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Mechanisms of release of granule enzymes from human neutrophils phagocytosing aggregated immunoglobulin.

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Mechanisms of Release of Granule Enzymes from Human
NeutrophiIs Phagocytosing Aggregated ImmunoglobuIin
An Electron Microscopic Study
Peter M. Henson
Neutrophils released a proportion of their potentially injurious granule
constituents during phagocytosis of particulate aggregated ?globulin.
Lactic dehydrogenase was not liberated and the cells were not lysed.
Electron microscopic observation revealed at least four related morphologic mechanisms for the release of granule enzymes. Extrusion of
granules was observed primarily into vacuoles. However, occasions were
noted when such vacuoles might be open t o the exterior: a) When a
vacuole apparently opened t o allow additional material t o be taken into
it; b) When a vacuole remained connected t o the exterior by a narrow slit;
c) When two cells were involved in phagocytosis of a single particle (aggregate); and d) When extrusion of granules preceded the complete closing
of pseudopods around the aggregates.
The reaction of polymorphonuclear neutro- cytoplasmic inclusions demonstrated to contain
phi1 leukocytes (neutrophils) with immunologic immunoglobulin and complement, are found in
complexes in tissues generally results in inflam- the synovial exudate (4,5). Neutrophil granules
matory tissue injury. In some instances the role injected into joints will themselves produce inof neutrophils has been directly demon- jury (6) and a clear role for neutrophils in an
strated (1) and evidence has accumulated that it experimental arthritis in rabbits has recently
is the injurious granule constituents which are been shown by De Shazo et a1 ( 7 , 8).
Human neutrophils react with immunologic
released from the neutrophils that produce the
damage (1 - 3 ) . This may be particularly true in complexes or aggregates by virtue of receptors
rheumatoid arthritis where neutrophils, with on their surface for both C3 and the Fc piece of
immunoglobulin (9-12). If the aggregates are of
a suitable size, phagocytosis ensues (12, 13), the
Publication 605 from the Department of Experimental
Pathology, Srripps Clinic and Research Foundation, La particles are engulfed and granules are released
into the phogocytic vacuole. During this process
Jolla Calif.
This study was supported by US Public Health Service a proportion of the granule constituents gain acGrant AI-07007 and the Council for Tobacco Research,
cess to the outside of the neutrophil by mechaUSA.
nisms which do not involve cell lysis (12-15).
PETER M. HENSON, PHD: Scripps Clinic and Research
Foundation, La Jolla, Calif, Recipient of the US Public T h e release appears to be a consequence of the
Health Service Career Development Award 3-K04-GM-42,
normal degranulation phenomenon. Thus, if for
567-01Sl.
Reprint requests should be addressed to: Dr. Henson, any reason the vacuole is open, or later becomes
Scripps Clinic and Research Foundation, 476 Prospect open to the outside, extracellular release of the
Street, La Jolla, Calif 92037.
Submitted for publication June 26, 1972; accepted Octo- granule enzymes can occur (16). In this study,
four possible mechanisms for this escape of vacber 23,1972.
208
Arthritis and Rheumatism, Vol. 16,No. 2 (March-April 1973)
RELEASE OF ENZYMES FROM NEUTROPHILS
Table 1. Release of Granule Constituents from Human
Neutrophils Phagocytosing Immunoglobulin Aggregates
Enzymes
Particulate
aggregated
Nonaggregated
HgG* (500 pg) HgG (500 p g )
Neutrophils
alone
~~
p-glucuronidase
13.4 + 0.4t
Myeloperoxidase 14.9 + 1.0
22.9 f 2.1
Lysozyme
Lactic
2.5 f 0.3
dehydrogenase
Al ka Iine
1.7 + 0.4
phosphatase
2.4 + 0.6
1.3 + 0.5
4.6 + 0.9
1.8 + 0.6
0.9 + 0.7
4.0 f 0.7
+ 0.3
2.1 A 0.4
2.5
1.5 + 0.3
2.1
+ 0.5
*HgG = Human 7-globulin.
7
tPercent release f SE from 1 x 10 neutrophils incubated for
30 minutes at 37°C. Four experiments with assays performed
in duplicate.
uole contents have been demonstrated in human
neutrophils phagocytosing aggregated y-globulin.
MATERIALS AND METHODS
Pure populations of human neutrophils were prepared
from the blood as described previously (12, 17). Human yglobulin (Pentex, Kankakee, 1. A,) was purified by column
chromotography on DEAE cellulose and aggregated, as described previously (12) with bisdiazotized benzidine (BDB)
according to the method of Ishizaka et af (18). Insoluble aggregates were achieved by use of 25 pg BDB/mg y-globulin.
Neutrophils (1 x 10') were incubated at 37°C in 2.5 ml
tris buffered Tyrode's solution with 0.25% bovine serum albumin at p H 7.2 with 500 pg of the aggregated or
nonaggregated human y-globulin (12). After 60 minutes the
cells were sedimented and the sopernatant assayed for released granule enzymes (8-glucuronidase, myeloperoxidase,
alkaline phosphatase and lysozyme) or a cytoplasmic enzyme (lactic dehydrogenase) by previously described technic (17). Total quantities of the enzyme were ascertained by
lysing the sedimented cells with 1 mg/ml Triton x 100.
For electron microscopic examination, the reactions were
terminated at varying times by addition of 5 ml 2% phosphate buffered glutaraldehyde at p H 7.3. T h e cells were
fixed in the glutaraldehyde for 1 hour at room temperature,
and then for 1 hour at 4" C in 1% osmic acid. They were
embedded in Vestopal W, sectioned on an LKB ultramicrotome, stained with uranyl acetate (2%) and lead citrate
(0.01%) and examined in a Hitachi HU 11A electron microscope.
RESULTS AND DISCUSSION
T h e release of granule enzymes from the
neutrophils phagocytosing the immunologic aggregates is shown in Table 1. Lactic dehydrogenase was not liberated, indicating the absence of cell lysis. Alkaline phosphatase was
also not released into the medium.
That phagocytosis was occurring is depicted
in Figure 1. Evidence of uptake is present with
developing vacuoles and the cell processes described by Weissmann et al (19). Degranulation
into vacuoles is apparent (Figure IA), and as
shown in Figure 1B, this eventually resulted in
a cell which lost most of its granules. T h e similarity in appearance of the cell to those seen in
the joints in rheumatoid arthritis (4, 5) is evident.
Neutrophils incubated with nonaggregated
immunoglobulin or alone did not release enzymes (Table 1) and after 30 minutes incubation had the normal appearance shown in
Figure 2.
Possible mechanisms for release of granule
enzymes from the vacuoles into which they have
been discharged are shown in Figures 3 to 6.
1. Instances have been noted in which
neutrophils, already containing many phago-
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
209
HENSON
Fig 1. Sections of human neutrophils phagocytosing aggregated human y-globulin
(Agg). A. After 5 minutes, developing vacuoles (v) are evident bordered by cell
processes. Discharge of granules into completed vacuoles is indicated by arrows.
B. After 30 minutes most of the granules have disappeared from this cell in which
many aggregate-containing vacuoles (v) may be seen.
210
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
RELEASE OF ENZYMES FROM NEUTROPHILS
Fig2. Section of neutrophil incubated alone for 30 minutes. The cytoplasm contains numerous
granules.
cytic vacuoles, are still phagocytosing more
aggregates. These are apparently being incorporated into preexisting vacuoles into which
degranulation has already occurred (Figure 3).
However, as the old vacuole opens to take in the
new material, the granule enzymes may escape.
This mechanism has been demonstrated in rabbit neutrophils phagocytosing zymosan particles (15, 16). Evidence for it was further
strengthened by finding that alkaline phosphatase which was liberated into the vacuole, could
not (unlike the other granule enzymes) be found
in the extFacellular medium (15, 16). It apparently adhered to the vacuole walls so that when
the vacuole later opened to the outside it was
not liberated along with the soluble enzymes. A
similar lack of release into the medium, of alka-
line phosphatase from human neutrophils has
been noted (Table 1).
2. A common finding in the neutrophils
phagocytosing immunologic aggregates was
membrane-bounded slits between vacuoles
(Figure 4). These were often found to be continuous with the external membrane of the cell and
were sometimes seen to connect vacuoles into
which degranulation was visible, to the outside
(Figure 4B). T h e reason why the membranes
lining the slit have not fused is as yet unclear.
Channels similar to this were described by Karnovsky et a1 (20) but from the pictures shown
may well have been examples of two neutrophils together phagocytosing a single particle.
Nevertheless, their demonstration of myeloperoxidase in the channel, whether it was be-
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
211
HENSON
Fig 3. Section of neutrophil incubated with aggregates for 30
minutes. Numerous vacuoles (v)
are apparent and the cell is apparently taking additional aggregates (Agg) into a pre-existing
vacuole (arrow). N = Nucleus:
g = granule.
tween two cells or of the type described above, is
a clear indication of enzyme release.
3. T w o neutrophils may not infrequently be
found adherent to one mass of aggregates. Discharge of granules into what is in fact a common vacuole formed between the two cells may
lead to escape of enzymes to the outside (Figure
5). A similar phenomenon has been described in
neutrophils phagocytosing zymosan particles (16, 21).
4. Examples are depicted in Figure 6 of discharge of granules into vacuoles which are
forming, but have not yet closed off. A similar
example has been presented by Zucker Franklin (14). While this might be expected to be
common, particularly in cells exposed to a massive stimulus, it was nevertheless not often seen
in these studies and those with rabbit neutrophils phagocytosing zymosan particles (1 6).
212
However, the transient nature of the event and
the small size of the granule would undoubtedly
reduce the likelihood of its visualization by
these technics.
Adherence of neutrophils to aggregated yglobulin or immune complexes on surfaces too
large to phagocytose results in release of enzymes (12, 17,22) by a direct discharge of granules along the stimulated portion of the cell
membrane (15, 16). This latter process is therefore merely an extension of the mechanisms described above in which, because of the size of
immune complex (in fact a surface), a vacuole
never forms.
From these observations, it would be expected that the larger the particle or aggregate,
the more readily would the vacuole open or remain open and the greater would be the release
of enzymes. T h e increased release of enzymes
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
RELEASE OF ENZYMES FROM NEUTROPHILS
Fig 4. Sections of single neutrophils incubated with aggregates. A. A system of
membrane bounded slits connecting aggregate-containing vacuoles (v) is apparent:
incubated for 30 minutes. 6. A slit is shown in this neutrophil which apparently
connects vacuoles into which granules are being extruded (arrows), with the outside
of the cell.
Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
213
HENSON
Fig 5. Section of two neutrophils
adherent to one aggregate. Discharge of granules into the vacuole
formed between the two cells (N1
and N2) is indicated by arrows. The
granule contents have clear access
to the external medium Incubated
for 30 minutes.
with increasing size of particle has been shown
to occur for both rabbit (16) and human neutrophils (12 ) .
ACKNOWLEDGMENTS
T h e author is indebted to .Mrs. I). Durham for preparing
the section for electron microsropy and Mrs. K . Presrott for
preparing the photographs for publication.
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Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
RELEASE OF ENZYMES FROM NEUTROPHILS
Fig 6 (A and 6). Two examples of discharge of granules from neutrophils into
developing phagocytic vacuoles which have not yet closed off. The released granules
are indicated by arrows. Figure 6B also shows a channel connecting a vacuole with
the outside as described previously (Figure 4). Agg = aggregates; incubated for 30
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Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
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Arthritis and Rheumatism, Vol. 16, No. 2 (March-April 1973)
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