:5
with distilled water, was stored for no longer

than 24 hr at 4°C. The pH was determined to be

7.3 to 7.4 before use. CFBG was made by dissolving
0.25 gm gelatin (General Foods Corporation, White
Plains, New York) in 200 ml distilled water. The
gelatin solution was mixed with 50 ml of stock CFB.
CFBG was stored for no longer than 24 hr at 4°C.

The pH was determined to be 7.3 to 7.4 before use.

0 Cell medium
1 pkg Medium 199 powder with Hank's salts
and L-glutamine (Grand Island Bio-
logical Company)

50 ml Fetal calf serum (Microbiological
Associates Inc., Bethesda, Maryland)

0.73 gm EDTA-trisodium salt

10 ml 200 mM L—glutamine (100x)
(Microbiological Associates)

3O

10 ml Penicillin (10,000 units per ml),
streptomycin (10,000 pg per ml)
(Grand Island Biological Company)
Cell medium reagents were diluted to l K with
distilled water, aliquoted into 100 m1 portions,
and stored at -20°C. Medium was thawed to room

temperature and the pH adjusted to 7.2 to 7.4

before use.

Turk's stain
5 ml Acetic acid
5 ml 1% Crystal violet
490 ml Distilled water.
Reagents were mixed and stored at room temperature.

Leukocytes at a concentration of 2 x 106

cells per
ml were mixed with an equal volume of stain. A

wet mount was made and examined microsc0pically.

RESULTS

Detection of Phagocytic
Leukocytes with Zymosan

 

 

Wright's stain differential examination of zymosan
treated and untreated whole blood stained prior to Ficoll-
Hypaque centrifugation resulted in cell types of comparable
frequencies (Table l). Basophils were seen in lowest fre-
quency; 0.2% for both zymosan treated and untreated blood.
Only minor variations in frequencies were seen with the
remaining cell types. All phagocytic leukocytes, with the
exception of baSOphilS, injested zymosan to some degree,
i.e., 90.0% of the monocytes, 79.0% of the neutrOphils,
and 86% of the eosinophils were seen with intracellular
zymosan. Extracellular zymosan was seen throughout the
blood smear.

Turk's stain examination of leukocytes, recovered
from Ficoll-Hypaque centrifugation, of the same blood sam—
ples yielded 15.3% phagocytes when blood was preincubated
with zymosan as compared to 11.3% for untreated blood
(Table 2). Of the phagocytes counted, 92.8% were seen
with intracellular zymosan. Extracellular zymosan was
only rarely seen. Leukocytes were greater than 90%

viable for all three untreated blood samples.

31

32

 

 

 

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o.o o.o m.oufl~.o m.o H~.o Haemommm
o.om H.m Hm.sa m.Hnfls.om ~.H Hm.m~ Harmocflmom
o.ms m.auflm.~m hm.H.nm.Hv n~.m.flo.em Handouusmz
0.0m o.Hnfln.m m.onflo.m m.o H5.m oumoocoz
.. o.o ~.H”fls.am m.einm.mm mumooemssq
mammoemu ammoewu mmuwooxsoa mmuhooxsma
umHsHHoomuucH umHsHHoomuucH ucooHom unmouom
sua3 mouwoommnm nuflz moumooxsoa
unmouom ucoouom cooHn
couwmuuca

 

UOOHQ poumouu :mmOE>N

 

 

vocab mH0£3 omumouucs
can Umummuu cmmoemu CH mmumooxsma mcfl>on mo :OHDMCHmem cflmgm m.u£mHH3

.H manna

33

Table 2. Turk's stain examination of bovine leukocytes
isolated from Ficoll-Hypaque centrifugation of
zymosan treated and untreated whole blood

 

 

 

 

Zymosan
Untreated treated
blood blood

Percent phagocytes ll.3i:2.3 15.3: 1.6
Percent leukocytes with
intracellular zymosan -- 14.2: 1.5
Percent phagocytes with
intracellular zymosana -- 92.8
Percent leukocytes b
viable 94.0::2.0 NT
aValue = (§% leukocytes with intracellular zymosan % §%
phagocytes) X 100.
b

NT = Not tested.

34

Antibody and Complement Titration
for Rosette Assays

 

 

An antibody titration study was performed so
that the Optimal anti-SRBC dilution, i.e., that dilution
resulting in the greatest frequency of rosette forming
cells, could be ascertained. The results are shown in
Table 3. Several dilutions between 1:50 and 1:1600 of
antiserum were used to sensitize sheep erythrocytes for
EA and EAC rosette studies. All EAC tests and an EC
control were performed with complement (mouse serum)
diluted 1:20. Dilutions of 1:50 and 1:100 resulted in
agglutination of erythrocytes after the 30 min incubation
making the assay difficult or impossible to read. The
lowest dilution without visible microscopic erythrocyte
agglutination (1:200) resulted in the highest mean fre-
quencies of EA and EAC rosette formation (31.0% and 41.5%,
respectively) and was chosen as the optimal antibody titer
for all subsequent EA and EAC assays. Rosette formation
decreased with increasing dilution of antiserum for both EA
and EAC assays. EAC rosette frequencies were consistently
higher than EA frequencies of the same antibody titer. An
EC control resulted in more cells forming rosettes than did
unsensitized erythrocytes but fewer cells forming rosettes
than all EA and EAC tests performed.

A similar study to assess the optimal complement

titer for EAC rosette studies was conducted. The results

35

Table 3. Rosette formation by bovine peripheral blood
lymphocytes and sheep erythrocytes sensitized
with variable dilutions of rabbit anti-sheep
erythrocyte serum

 

 

Percent rosettes with diluted
mouse serum and no serum

 

Rabbit antiserum

 

dilution 1:20 No serum
1:50 Agga Agg
1:100 Agg Agg
1:200 41.5: 3.1 31.0::5.6
1:400 32.5: 3.5 27.5::2.0
1:800 ll.5::l.3 ‘ 6.0: 1.0
1:1600 9.0: 1.0 3.52:2.1
No antiserum 3.0: 0.5 0.3: 0.3

 

aAgg = Agglutination.

36

are shown in Table 4. A11 EAC assays and an EA control
were performed with anti-SRBC serum diluted 1:200. Incu-
bation of antibody sensitized erythrocytes with undiluted
mouse serum resulted in packed erythrocytes upon even gentle
washing. The packed erythrocytes required prolonged mixing
before the cells could be resuspended making undiluted mouse
serum an impractical source of complement. The first mouse
serum dilution (1:5) minimized this packing phenomenon con-
siderably and resulted in the highest mean frequency of EAC
rosettes of all dilutions studied. EAC rosette formation
decreased with increasing complement dilution. EAC fre-
quencies were consistently higher than EC controls of the
same complement titer. An EA control resulted in fewer
cells forming rosettes than all EAC tests and more cells
forming rosettes than that seen with the E control. Unsen-
sitized erythrocytes resulted in the lowest mean frequency
of rosette formation with the exception of erythrocytes
sensitized with mouse serum diluted 1:20. Mouse serum
diluted 1:5 was chosen as the most appropriate complement
titer for the EAC rosette assay and was used in all
subsequent EAC rosette studies.

Analysis of Enriched Rabbit
Anti-Bovine IgG

 

 

Electr0phoretic analysis of immune rabbit serum
and enriched anti-bovine IgG antibody (Figure 1) resulted

in a demonstrated reduction in non-gamma globulin protein

37

Table 4. Rosette formation by bovine peripheral blood

lymphocytes and sheep erythrocytes sensitized
with variable dilutions of mouse serum

 

Percent rosettes with diluted
rabbit antiserum and no antiserum
Mouse serum

 

 

 

dilution 1:200 No antiserum
Undiluted EPa NTb
1:5 42.8:.6.5 2.31:0.3
1:10 42.0::4.3 1.31:0.6
1:20 37.81.l.5 0.81:0.6
1:40 32.8:2.3 l.2:0.3
1:80 31.71 4.5 1.31 0.8
No serum 30.2: 3.6 1.01:0.5
aEP = Erythrocyte packing.
bNT = Not tested.

38

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

r
I:
i
i

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

“‘4

 

 

f

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure l.

 

 

 

[F7] “~T::aua-J:£— ——-

Electrophoretic patterns of rabbit anti-bovine
196 serum (A) showing a high albumin peak (far
right) and a relatively low gamma globulin peak
(far left) and enriched anti-IgG (B) showing
considerable reduction in non-gamma globulin
protein.

39

as a result of the enrichment technique. Gamma globulin
protein accounted for 13.22% of the total protein of immune
serum and 82.79% of the purified product according to
densitometer computations. The highest contaminating
protein of the enriched product was albumin (8.18%).
Ouchterlony gel diffusion of immune serum and enriched
antibody against IgG showed a retention of precipitin

activity following antibody enrichment.

Fractionation of Bovine Leukocytes

 

Bovine leukocyte fractionation studies were
performed with leukocytes isolated from zymosan treated
peripheral whole blood. The number of cells recovered
from cell medium and cell medium containing bovine gamma
globulin elution reflects counts of both leukocytes with
intracellular zymosan, henceforth referred to as phagocytes,
and leukocytes without intracellular zymosan, henceforth
referred to as lymphocytes. Viabilities, surface immuno-
globulin, and rosetting assays are reported as percentages
determined from lymphocyte counts.

The results of the first fractionation experiment
are shown in Table 5. The column was adjusted to a flow
rate 0.5 ml per min and 9.04 x 107 leukocytes were applied.
The entire procedure was performed at room temperature.

Of the leukocytes applied to the column 58.6 were recovered.

Elution with cell medium resulted in a decrease in the

40

Table 5. Fractionation of 9.04 x 107 bovine leukocytes at
25°C and flow rate of 0.5 ml per min

 

 

Leukocytes eluted with

 

Cell medium
with bovine

 

 

Unfractionated Cell medium gamma globulin
leukocytes (45 ml) (45 m1)

NRa x 106 -- 49.6 3.4

% vb 75.5 89.0 76.3

% z+C 9.0 0.5 11.5

% sIgd 23.0 7.0 44.0

% EACe 14.2:t 11.21:6.8 28.31 3.3

% EAf 8.3:2.8 8.2i0.6 13.7:2.5
:rfl m3: fl m3t0£ m7i03

a

NR = Number of leukocytes recovered.
% V = Percent lymphocytes viable.

% 2+ = Percent leukocytes with intracellular zymosan.

% sIg Percent lymphocytes with surface immunoglobulin.

% EAC Percent lymphocytes forming EAC rosettes.
% EA = Percent lymphocytes forming EA rosettes.

9% E = Percent lymphocytes forming E rosettes.

41

number of lymphocytes with surface immunoglobulin to 7.0%
as compared to 23.0% seen in unfractionated cells. Elution
with cell medium containing gamma globulin resulted in a
cell population with 44.0% of the lymphocytes containing
surface immunoglobulin. Phagocytes were markedly reduced
from precolumn cells (9.0%) to 0.5% in cell medium eluted
cells and enhanced to 11.5% of the gamma globulin eluted
cells. Viabilities ranged from 75.5% (unfractionated cells)
to 89.0% (cell medium eluted cells). EAC and EA rosette
formation increased with increasing percentages of lympho-
cytes with surface immunoglobulin.

The results of the second leukocyte fractionation
experiment are shown in Table 6. Several changes were made
in the Operation of this column, including: a reduction in
the number of cells applied from 9.04 to 6.27 x lo7 leuko-
cytes; operation of the column at 37°C instead of 25°C; a
decrease in flow rate from 0.5 to 0.3 ml per min. In
addition, the cell medium elution was divided into 30 m1
portions. Cells recovered from the first 30 ml portion,
as well as unfractionated and gamma globulin eluted cells,
were analyzed as in the previous study. Due to the low
recovery of cells from the second and third 30 ml portions
of cell medium, only viability and surface immunoglobulin
determinations were made. Because of the prolonged duration

of operation of this column, recovered and retained cells

42

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.mouuomou mm mcwfinom moumoonmsma unmouom H mm m
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.cflasnonocaEEfl oomwusm apex moumoonmewa unmoumm n mHm w
.cmmOE>N Hoasaamomuucfl nufl3 mmuwooxsoa unwouom u +N w
.OHQMH> mmumoosm8>a unmouom n > w

.cono>ooou mwuaooxsma mo Honasz n mz

 

 

 

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e.em o.e~ o.e 0.5 o.e~ emHm e
e.m 92 :92 o.e o.o 0+N w
e.em m.em e.mm o.am m.em n> e
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m m
as me He em as on He om mwumooxsma
Ghana pcooom umnflm Umumcofluomnmco

 

cflasnoam mEEmm
oafl>on SDHB Esflcoe Haou
EsHUoE HHoU

 

nuHB copuao nonwooxsoq

 

 

CHE you as m.o

mo mung 30am cam Uohm um mouwooxsoa ocH>on boa x >~.w mo :oflumcofluomum .m OHQMB

43

were periodically washed and resuspended in fresh cell
medium while waiting for the remaining cells to elute from
the column. All cells were kept at 37°C until analyzed.
Although this column took considerably longer to
run cell Viabilities improved (greater than 80% for all
fractions). Cell recovery improved slightly (67.2%).
Nearly all of the recovered leukocytes from cell medium
were recovered from the first 30 ml. Phagocytes were
observed only in the gamma globulin eluate fraction (3.0%).
The first 30 ml of cell medium elution yielded a population
of cells with 7.0% of the lymphocytes containing surface
immunoglobulin as compared to 26.0% seen in unfractionated
lymphocytes. Eighty percent of the lymphocytes eluted with
gamma globulin contained surface immunoglobulin. EAC and
EA rosette formation increased with increasing percentages

of lymphocytes with surface immunoglobulin.

DISCUSSION

The isolation of bovine lymphocytes by gradient
centrifugation of peripheral blood results in contamination
with phagocytes as has been seen with the isolation of
human lymphocytes using the same method (10). The con-
taminants are largely monocytes and on the average account
for approximately 20% of the cells isolated when bovine
leukocytes are isolated from normal animals (68). Monocytes
are sometimes difficult to distinguish from lymphocytes
using cell morphology alone. An investigation of lympho-
cytes should then include some means of phagocyte removal
or make differentiation of these cells more apparent by
some means of phagocyte detection. This is generally
accomplished in human studies by removal with iron carbonyl
(38) or detection with latex (16).

Several attempts to remove bovine phagocytes with
iron carbonyl failed despite previous reports of success
with this technique (43, 85). The discrepancy in results
may be due to the source of iron particles used in this
study. Microsc0pic examination of leukocytes incubated
with iron carbonyl prior to gradient centrifugation showed
no phagocytes with intracellular iron although most of the

particles were quite large as compared to the average size

44

45

of the leukocytes. Smaller particles may allow for
phagocyte removal with iron.

Attempts to detect bovine phagocytes with latex
gave ambiguous results although Paul et a1. (68) have
since reported success with this technique. The problem
encountered in this laboratory with respect to this tech—
nique was the frequent inability to distinguish between
surface adherence and actual ingestion of latex.

The problem of phagocyte removal or detection was
overcome by treating freshly drawn blood with zymosan.
Examination of blood immediately following zymosan treatment
showed that 90% of the monocytes ingested the particles
(Table 1). Slightly lower percentages were seen with
neutrOphil and eosinophil populations although the principle
contaminating phagocytes after centrifugation are mono-
nuclear cells. Phagocytes accounted for 15.3% of the cells
isolated upon centrifugation (Table 2). Of the phagocytes
encountered in this cell isolate, 92.8% were seen with
intracellular zymosan. Thus only 1.1% of the leukocytes
counted were phagocytic without intracellular zymosan.
Zymosan tagging of phagocytes proved successful for this
study in that rosetting assays and fluorescent antibody
test for sIg could be performed allowing for data collection
with respect to lymphocytes only (Figure 2).

The results of the antibody (Table 3) and complement

(Table 4) titration studies showed that anti-erythrocyte

46

 

 

 

Figure 2. Photomicrograph of two phagocytes with
intracellular zymosan and a rosetting
lymphocyte at 400x.

47

serum diluted 1:200 and mouse serum diluted 1:20 resulted

in the greatest frequency of EAC rosette formation. Both
EA and EAC rosetting frequencies increased with decreasing
dilution of antiserum. Similarly, EAC rosetting frequencies
increased with decreasing dilutions of mouse serum. These
results differ somewhat from those of Higgins and Stack (43)
where EAC rosetting frequencies reached a peak value and
then declined substantially with decreasing dilutions of
both antibody and complement.

From a theoretical standpoint one would expect to
see increasing rosetting cells with decreasing antibody and
complement dilutions since the erythrocyte surface would
accumulate an increasing amount of antibody and complement.
With more antibody and complement on the erythrocyte surface
the EAC assay should become more sensitive in detecting 19
and C lymphocyte receptors. It would be the hope, with any
titration such as this, that a plateau would be reached.

If such a plateau is observed, dilutions well into the
plateau can be used and thereby assure that the assay is
operating at its maximum capacity. No such plateau was
demonstrated with respect to the titrations performed here.
This would suggest that both the EA and EAC assays, as used
in this study, are not sensitive enough to detect all
lymphocytes with 19 or C receptors.

EC controls performed during the complement titra-

tion study resulted in low rosetting frequencies for all

48

mouse serum dilutions (range 0.8 to 2.3%). Little
difference is seen from that obtained from the E control
(1.0%). Since EC's are not sensitized with antibody,
activation of complement is unlikely as is adherence of
complement components to the erythrocyte surface. It is
logical to assume then that EC rosetting is due to the
erythrocyte receptors on lymphocytes.

Sheep erythrocytes prove to be an appropriate
choice for performing EA and EAC assays since E rosetting
is minimal. Thus, most of the EA rosettes should be due
to the presence of lymphocyte Ig receptors. Some rosettes
formed with EAC's may be due to the presence of lymphocyte
Ig receptors as well as C receptors. This is supported by
the observation that EAC rosetting frequencies are always
higher than EA rosetting frequencies.

Results from the first fractionation experiment
(Table 5) show that passage of bovine lymphocytes through
an immunoabsorbent column allows for the isolation of two
lymphocyte subpopulations: one enriched in Ig-bearing cells
and the other enriched in non-Ig-bearing cells. Lymphocytes
isolated from cell medium elution resulted in a lymphocyte
subpopulation with only 7.0% of the lymphocytes positive
for $19. Lymphocytes isolated due to elution with cell
medium containing bovine gamma globulin resulted in an

enrichment of sIg positive lymphocytes to 44.0%. This

49

experiment also shows that both EA and EAC rosetting
frequencies increase with an increase in lymphocytes with
sIg. This observation is in agreement with that reported
by others where both EA (43) and EAC (43, 50) rosetting
frequencies decreased when these assays were performed

on bovine lymphocytes passed through nylon wool.

In hopes that a more effective separation of sIg
positive and sIg negative lymphocytes could be accomplished,
a second fractionation study was undertaken involving
several changes. Contamination of Ig-bearing lymphocytes
in the cell medium eluate of the first column may have been
due to the presence of too many sIg positive cells for the
column to retain. Thus the number of cells applied to the
second column was reduced to almost two-thirds of that
applied to the first column. Furthermore, it was believed
that by reducing the flow rate from 0.5 to 0.3 ml per min
more time would be allowed for column antibody to react
with 319 and possibly reduce 519 positive cells in the cell
medium eluate. In running the second column at 37°C
instead of room temperature it was hoped that cell viability
would improve. By increasing the cell medium elution volume
from 45 to 90 ml it was hoped that sIg negative cells would
be more thoroughly eliminated from the column so that 519
positive cells could be obtained in greater purity. By

dividing the cell medium elution into three-30 ml portions

50

and analyzing those cells eluted with each portion, it
was hOped that some insight might be gained relating to
the effectiveness of increasing the cell medium elution.

The net effect of these changes resulted in the
isolation of sIg positive lymphocytes in higher purity
upon elution with cell medium containing gamma globulin
(80% positive for sIg). Cells eluted with the first 30 ml
of cell medium resulted in a cell isolate similar to that
obtained in the first fractionation study. Elution with
cell medium beyond the first 30 m1 further reduced the
number of sIg negative cells in the column although loss
of sIg positive cells increased with the last 30 ml as
compared to the second 30 ml. Cell viability improved
slightly. Again both EA and EAC rosetting frequencies
increased with increasing enrichment of Ig-bearing
lymphocytes suggesting that Ig and C receptors, as
detected by these assays, are largely cell markers
found on 519 positive cells.

Although some caution is warranted in equating
ontogenetically defined lymphocytes with marker defined
lymphocytes, there is general agreement that B cells, in
contradistinction to T cells, possess readily detectable
sIg. Separation of Ig-bearing (B) and non-Ig-bearing (T)
lymphocytes of bovine origin with an immunoabsorbent column

yielded results inferior to those obtained by Chess et a1.

51

(17) for human lymphocytes. A more thorough and controlled
investigation into the dynamics of such a column may be

necessary before comparable results are obtained.

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