Download Cellular Recognition and Activation within the Lymphoid System

Cellular Recognition and Activation within
the Lymphoid System
J O H N H. K E R S E Y , M.D.,
A N D G L E N J. B O O T H
Departments of Laboratory Medicine and Pathology and Pediatrics,
University of Minnesota, Minneapolis, Minnesota 55455
ABSTRACT
Kersey, John H., and Booth, Glen J.: Cellular recognition and activation within
the lymphoid system. Am J Clin Pathol 63: 6 2 9 - 6 3 5 , 1975. The present
study is concerned with cellular recognition and activation within the lymphoid
system. Data presented indicate that in human peripheral blood phagocytes
recognize and preferentially bind phagocytes, and thymus-derived (T)
lymphocytes recognize and preferentially bind other T lymphocytes rather
than thymus-independent (B) lymphocytes. Recognition of non-self results in
activation of lymphoid cells; recent data using a calcium ionophore, A23187,
suggest that calcium may act as an intracellular messenger by which signals
for cellular activation are transmitted from the cell surface to the nucleus.
(Key words: Lymphocytes; Cellular recognition.)
cellular activation, and the immune response are
not new to scientific investigators. For
example, Paul Ehrlich, in 1900, described
cell membrane receptors in a manner that
in many ways seems satisfactory to us
today. 12 He stated that cell receptors were
the "catching arms" of cells that would
bind specific substances. If the substance is
toxic, the cell will be killed. "If the cell is
not killed the receptors would be generated
in excess, some would float off into the
serum and there function as a specific
antibody for the foreign substance." Similarly, hypotheses popular today suggest
that cellular receptors specifically bind
STUDIES O F CELLULAR RECEPTORS,
Received October 29, 1974; accepted for publication December 9, 1974.
Supported in part by Virus Cancer Program contract number N01-CP-33357 and Grants no. CA-7306
and CA-16228 from the U.S. Public Health Service.
Reprints of this entire Research Symposium are
available from the ASCP Meeting Services Department, 2100 West Harrison St., Chicago, Illinois 60612,
for $3.00 per copy.
629
substances that may be toxic (e.g., other
antibodies), friendly (e.g., other similar cell
types) or hostile (e.g., histoincompatible
cells or tumor cells).
From another perspective, when one
looks at the phylogenetic tree one also
gets the impression that there's not much
new in cellular recognition and cellular
receptors. Today, I would like to review
briefly some aspects of the phylogenetic
development of cellular recognition and
cellular communication. I would then like
to present some data which suggest that,
contrary to some fondly held beliefs, cellular recognition processes may play only a
limited role in the development of malignancy.
It is of some interest that animals that
are rather early in phylogenetic development often have the capacity to distinguish
differing members of the species from
themselves. In one of the earliest multicellular organisms, the sponge, Humphreys showed clearly that red sponge
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KERSEY AND BOOTH
A,J.C.P.— Vol. 63
cells recognize and bind red sponge cells, containing iron or latex were considered to
and lavender sponge cells recognize and be phagocytes; the remaining mononuclear
bind lavender sponge cells.6 It is interesting cells were identified as lymphocytes.
that tumors are almost unheard of in
E-Rosette Assay
these early ancestors who have no specialized immune system. With the developFor experiments characterizing lymment of tissue and organ systems in more phoid subpopulations, lymphocytes were
specialized animals, we see evidence of purified by incubating the leukocyte-rich
more specific cellular interactions. Classic plasma with an equal volume of carbonyl
experiments by Moscona and his students, iron-latex suspension for 60 minutes at
for example, indicate that kidney cells 37 C. on a rocker platform. This mixture
will preferentially bind kidney cells and was layered on a Ficoll-Hypaque gradient
liver cells will bind liver cells when the and centrifuged at 400 X g for 45 minutes.
two are mixed together. This type of organ- The cells at the interface were collected,
specific recognition can be best demon- washed three times with D-PBS, and ad6
strated in embryonic cells.13 We recently justed to a concentration of 4 X 10 per cu
demonstrated what may be similar type- cm in D-PBS plus 10% heat-inactivated
specific interactions in mononuclear cells fetal calf serum. Suspended cells were
centrifuged at 200 X g for 3 minutes to inof human peripheral blood.
crease cell-to-cell contact. The lymphocytes
were gently resuspended and mixed
Materials and Methods
with an equal volume of washed sheep
H u m a n venous blood collected in erythrocytes, centrifuged at 200 X g for 3
heparin and sedimented in 5% dextran minutes, and incubated at 4 C. for one
was prepared according to the two follow- hour. The pellet was gently resuspended
ing protocols.
and counted on a standard hemocytometer.
Phagocytosis Experiment
Fifty pairs of cells in which both of the
T h e leukocyte-rich plasma obtained
from dextran sedimentation was treated
with tris-ammonium buffer for 10 minutes
at 37 C. T h e cells were washed and incubated with an equal volume of carbonyl
iron-latex suspension for 60 minutes at
37 C. on a rocker platform. They were
washed three times with Dulbecco's phosphate-buffered saline solution (D-PBS).
After the final wash, the cells were resuspended at a concentration of 4 X 106
per cu cm in D-PBS plus 10% heat-inactivated fetal calf serum and incubated at
4 C. for one hour. Finally, the cells were
gently resuspended and counted in a
standard hemocytometer.
In each preparation, 50 clearly recognizable pairs of cells in contact were
counted under a light microscope. Cells
cells in contact could be clearly identified
were counted under a light microscope.
Cells that phagocytized latex were not
counted. Latex-negative lymphocytes binding two or more erythrocytes were considered to be T cells; latex-negative
lymphocytes not binding erythrocytes were
considered to be either B cells or "null"
cells.
Results
Approximately 2 to 5% of cells prepared
according to the above protocols were
observed to form pairs or larger multiples.
Cell contact generally remained stable
throughout the period of observation. In
many instances a slight indentation of the
surface of one or both of the paired cells
was noted in the area of contact. Experi-
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LYMPHOCYTES AND CELLULAR RECOGNITION
631
40-
|
FlG. 1. Percentage observed versus predieted pairings of lymphocytes (L) and
phagocytes (P) in human peripheral blood.
»•
30
'
20-
10-
L-P
0
•
Observed
Predicted
ments performed at 4, 20, and 37 C.
established that the extent of pairing was
the same at these temperatures. Further
experiments were conducted to determine
whether these pairings were based on
random interactions between cells or
whether contacts were established between
specific cell types.
The shaded bar graphs in Figure 1
represent the percentages of lymphocyte
plus lymphocyte (L + L), lymphocyte plus
phagocyte (L + P), and phagocyte plus
phagocyte (P + P) contacts observed in
phagocytosis experiments. Interpopulation (P + P, L + L) contacts accounted for
83% (P + P = 45%, L + L = 38%) of all
observed pairings. The unshaded bar
graphs represent the predicted percentages of the various pairs, assuming random
association using the formula fL2 + 2fL • fp
+ fP2 = 1. T h e most significant variation
between observed and predicted percentages was found in interpopulation
(L + P) contacts. Calculations based on the
assumption of random association between all cells in contact predicted that
48% of the pairs would be L + P. In
our experiments, only 17% of the observed
pairs were L + P.
Subsequent investigations of the nature
of the interaction between subpopulations
of the lymphoid population were accomplished by performing E-rosette assays
on L + L contacts. T h e shaded bar graphs
in Figure 2 represent the percentages of
T-lymphocyte plus T-lymphocyte (T + T),
T-lymphocyte plus B-lymphocyte (T + B),
and B-lymphocyte plus B-lymphocyte
(B + B) contacts observed in the E-rosette
assay experiments. Contacts between members of the same lymphoid subpopulation
(T + T, B + B) accounted for 77% (T + T
= 18%, B + B = 59%) of all observed
pairings. T h e unshaded bar graphs represent the predicted percentages of the
various pairs, assuming random association of the lymphocytes in contact. The
most significant variation between observed and predicted percentages was
found in contacts between members of
different lymphoid subpopulations. Assuming random pairing, intersubpopu-
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KERSEY AND B O O T H
A.J.C.P. —Vol. 63
FIG. 2. Percentage observed versus predicted pairings of sheep erythrocyte-binding (T) and non-erythrocyte-binding (B)
lymphocytes in human peripheral blood.
T.T
E2 ObsarvM
• Predicted
lation pairing (T + B) was predicted to
occur in 40% of all pairs. T + B pairing
was observed in 23% of all pairs.
Discussion
Like-like relationships may be demonstrated in the sponge, in the liver and
the kidney, and now with human phagocytes, T lymphocytes, and B lymphocytes.
T h e relationships demonstrated may be
important for cellular communication and
the development of specialized organ systems throughout phylogeny.
Recognition of non-self clearly results
in destruction of the non-self component in many instances; it is of interest
that this destruction is demonstrable in
phylogeny before evidence of a specialized lymphoid system. For example,
in the marine colonial hydroids, Theodor
demonstrated that mutual destruction
results from the interaction of incompatible members of the same species. 5 In
these early non-immunologic recognition
systems the presence of one common component is sufficient to result in recognition of another as friend rather than foe.
In other words, if two marine forms are
in any way related, they will recognize
each other as friends and not destroy
each other. It seems likely that these early
recognition systems, while maintaining
integrity of self, would not be very effective in eliminating malignant cells. Such a
statement seems relatively safe since it is
likely that while malignant transformation
may result in new surface antigens, many
of the old familiar antigens of the nontransformed cells will remain. If such is the
case, we have difficulty understanding how
a normal coral cell could recognize and
destroy a malignant coral cell or a normal
kidney cell a malignant kidney cell.
Perhaps it is the lymphoid system which
is capable of recognizing and destroying
malignant cells. Let us examine the evidence.
The development of a system that
specializes in cellular recognition {i.e., the
lymphoid system) appears to have beginnings rather early in phylogeny. Our
spiny-skinned ancestors, the echinoderms,
e.g., the starfish, are found to have circulating lymphoid cells.19 The starfish and
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1975
LYMPHOCYTES AND CELLULAR RECOGNITION
633
another ancestor, the sea cucumber, have Table I. Possible Causes of Lymphoreticular
Malignancies in Immunodeficient
the ability to reject grafts of foreign skin
Patients
(albeit rather slowly, since the job often
19
takes several months).
Increased malignant transformation of lymphoid cells
Certain evidence suggests that the phylo- due to:
genetic developments of specialized im(a) Intrinsic defects in lymphoid cells (e.g., chromune systems occurred at about the same
mosomal breaks)
(b) Increased activity of exogenous and endogenous
time as animals began to bear living young.
oncogenic viruses
Speculation of Burnet and others runs that
specialized immunocytes assisted in the
Decreased ability of the lymphoid system to recognize
defense against parasitism of parents by and destroy malignant lymphoid cells:
their own offspring. 3 It is rather unlikely
(a) Defective recognition
that development of this specialized im(b) Defective response following recognition
mune system was especially useful in host
defenses against microorganisms, since
the plants and early animals without im- Such anterior mediastinal lymphomas, we
mune systems thrived side by side with and know today, almost invariably involve T
often in symbiosis with their microorgan- cells, in that they have the capacity to
isms. Similarly, from the phylogenetic per- form rosettes with sheep erythrocytes, a
spective, it is difficult to imagine that the reliable marker of human T cells.7 Studies
system provided any new defense against of children with the various immunodemalignant cells, since those ancestors with- ficiency syndromes continue through an
out immune systems appear not to be Immunodeficiency-Cancer Registry esparticularly plagued by neoplasms.
tablished at Minnesota. 9 We now know of
Another approach to the question of the almost 200 persons with immunodeficiency
possible role of the immune system in the who developed cancers; the risk of dedefense against malignant cells is to ex- velopment of these cancers is about 100
amine the effect of elimination of the times that of the general population. It
immune system. Our studies involved is of interest that the types of malighuman individuals and date back to the nancies are quite different from those of
early 1960's. At that time, reports by Page, an unselected population. About 8% of
ten Bensel, Krivit, Good and others at tumors in unselected children are lymphoMinnesota indicated that children with reticular; in contrast, 67% of the maliggenetically determined immunologic de- nancies are lymphoreticular solid tumors
ficiency syndromes that involved the im- (including l y m p h o m a , reticulum-cell
mune system were at high risk for develop- sarcoma) or leukemia in children with
ment of cancer. 4 These syndromes may, primary immunodeficiency disorders. 10
as you know, be classified into those inAnother group of immunodeficient
volving the lymphocytes that are thymus- patients, those who are immunosuppressed
derived (T) cells, and those that are with drugs for renal transplantation, also
thymus-independent and involve (B) often develop lymphoreticular tumors.
bursa-equivalent or bone marrow cells. These lymphoreticular tumors frequently
An early example was a child with repeated involve the brain. 15 T h e reasons for the
infections and the syndrome of X-linked frequent lymphoid malignancies in imagammaglobulinemia known to be asso- munodeficient individuals could be several,
ciated with B-cell deficiency. This child and include those listed in Table 1. Our
developed a large anterior mediastinal findings and those of others indicate that
lymphoma, presumably in the thymus. 14 there is only a slight increase in non-
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KERSEY AND BOOTH
lymphoid malignancies in immunodeficient patients. 10,15 In the same vein, Stuman observed that nude mice (i.e., mice
with a severe genetically determined
immunodeficiency) were not at increased
risk for spontaneously occurring or
chemically-induced neoplasms. 18 These
results are, of course, in contrast to some
earlier reports indicating that laboratory
infections of immunodeficient mice
with some oncogenic viruses resulted in
increased tumor incidence. 8 Because of
these recent observations, we are somewhat skeptical of hypotheses suggesting
that a major function of the lymphoid
system is to provide surveillance against
malignant cells that develop within the
liver, kidney, brain, and other organs.
In fact, it is remarkable that cancer is a
relatively infrequent disease in both normal
and immunodeficient individuals, despite
daily exposure of billions of cells to dozens
of environmental carcinogens.
To turn to a different but related subject, we have recently been interested in
the membrane consequences of the interaction of non-self components with specific
cellular receptors. In particular, we were
interested in signals that might be generated at the cell surface that could be
translated to the cytoplasm and cell nucleus and result in cell activation and cell
division. Some evidence suggests that calcium may act as such a signal. Data presented by Alford, 1 Allwood and colleagues, 2 and Whitney and Sutherland 20
indicated that calcium was required for
activation of lymphocytes by nonspecific
mitogens, e.g., phytohemagglutinin. Additionally, Whitney and Sutherland showed
that activation resulted in significant influx
of calcium from the external medium. 21
We approached the problem using an
antibiotic derived from cultures of a type
of Streptomyces known as A23187. This
antibiotic is known as an ionophore because it selectively transports ions across
cell membranes. This particular ionophore
A.J.C.P.—Vol.63
selectively transports divalent cations with
highest affinity for calcium. 16 It is of note
that this ionophore affects the eggs of one
of our echinoderm friends, the sea urchin.
Steinhardt and Epel noted that the ionophore stimulated calcium-dependent protein and DNA synthesis in the eggs of
this species.17 Our studies indicate that the
ionophore will stimulate blast transformation and DNA synthesis in human lymphocytes.11 Activation of lymphocytes requires
calcium and, to a lesser extent, magnesium,
in the external medium. More than two
hours of contact of ionophore with lymphocytes is necessary for optimal activity.11
In summary, the cell membrane appears
to be the site of specific interaction of
friend and foe in all species and in many
cell types throughout phylogeny. These
interactions, which require specific membrane receptors, appear to be important
in tissue and organ formation and in interactions with microorganisms, even prior to
development of specific i m m u n e responses.
Lymphocytes and other cells that mediate these specific immune responses, which
are found as early as the starfish and other
echinoderms, are rather latecomers in the
evolutionary scheme of things. We later
vertebrates are clearly dependent in this
respect, however, as death from infection
quickly ensues when they are defective.
The role of these specialized lymphoid
cells in the defense against malignancy
remains a matter of continuing controversy, and I suspect the data will show
that they are less important than we had
previously suspected.
One consequence of cell-to-cell interaction may be cellular proliferation. Certain evidence suggests that calcium may
be involved as an intracellular mediator
to communicate a membrane signal to
the cytoplasm and the nucleus. Ongoing
studies of signal formation may assist in
the understanding of both normal and
malignant cells.
May
1975
LYMPHOCYTES AND CELLULAR RECOGNITION
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