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Cellular reaction to injury in the anthozoan Anthopleura elegantissima
,IOURKAI.
OF ISVERl‘EBK~T’E
Cellular
PATHOLOGY
Reaction
33, 189-196 ( 1979)
to Injury in the Anthozoan elegan tissima
MICHAEL J. PATTERSON AND MARSHAL.
Anthopleura
LANDOLT
College of Fisheries, University of Washington, Seattle, Washington 98105 Received July 6, 1978 Small thermal injuries were used to examine the ability ofAnthop/eura elegantissima (Cnidaria: Anthozoa) to establish a successful inflammatory response. Experimental animals were seen to react by an initial influx of phagocytes derived from resident amoebocytes. Within 72 hr, a zone of repair formed which contained distinctly atypical cells morphologically suited for the production and secretion of unknown substances. At all times the wound remained infection free and was rapidly repaired by the passive influx of cells from the surrounding epithelium as well as progressive replacement of lost tissue by the mesogleal repair zone. KEY WORDS: Anthopleura; antbozoan; Cnidaria; inflammation; wound repair.
observed behavior or induced to employ mechanisms associated with asexual reproduction. The main purpose was to determine if anthozoans were structurally capable of responding to local injury with a multifaceted reaction or if they were limited to the simple phagocyte response previously observed.
INTRODUCTION
Reaction to injury in cnidarians has been a popular field of study for at least 100 years. The motives and success of these projects have been significantly more varied than the subject, which has usually been Hydra. This species, while ideal for some experiments, is far from typical of the phylum and is difficult to correlate with more complex animals. As noted in the review by Sparks (1972), the consensus of opinion has been that cnidarians do not exhibit a true inflammatory response since the only immediate reaction to injury is a phagocytic response by all cells. Young (1974), working with the anthozoan Calliactis parasitica, demonstrated the influx of amoebocytes into a wound, but, as in earlier studies, these cells appeared to be uniformly phagocytes. In general, more recent work has failed to add significantly to the early work of Metchnikoff (1893), whose conclusion was that cnidarians are capable of only a simple homogenous response. This places in question the relevance of their use as models for the reactive pathology of more complex animals. In this work, small, well-defined lesions were used to examine the response in animals which were not altered significantly in
METHODS
Anthopleura efegantissima, l-2.5 cm in diameter, were collected from the intertidal zone of Puget Sound near Alki Point. These animals were usually attached to small rocks buried in a sandy substrate. To avoid injury, the animal was removed with the associated rock and transported directly to the large-volume recirculating aquarium at the University of Washington, College of Fisheries. Upon arrival, the animals were placed in individual glass containers substantially larger than the attached rocks. Within a few days, most animals had moved off the rock surface onto the glass containers from which they could be removed without injury to the pedal disc. During this time, animals were fed daily with frozenArtemia. Experimental animals were injured by briefly touching the mid-column region with
189 0022-201 l/79/020189-08$01.00/O Copyright Q 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
190
PATTERSON
AND
LANDOLT
a heated blunt probe. This produced a uni- translucent circular area. This feature was form circular wound approximately Imm in clearly visible in animals injured up to 5 diameter. Immediately upon return to flow- days before fixation, but was generally not ing seawater, these animals resumed a feed- seen after longer periods of repair. Animals ing posture and actively consumed food if were sectioned through the center of this presented. At no time during this procedure wound, producing two identical cross secwere animals handled directly, and they tions of the area. One of these pieces was remained attached to the container. directly dehydrated in a graded methanol After various periods of time, animals series and embedded in paraffin for light were fixed within their glass chambers with microscopy. The remaining tissue was rea solution of paraformaldehyde and duced in size to an area encompassing the entire wound and an equal amount of ungluteraldehyde buffered to p H 7.2 (Karnovsky, 1965). Occasional flooding of the damaged surrounding tissue. This section gastric cavity assured complete access of was postfixed in 2% osmium tetroxide bufthe fixative. Due to the rapidity of this fura- fered with bicarbonate, followed by dehytive, prior relaxation was not required to dration in a graded ethanol series and propreserve the animals in the expanded pos- pylene oxide. After dehydration, the tissue ture, as had been necessary with other fixa- was embedded in Epon 812 (Polysciences) tives. Fixation was continued for 24 hr at and oriented to match the light microscope 4°C. section. Using 6-pm sections stained with After fixation the wound was located as a
FIG. 1. Cross section through the column of Anthupleura characteristic microvilli, is shown resting on the cellular collagen. Richardsons stain; lpm Epon section x 2,200.
eleganfissima. mesoglea (M)
The epidermis (E), composed primarily
with of
ANTHOZOAN
REACTION
hematoxylin and eosin as a guide, specific areas of the Epon blocks were selected for subsequent thin-section preparation. After staining with saturated uranyl acetate in 50% methanol and lead citrate (Reynolds, 1963), sections were viewed in a JEOL 1OOBelectron microscope. Measurements of cell population changes were accomplished with 6-pm sections viewed through a calibrated grid fitted to the microscope eyepiece. Values reported are the mean and standard deviation of cell counts derived from 3,920+m2 areas in each specimen. Ten samples were examined for each time period presented. RESULTS
The normal anthozoan surface is a pseudostratified epithelium resting on a connective tissue layer containing cellular elements and a network of collagenous mi-
L &,w .,a
191
TO INJURY
crofilaments (Fig. 1). The epithelial layer is characteristically separated from the connective tissue by a dense layer of filaments distinct from the collagen microfilaments. In the experimentally injured animals, only the epithelium and connective tissue were affected. In normal animals, the connective tissue contains a homogenous population of amoebocytes (Fig. 2) which have numerous specific granules. In hematoxylin and eosin preparations, these granules may be eosinophilic or neutrophilic. Under control conditions, the Golgi and endoplasmic reticular structures are absent or poorly developed in these cells and they are not seen to be mitotically active. In control animals, the mesogleal cell populations were determined to be 4.9 x lo3 cells/mm2 and appeared to be stable throughout the column. By 24 hr following injury, the area was seen as a cratered region (Fig. 3) devoid of
_
FIG. 2. Normal configuration of anthozoan mesogleal cells. Numerous acteristic. (M), mesoglea; (E), epithelium; arrows indicate the basal epithelium from mesoglea. Uranyl acetate and lead citrate x 8500.
granular complex
inclusions are charwhich separates the
FIG. 3. Anfhopleuru 24 hr after injury. The wound surface (W) appears as a concave crater surrounded by surviving epithelium (E). Hematoxylin and eosin stain x 1200. FIG. 4. Expanded, diffuse mesoglea with numerous phagocytes 48 hr after experimental injury. (W), wound surface; (E), epithelium. Hematoxylin and eosin stain x 1500. FIG. 5. Wound after 72 hr of repair. The size of the wound (W) is reduced by encroaching epithelium (E). Arrows indicate a region of mesogleal repair in which several cell types are found. Hematoxylin and eosin stain X 1500.
ANTHOZOAN
REACTION
epithelial covering. In the wound region the cell population had not significantly exceeded control levels (5.1 x lo3 cells/mm2). Cellular elements of the wound retained the morphology of control cells with the exception that some were seen to contain secondary lysosomes. After 48 h the cell population had risen to 8.0 x lo3 cells/mm2; however, there was little evidence of any change in the composition of cellular elements. The mesogleal matrix was seen to swell (Fig. 4) substantially, and this was reflected ultrastructurally by a dispersion of filaments and a loss of orientation. The cells present in this material appeared to be limited to secondary phagocytes which could be seen at the surface of the wound where they ruptured, discharging their contents to the surrounding media. In these cells, the mitochondria and various granules were the only organelles present in the cytosol with regularity. At no time was there evidence of these cells forming organized layers, and their presence in increased numbers appeared to represent an active migration through the wound area. By 72 hr, the surrounding epithelial cells were seen to be encroaching on the wound area (Fig. 5) and there was significant reduction of the wound size. The cell population had increased to 1.2 x 104 cells/mm* within the reduced area of damage. In the central wound area, the cell type was still secondary phagocytes (Fig. 6) and diapedesis continued. Throughout the mesoglea, cells could be seen to form an area of increased cellular density in a circular pattern around the region of damage. In this region numerous cells resembling amoebocytes were detected and transition from the disordered filament arrangement of wound tissue to a regular pattern typical of normal tissue was seen. In addition, atypical cells were present which were detected only in this repair zone. One cell type (Fig. 7) had a basophilic cytoplasm, which was seen ultrastructurally to be caused by a proliferation of rough endo-
193
TO INJURY
plasmic reticulum. These cells typically had an increased number of mitochondria, a round nucleus with dispersed chromatin, and a general lack of specific granules. The dilated endoplasmic reticulum invariably contained material which was more electron dense than the surrounding cell sap. These cells could be detected light microscopically as small, basophilic cells in the mesoglea. They comprise less than loo/o of the cellular infiltrate at 72 hr and they were not present by 7 days postinjury. The third member of the cellular infiltrate was identified only by electron microscopy and appeared to be part of the neutrophil population in hematoxylin and eosin preparations. These cells (Fig. 8) showed a proliferation of the Golgi apparatus and smooth endoplasmic reticulum far beyond any other cell type in this species. The nucleus was large and pleomorphic although the lobed nuclei characteristic of many active phagocytes was not seen. There was a total lack of specific granules and there were few mitochondria relative to the number seen in basophils and phagocytes. An estimate of the contribution to the overall cell population was not made for these cells; however, they were seen in specimens as late as 240 hr postinjury, though only rarely past 120 hr. DISCUSSION
In anthozoans responding to small lesions a rapid influx of phagocytes derived from resident cells in the mesoglea was noted. As the reaction progressed, a relatively small number of cells exhibited distinctive features not seen in control animals. The predominant change in these cells was a proliferation of specific cellular organelles, and their accumulation in a zone of repair surrounding the damaged tissue. At no time was there any indication of secondary infection despite the failure of the infiltrate to form continuous protective cell layers as was reported by Young (1974) in Calliactis. The overall cell population increased
.1
FIG. 6. the repair lysosomes FIG. 7. developed citrate y FIG. 8. extensive
-.
.
c-
_.
‘
Various derivatives of resident amoebocytes compose the major cell population throughout process. After entering the wound these cells are characterized by dense inclusions resembling (L). Uranyl acetate and lead citrate Y 11.000. the repair zone at 72 hr postinjury contains cells with well In addition to phagocytes. rough endoplasmic reticulum (R) and numerous mitochondria CM). Uranyl acetate and lead 18.000. A small group OF cells in the 72.hr repair zone develop large Golgi complexes (arrow) and agranular reticulum. Uranyl acetate and lead citrate ‘. 23.000.
ANTHOZOAN
REACTION
TABLE CELL
Postinjury
DENSITIES
DURING
TO
1
ANTHOZOAN
REACTION
(HR)
Control 24 48 12 96 120
with time; however, this appeared to be due to an increased rate of cell migration through the wound to the surface, where they discharge debris through the same mechanism seen in molluscs (Stauber, 1950; Pauley and Sparks, 1965). Diffuse collagen microfilaments were seen in the area of injury almost immediately and appeared to be the result of exposure to sea water, rather than the early synthesis of new material. This theory was supported by the absence of larger cell populations at 48 hr postinjury, when mesogleal expansion was significant and the microfilaments were widely dispersed (Patterson and Landolt, 1978). During the initial stages of the repair process, the cellular infiltrate consisted of a homogenous population of primary and secondary phagocytes. Between 48 and 72 hr postinjury, a zone of repair was formed. On the distal side of the region collagen filament arrangement appeared normal and most cells in the region contained specific granules with no evidence of secondary lysosomes, the predominant feature of cells within the wound area. At the margin between normal and damaged tissue was the region of atypia and high cell density. The most numerous of these altered cells showed a prolific rough endoplasmic reticulum and evidence of high energy-dependent activity. The function of these cells was undetermined; however, their morphology was suggestive of intense protein production. Absence of a Golgi apparatus and a lack of storage granules suggested that this material was released locally in uncomplexed form. The occurrence of
195
INJURY
TO INJURY
Cell number/mm2 4.9 5.1 8.0 1.4 1.3 7.0
x x x x x x
103 103 lo3 104 104 103
(SD) (1.5 (1.5 (2.1 (3.4 (2.4 (3.2
x x x x x x
IO’) 103) 103) IO’) 10)) 10))
injury-related proteins in cnidarians has not been documented; however, the small number of cells and their local occurrence would be consistent with very low total protein levels. Accumulation in a small, welldefined area would allow small levels of protein to reach significant concentrations in a restricted zone. In addition to protein cells, a few cells showed massive proliferation of the agranular endoplasmic reticulum and the Golgi. Like the basophils, these cells were localized in the transition zone and were not seen in other regions. The function of smooth endoplasmic reticulum is not as clear as rough endoplasmic reticulum, and functional correlations depend on enzymatic patterns. The low cell number and an inability to discriminate these cells from phagocytes using conventional light microscopy has hampered investigation of their role in the reaction to injury. Their structural features would be compatible with such cell lysins as saponins, where a lipid derivative is coupled to a series of sugar residues and released. Hemolysins are known to exist in the cnidarians (Keen and Crone, 1969), but their chemical identity or possible induction by injury remains unknown. The cells in the reactive zone, regardless of morphology, did not appear mitotically active. Centrioles were occasionally seen, but no mitotic figures were discernable by light or electron microscopy. No wellorganized storage site has been reported in these animals; however, proliferation sites in the gastrodermis have been suggested by Young (1974), who also demonstrated some
196
PATTERSON
sensitivity of the reaction to colchicine, an inhibitor of cell multiplication. Lack of intermediate cell forms leading from normal amoebocytes to altered forms implied the production of modified cells at some site distant from the injury, followed by their migration or accumulation in the zone of repair. The migration of specific cells from a distal proliferation or differentiation site to the wound implies a degree of control, possibly chemical, which is not needed to explain the presence of normally motile resident amoebocytes. Injuries used in this study were small and did not alter the behavior patterns of experimental animals. The extensive mechanisms for asexual reproduction did not appear to be employed. Rather, these local injuries elicited an immediate influx of amoebocytes involved in phagocytizing necrotic tissue and eliminating it by ejection into the surrounding water. This was followed by a progressive zone of repair, which proceded through the wound area and reestablished tissue integrity. The repair zone contained cells which were unique to the reaction to injury and appeared to be active in the synthesis of various substances. The lack of a circulatory system or any specific organs makes the concept of an inflammatory response difficult. There was, however, a distinct series of cellular events following injury. These events, and the component cells, appeared to be distinct members of an organized reaction to injury which prevented secondary infection, isolated the damaged region,
AND LANDOLT
and initiated tissue repair. This ancient group of animals appeared to have developed a functional inflammatory response which predates the origin of a circulatory system or specialized organs. ACKNOWLEDGMENTS We are grateful for the assistance ofDr. M. C. Lowe, Dr. G. B. Pauley, and Dr. A. K. Sparks. This project was supported in part by a research grant from the University of Washington Graduate School Research Fund.
REFERENCES KARNOVSKY, M. J. 1965. A formaldehydegluteraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell Bin/., 27, 137A. KEEN, T. E. B., AND CRONE, H. D. 1%9. The hemolytic properties of extracts of tentacles from the cnidarian Chironexjleckeri. Toxicon, 7,55-63. METCHNIKOFF, E. 1893. Lecons sur la pathologic comparee l’inflammation. In “Faites a 1’ Institute Pasteur en Avril et Mai 1891.” Masson, Paris. PATTERSON, M. J., AND LANDOLT, M. L. 1978. Primary events in the anthozoan reaction to injury. In “Pacific Division, A.A.A.S., 59th Annual Meeting,” p. 43 (Abstract). PAULEY, G. B., AND SPARKS, A. K. 1%5. Preliminary observations on the acute inflammatory reaction in the Pacific Oyster Crussostrea gigas (Thunberg). J. Znvertebr. REYNOLDS,
Pathol.,
7,248-2X
E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. .I. Cell Biol., 17,201212. SPARKS, A. K. 1972. “Invertebrate Pathology,” 387 pp., Academic Press, New York. STAUBER, L. A. 1950. The fate of india ink injected intracardially into the oyster, Ostrea virgin& (Chelin) Biol.
Bull.,
98, 227-241.
YOUNG, J. A. C. 1974. The nature oftissue regeneration after wounding in the sea anemone Calliactis parasitica (Couch). J. Mar. Biol. Ass. U. K., 54, 599-617.
OF ISVERl‘EBK~T’E
Cellular
PATHOLOGY
Reaction
33, 189-196 ( 1979)
to Injury in the Anthozoan elegan tissima
MICHAEL J. PATTERSON AND MARSHAL.
Anthopleura
LANDOLT
College of Fisheries, University of Washington, Seattle, Washington 98105 Received July 6, 1978 Small thermal injuries were used to examine the ability ofAnthop/eura elegantissima (Cnidaria: Anthozoa) to establish a successful inflammatory response. Experimental animals were seen to react by an initial influx of phagocytes derived from resident amoebocytes. Within 72 hr, a zone of repair formed which contained distinctly atypical cells morphologically suited for the production and secretion of unknown substances. At all times the wound remained infection free and was rapidly repaired by the passive influx of cells from the surrounding epithelium as well as progressive replacement of lost tissue by the mesogleal repair zone. KEY WORDS: Anthopleura; antbozoan; Cnidaria; inflammation; wound repair.
observed behavior or induced to employ mechanisms associated with asexual reproduction. The main purpose was to determine if anthozoans were structurally capable of responding to local injury with a multifaceted reaction or if they were limited to the simple phagocyte response previously observed.
INTRODUCTION
Reaction to injury in cnidarians has been a popular field of study for at least 100 years. The motives and success of these projects have been significantly more varied than the subject, which has usually been Hydra. This species, while ideal for some experiments, is far from typical of the phylum and is difficult to correlate with more complex animals. As noted in the review by Sparks (1972), the consensus of opinion has been that cnidarians do not exhibit a true inflammatory response since the only immediate reaction to injury is a phagocytic response by all cells. Young (1974), working with the anthozoan Calliactis parasitica, demonstrated the influx of amoebocytes into a wound, but, as in earlier studies, these cells appeared to be uniformly phagocytes. In general, more recent work has failed to add significantly to the early work of Metchnikoff (1893), whose conclusion was that cnidarians are capable of only a simple homogenous response. This places in question the relevance of their use as models for the reactive pathology of more complex animals. In this work, small, well-defined lesions were used to examine the response in animals which were not altered significantly in
METHODS
Anthopleura efegantissima, l-2.5 cm in diameter, were collected from the intertidal zone of Puget Sound near Alki Point. These animals were usually attached to small rocks buried in a sandy substrate. To avoid injury, the animal was removed with the associated rock and transported directly to the large-volume recirculating aquarium at the University of Washington, College of Fisheries. Upon arrival, the animals were placed in individual glass containers substantially larger than the attached rocks. Within a few days, most animals had moved off the rock surface onto the glass containers from which they could be removed without injury to the pedal disc. During this time, animals were fed daily with frozenArtemia. Experimental animals were injured by briefly touching the mid-column region with
189 0022-201 l/79/020189-08$01.00/O Copyright Q 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
190
PATTERSON
AND
LANDOLT
a heated blunt probe. This produced a uni- translucent circular area. This feature was form circular wound approximately Imm in clearly visible in animals injured up to 5 diameter. Immediately upon return to flow- days before fixation, but was generally not ing seawater, these animals resumed a feed- seen after longer periods of repair. Animals ing posture and actively consumed food if were sectioned through the center of this presented. At no time during this procedure wound, producing two identical cross secwere animals handled directly, and they tions of the area. One of these pieces was remained attached to the container. directly dehydrated in a graded methanol After various periods of time, animals series and embedded in paraffin for light were fixed within their glass chambers with microscopy. The remaining tissue was rea solution of paraformaldehyde and duced in size to an area encompassing the entire wound and an equal amount of ungluteraldehyde buffered to p H 7.2 (Karnovsky, 1965). Occasional flooding of the damaged surrounding tissue. This section gastric cavity assured complete access of was postfixed in 2% osmium tetroxide bufthe fixative. Due to the rapidity of this fura- fered with bicarbonate, followed by dehytive, prior relaxation was not required to dration in a graded ethanol series and propreserve the animals in the expanded pos- pylene oxide. After dehydration, the tissue ture, as had been necessary with other fixa- was embedded in Epon 812 (Polysciences) tives. Fixation was continued for 24 hr at and oriented to match the light microscope 4°C. section. Using 6-pm sections stained with After fixation the wound was located as a
FIG. 1. Cross section through the column of Anthupleura characteristic microvilli, is shown resting on the cellular collagen. Richardsons stain; lpm Epon section x 2,200.
eleganfissima. mesoglea (M)
The epidermis (E), composed primarily
with of
ANTHOZOAN
REACTION
hematoxylin and eosin as a guide, specific areas of the Epon blocks were selected for subsequent thin-section preparation. After staining with saturated uranyl acetate in 50% methanol and lead citrate (Reynolds, 1963), sections were viewed in a JEOL 1OOBelectron microscope. Measurements of cell population changes were accomplished with 6-pm sections viewed through a calibrated grid fitted to the microscope eyepiece. Values reported are the mean and standard deviation of cell counts derived from 3,920+m2 areas in each specimen. Ten samples were examined for each time period presented. RESULTS
The normal anthozoan surface is a pseudostratified epithelium resting on a connective tissue layer containing cellular elements and a network of collagenous mi-
L &,w .,a
191
TO INJURY
crofilaments (Fig. 1). The epithelial layer is characteristically separated from the connective tissue by a dense layer of filaments distinct from the collagen microfilaments. In the experimentally injured animals, only the epithelium and connective tissue were affected. In normal animals, the connective tissue contains a homogenous population of amoebocytes (Fig. 2) which have numerous specific granules. In hematoxylin and eosin preparations, these granules may be eosinophilic or neutrophilic. Under control conditions, the Golgi and endoplasmic reticular structures are absent or poorly developed in these cells and they are not seen to be mitotically active. In control animals, the mesogleal cell populations were determined to be 4.9 x lo3 cells/mm2 and appeared to be stable throughout the column. By 24 hr following injury, the area was seen as a cratered region (Fig. 3) devoid of
_
FIG. 2. Normal configuration of anthozoan mesogleal cells. Numerous acteristic. (M), mesoglea; (E), epithelium; arrows indicate the basal epithelium from mesoglea. Uranyl acetate and lead citrate x 8500.
granular complex
inclusions are charwhich separates the
FIG. 3. Anfhopleuru 24 hr after injury. The wound surface (W) appears as a concave crater surrounded by surviving epithelium (E). Hematoxylin and eosin stain x 1200. FIG. 4. Expanded, diffuse mesoglea with numerous phagocytes 48 hr after experimental injury. (W), wound surface; (E), epithelium. Hematoxylin and eosin stain x 1500. FIG. 5. Wound after 72 hr of repair. The size of the wound (W) is reduced by encroaching epithelium (E). Arrows indicate a region of mesogleal repair in which several cell types are found. Hematoxylin and eosin stain X 1500.
ANTHOZOAN
REACTION
epithelial covering. In the wound region the cell population had not significantly exceeded control levels (5.1 x lo3 cells/mm2). Cellular elements of the wound retained the morphology of control cells with the exception that some were seen to contain secondary lysosomes. After 48 h the cell population had risen to 8.0 x lo3 cells/mm2; however, there was little evidence of any change in the composition of cellular elements. The mesogleal matrix was seen to swell (Fig. 4) substantially, and this was reflected ultrastructurally by a dispersion of filaments and a loss of orientation. The cells present in this material appeared to be limited to secondary phagocytes which could be seen at the surface of the wound where they ruptured, discharging their contents to the surrounding media. In these cells, the mitochondria and various granules were the only organelles present in the cytosol with regularity. At no time was there evidence of these cells forming organized layers, and their presence in increased numbers appeared to represent an active migration through the wound area. By 72 hr, the surrounding epithelial cells were seen to be encroaching on the wound area (Fig. 5) and there was significant reduction of the wound size. The cell population had increased to 1.2 x 104 cells/mm* within the reduced area of damage. In the central wound area, the cell type was still secondary phagocytes (Fig. 6) and diapedesis continued. Throughout the mesoglea, cells could be seen to form an area of increased cellular density in a circular pattern around the region of damage. In this region numerous cells resembling amoebocytes were detected and transition from the disordered filament arrangement of wound tissue to a regular pattern typical of normal tissue was seen. In addition, atypical cells were present which were detected only in this repair zone. One cell type (Fig. 7) had a basophilic cytoplasm, which was seen ultrastructurally to be caused by a proliferation of rough endo-
193
TO INJURY
plasmic reticulum. These cells typically had an increased number of mitochondria, a round nucleus with dispersed chromatin, and a general lack of specific granules. The dilated endoplasmic reticulum invariably contained material which was more electron dense than the surrounding cell sap. These cells could be detected light microscopically as small, basophilic cells in the mesoglea. They comprise less than loo/o of the cellular infiltrate at 72 hr and they were not present by 7 days postinjury. The third member of the cellular infiltrate was identified only by electron microscopy and appeared to be part of the neutrophil population in hematoxylin and eosin preparations. These cells (Fig. 8) showed a proliferation of the Golgi apparatus and smooth endoplasmic reticulum far beyond any other cell type in this species. The nucleus was large and pleomorphic although the lobed nuclei characteristic of many active phagocytes was not seen. There was a total lack of specific granules and there were few mitochondria relative to the number seen in basophils and phagocytes. An estimate of the contribution to the overall cell population was not made for these cells; however, they were seen in specimens as late as 240 hr postinjury, though only rarely past 120 hr. DISCUSSION
In anthozoans responding to small lesions a rapid influx of phagocytes derived from resident cells in the mesoglea was noted. As the reaction progressed, a relatively small number of cells exhibited distinctive features not seen in control animals. The predominant change in these cells was a proliferation of specific cellular organelles, and their accumulation in a zone of repair surrounding the damaged tissue. At no time was there any indication of secondary infection despite the failure of the infiltrate to form continuous protective cell layers as was reported by Young (1974) in Calliactis. The overall cell population increased
.1
FIG. 6. the repair lysosomes FIG. 7. developed citrate y FIG. 8. extensive
-.
.
c-
_.
‘
Various derivatives of resident amoebocytes compose the major cell population throughout process. After entering the wound these cells are characterized by dense inclusions resembling (L). Uranyl acetate and lead citrate Y 11.000. the repair zone at 72 hr postinjury contains cells with well In addition to phagocytes. rough endoplasmic reticulum (R) and numerous mitochondria CM). Uranyl acetate and lead 18.000. A small group OF cells in the 72.hr repair zone develop large Golgi complexes (arrow) and agranular reticulum. Uranyl acetate and lead citrate ‘. 23.000.
ANTHOZOAN
REACTION
TABLE CELL
Postinjury
DENSITIES
DURING
TO
1
ANTHOZOAN
REACTION
(HR)
Control 24 48 12 96 120
with time; however, this appeared to be due to an increased rate of cell migration through the wound to the surface, where they discharge debris through the same mechanism seen in molluscs (Stauber, 1950; Pauley and Sparks, 1965). Diffuse collagen microfilaments were seen in the area of injury almost immediately and appeared to be the result of exposure to sea water, rather than the early synthesis of new material. This theory was supported by the absence of larger cell populations at 48 hr postinjury, when mesogleal expansion was significant and the microfilaments were widely dispersed (Patterson and Landolt, 1978). During the initial stages of the repair process, the cellular infiltrate consisted of a homogenous population of primary and secondary phagocytes. Between 48 and 72 hr postinjury, a zone of repair was formed. On the distal side of the region collagen filament arrangement appeared normal and most cells in the region contained specific granules with no evidence of secondary lysosomes, the predominant feature of cells within the wound area. At the margin between normal and damaged tissue was the region of atypia and high cell density. The most numerous of these altered cells showed a prolific rough endoplasmic reticulum and evidence of high energy-dependent activity. The function of these cells was undetermined; however, their morphology was suggestive of intense protein production. Absence of a Golgi apparatus and a lack of storage granules suggested that this material was released locally in uncomplexed form. The occurrence of
195
INJURY
TO INJURY
Cell number/mm2 4.9 5.1 8.0 1.4 1.3 7.0
x x x x x x
103 103 lo3 104 104 103
(SD) (1.5 (1.5 (2.1 (3.4 (2.4 (3.2
x x x x x x
IO’) 103) 103) IO’) 10)) 10))
injury-related proteins in cnidarians has not been documented; however, the small number of cells and their local occurrence would be consistent with very low total protein levels. Accumulation in a small, welldefined area would allow small levels of protein to reach significant concentrations in a restricted zone. In addition to protein cells, a few cells showed massive proliferation of the agranular endoplasmic reticulum and the Golgi. Like the basophils, these cells were localized in the transition zone and were not seen in other regions. The function of smooth endoplasmic reticulum is not as clear as rough endoplasmic reticulum, and functional correlations depend on enzymatic patterns. The low cell number and an inability to discriminate these cells from phagocytes using conventional light microscopy has hampered investigation of their role in the reaction to injury. Their structural features would be compatible with such cell lysins as saponins, where a lipid derivative is coupled to a series of sugar residues and released. Hemolysins are known to exist in the cnidarians (Keen and Crone, 1969), but their chemical identity or possible induction by injury remains unknown. The cells in the reactive zone, regardless of morphology, did not appear mitotically active. Centrioles were occasionally seen, but no mitotic figures were discernable by light or electron microscopy. No wellorganized storage site has been reported in these animals; however, proliferation sites in the gastrodermis have been suggested by Young (1974), who also demonstrated some
196
PATTERSON
sensitivity of the reaction to colchicine, an inhibitor of cell multiplication. Lack of intermediate cell forms leading from normal amoebocytes to altered forms implied the production of modified cells at some site distant from the injury, followed by their migration or accumulation in the zone of repair. The migration of specific cells from a distal proliferation or differentiation site to the wound implies a degree of control, possibly chemical, which is not needed to explain the presence of normally motile resident amoebocytes. Injuries used in this study were small and did not alter the behavior patterns of experimental animals. The extensive mechanisms for asexual reproduction did not appear to be employed. Rather, these local injuries elicited an immediate influx of amoebocytes involved in phagocytizing necrotic tissue and eliminating it by ejection into the surrounding water. This was followed by a progressive zone of repair, which proceded through the wound area and reestablished tissue integrity. The repair zone contained cells which were unique to the reaction to injury and appeared to be active in the synthesis of various substances. The lack of a circulatory system or any specific organs makes the concept of an inflammatory response difficult. There was, however, a distinct series of cellular events following injury. These events, and the component cells, appeared to be distinct members of an organized reaction to injury which prevented secondary infection, isolated the damaged region,
AND LANDOLT
and initiated tissue repair. This ancient group of animals appeared to have developed a functional inflammatory response which predates the origin of a circulatory system or specialized organs. ACKNOWLEDGMENTS We are grateful for the assistance ofDr. M. C. Lowe, Dr. G. B. Pauley, and Dr. A. K. Sparks. This project was supported in part by a research grant from the University of Washington Graduate School Research Fund.
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