Studies of lymphocytes: Relationship to mononuclear cells of inflammatory exudates

Biochemical Pharmacology, Supplement, pp. 185-196. Pergamon Press. 1968. Printed in Great Britain

STUDIES OF LYMPHOCYTES: RELATIONSHIP TO M O N O N U C L E A R CELLS OF I N F L A M M A T O R Y EXUDATES* N. B. EVERETT and R. W. TYLER Department of Biological Structure, University of Washington, Seattle, Washington Abstract--Lymphocytes are considered with respect to morphology, tissue origin, life-span, developmental potential, and function. The data presented are based primarily on in vivo kinetic studies in rats by means of tritiated thymidine and radioautog~aphy. Evidence is given supporting the concept of heterogeneity of lymphocytes, more particularly of the small cells for which two major populations, long- and short-lived varieties, are now generally recognized. In addition, results of in vitro studies of lymphocytes, by the monolayer culture system, are reviewed, which show that long-lived cells enlarge and divide in response to antigenic stimulation. The questions of identity and origin of the mononuclear cells which appear in subcutaneous inflammatory exudates are considerea. The results of further studies concerning these cells are presented. AS WE learn more about the mononuclear, agranular leucocytes to which the term "lymphocyte" is usually applied, it becomes increasingly difficult to provide an acceptable definition for this group of cells on the basis of conventional morphological criteria. Included in the lymphocyte category are cells of the thymus, bone marrow, and connective tissue as well as cells of spleen, lymph nodes, lymph, and blood. Many writers have expressed the belief that these cells may not all have the same origin, fate, or function by referring to cells of bone marrow as "lymphocyte-like" cells and to cells of thymus as "thymocytes", thereby reserving the term "lymphocyte" for those cells present in lymph and lymph nodes. From a practical point of view we must accept a morphological definition to identify a lymphocyte, although this does not mean that we must consider these cells as a homogeneous group. It is becoming increasingly evident that when speaking of the lymphocyte we are speaking of a heterogeneous population of cells grouped together only by their similarities in morphology and that what may be true of one small portion of the total population should not be applied as a general rule to all of it. The problem with the morphological definition for the lymphocyte (as Yoffey so adequately stated) is that the lymphocyte must be identified with respect to the absence of characteristics which other leucocytes possess rather than by positive attributes of its own. 1 The gradation in sizes of lymphocytes is illustrated by smears of rat thoracic duct lymph (TDL) (Fig. 1). A distinguishing feature of medium and small cells is the high ratio of nucleus to cytoplasm. Moreover, the nucleus of the small lymphocyte is typically pachychromatic. Some of the larger cells, however, have * Based upon studies supported by research grant GM-06309 from the National Institutes of Health, Public Health Service, and by U.S. Atomic Energy Commission reseazch contract AT(45-1)Â377. 185

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abundant cytoplasm while others show only a basophilic rim. Cells with essentially identical morphology are present in the thymus although they tend to be slightly smaller than cells of the T D L and the majority of the large and medium thymocytes have only a basophilic rim of cytoplasm. Likewise, the small lymphocytes of the bone marrow may be slightly smaller than T D L cells and again large and medium forms are present, which have a basophilic rim of cytoplasm. The medium-large cells in bone marrow have been termed "transitional" cells by Yoffey2 who has considered them to be intermediate forms between the small lymphocytes of the bone marrow and cells of the erythrocytic and myelocytic series. However, recent experiments using bone marrow occlusion and local marrow labeling techniques ~, 4 have shown that small lymphocytes are formed in the marrow and that the so-called transitonal cell is their most likely precursor. Furthermore, at least some of the small lymphocytes formed within the marrow enter the blood, and it would be impossible to distinguish them morphologically from those entering blood by way of the TDL. The similarity in structure of small lymphocytes from different tissues is also applicable at the electron microscope level of resolution. Fig. 2 shows the characteristic fine structure of the small lymphocyte. Whether, in the future, the lymphocyte retains a strictly morphological definition or acquires a functional one will probably depend on the establishment of relationships between the ceils at the various hemopoietic and lymphopoietic sites. Considerable progress has been made in recent years toward clarifying these relationships. In this paper some of the evidence relating to the heterogeneity of the lymphocyte population is presented along with more detailed considerations of studies concerning the origin and life-span of small lymphocytes. Results of these studies are related to advances that have been made in our knowledge of lymphocyte function. The results of preliminary studies concerning the origin of mononuclear cells in inflammatory exudates are also reported.

Large, medium and small lymphocytes It is conventional to consider three size categories of lymphocytes--large, medium and small, although this distinction is generally made subjectively. It is possible, however, to correlate lymphocyte size with proliferative behavior. Such a study was carried out in our own laboratory ~ in the rat with the following results: (1) All (100%) of the large lymphocytes (more than 10/~ in nuclear diameter) in T D L and lymph nodes were labeled by 36 hr of continuous infusion of thymidine-H 3 (TTH). (This observation was also true in blood, bone marrow, and spleen but not in thymus.) No labeled large lymphocytes were observed in any tissue at intervals greater than 2 weeks after single or multiple TTH-injections, due apparently to division to form smaller cells. (2) Small lymphocytes (7 tz or less in nuclear diameter) did not incorporate T T H but became labeled by division of labeled precursors. A large percentage of these cells in nodes, TDL, and spleen were labeled at prolonged intervals after multiple injections. (3) Some of the medium lymphocytes (nuclear diameter between 8 and 10/~) incorporated TTH. Unlike the large lymphocytes, not all of these cells could be labeled by prolonged administration of TTH. At 2 weeks after multiple injections, a minor percentage of medium cells remained labeled.

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B FIci. 1. Cclls from thoracic duct lymph of y o u n g adult rats, showing large, mediuna, and small lymphocytes. Note the relatively n a r r o w basophilic rim of cytoplasm in the large cell in A and the more e×tensive, less basophilic cytoplasm of the upper large cell in B. (A , 1660; B 21001.

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PIc;. 2. Small lymphocyte from the lhoracic duct of a normal ral; fixed in glutaraldeh)dc and poslfixed ~filh osmic acid, both with tissue culture naedium 199 as vehicle. The condensed chromalin of tile nucleus is typical of gluiaraldehyde fixation. Irregular microvilli extend from the cell surl'acc and lhere is a prominent Qolgi region near tile nuclear indentation. Several milochondri~ arc seen a.~ \veil as free ribosomes bul rio rough endoplasmic reticulum is evidem. ( 20,000, laken \~iih 3(i elcclrc, n nlicroscopc). (Courlcsy of Dr. Jaille~, K. Kochlcr).

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FiG. 3. R a d i o a u t o g r a p h s showing distinctly labeled small lyrnphocytes recovered l'rom T I ) L o f a ra~ l yr after administration of multiple doses of thynlJdine-H :~ I 1660}.

F-I~.~. 4. Representative cells from a 2-day m o n o l a y e r culture of lymphocytes from rat T D L . Tile enlarged cells arose from small lymphocytes. Tile cell in B is in file telophase stage of mitosis ( 1 6 6 0 ) . Fl(~. 5. R a d i o a u l o g r a p h of cells from nlonolayer culture of lymphocytes frolll T D L ; only cells labeled at the beginning of the culture period were Iong-li~ed I)nlphocyles ( 1660}.

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D Fi(i. 6. M o n o n u c l e a r celE in smears of rat tissue e x u d a l e s al intcrxal~ after placing coverslip~ ~.c. ( 1 6 6 0 ) . A and B, 3 hr interval. Tile cell at the right ill A is a typical nlonocyle. Tile o l h e r mononuclear cells ate considered to be nlonocytoi d in I)pe. (', 6 hr inter~al. 1). 24 hr intcrxal.

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The results of these kinetic experiments, carried out in vivo, are representative of the vast majority of the lymphocytes. It is now well established, however, that at least some small lymphocytes can, when properly stimulated, enlarge to cells greater than 10t~ in nuclear diameter, but it should be emphasized that this reaction is limited to a very small fraction of an animal's total small lymphocyte population as measured at any one time. This phenomenon is of such low frequency that it is apparently not encountered in the unstimulated animal, and it is not common even under the proper conditions of stimulation. 6

Kinetics of small-lymphocyte populations Studies in our own laboratory, using T T H and radioautography, have shown that the small-lymphocyte population is comprised of two major groups with respect to life_span.7, 8 Moreover, except for bone marrow, the various lymphocyte compartments contain a mixture of the two groups which cannot be distinguished from each other by morphological criteria. Bone marrow has been shown to contain only the short-lived variety. It is not implied, however, that there are only two groups of small lymphocytes, and it would not be surprising that physiological studies would lead to subdivision of each of the established categories. Two basic types of experiments, using multiple injections of TTH, established that there are two populations of small lymphocytes in the rat. 8, 9 The first measured the rate of disappearance of labeled small lymphocytes subsequent to a 2-week period of multiple injections. The second measured the rate at which small lymphocytes appeared during an l 1-day period in which T T H was injected at intervals frequent enough to label all cells entering DNA synthesis. Rate of disappearance of labeled small lymphocytes. In this series, each rat was given twelve injections of T T H over a 16-day period prior to start of the experiment. (Each injection was 0.5/zc/g body wt.) The animals were sacrificed at intervals of 2 weeks or less after the last T T H injection. The decrease in percentage of labeled small lymphocytes was greater during the first 2-week period after T T H than in any of the subsequent 2-week intervals. This was interpreted as indicative of two populations of small lymphocytes, one with a life-span of more than 2 weeks and the other with a life-span of less than 2 weeks. Not all of the short-lived cells were labeled at zero time in this series of animals because the frequency of T T H injections did not provide for labeling all cells entering D N A synthesis. Thus, the percentage of labeled lymphocytes in the thymus and bone marrow was not 100 per cent at zero time as in the case of animals receiving more frequent injections. It may be pointed out, however, that the initial percentage of labeled cells is not pertinent to the consideration since the rate of decrease in the percentage of labeled cells during the first 2 weeks cannot be used to measure life-span of the short-lived cells. This is the case because label is not cleared sufficiently from precursor elements through cell division until 2 weeks postinjection. Moreover, during the first 2 weeks, there is considerable reutilization of label. 1°-a2 At periods longer than 2 weeks postinjection, however, the rate of decrease in percentage of labeled cells was interpreted as being proportional to the rate of long-lived lymphocyte formation since label was no longer evident in the large precursor cells and there was no detectable reutilization of TTH. The rate of formation of long-lived lymphocytes was essentially proportional to the body growth of the animals 7 and significant numbers of these labeled cells were recovered from rats BIO. SUPPL.~N

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sacrificed at periods up to 1 yr postinjection with no apparent reduction in grain count.la, 14 Fig. 3 shows labeled small lymphocytes from T D L of an animal 1 yr postinjection. Labeled lymphocytes were no longer present in thymus and bone marrow by 2 weeks postinjection, and it was concluded that most, if not all, of the cells in these organs are of the short-lived variety. The decreasing order for percentages of short-lived cells in other tissues was: spleen > blood > nodes > TDL. Rate Of appearance of labeled small lymphocvtes. In this series each animal was given 0.75/xc of T T H / g body wt. every 6 hr until sacrificed. This injection schedule was believed to be adequate for labeling all cells entering D N A synthesis because the resultant percentages of labeled cells were comparable to those of animals receiving continuous T T H infusions. 8 Labeling of small lymphocytes in bone marrow approached 100 per cent by day 4 of the 6-hr injection schedule, giving a calculated half-time renewal rate of 24 hr. This turnover rate exceeded that for small lymphocytes of all other tissues. The halftime renewal rate of small lymphocytes in the thymus, generally considered to be perhaps the highest rate of lymphocyte turnover, ~ was 36 hr. Spleen had the next highest rate of small lymphocyte turnover, and following in decreasing order were blood, mesenteric lymph node, and TDL. This order of lymphocyte turnover was the same as that determined by the experiments measuring the rate of disappearance of labeled cells. Blood showed a sharp break in the slope of the labeling curve for small lymphocytes after 4-5 days of injections, which was attributable to essentially 100 per cent of the short-lived cells being labeled by that time. Thus, the life-span of the short-lived population appeared to be of this order, 4-5 days. It is to be noted, however, that the disappearance of nonlabeled small lymphocytes from the bone marrow and thymus was a logarithmic function of time, which indicates that the life-span of the short-lived lymphocytes is probably not finite. It was estimated that approximately one third of the small lymphocytes in blood at any one time were of the short-lived variety. By considering the slope of the blood curve after 5 days to indicate the rate of long-lived lymphocyte formation, it was possible to estimate the percentages of tong- and short-lived cells in other tissues. This consideration indicated that approximately 90 per cent of the small lymphocytes in T D L were long-lived and 10 per cent were short-lived.

Percentages of labeled small lymphocytes in blood and TDL Comparison of blood and T D L with respect to appearance and disappearance of labeled small lymphocytes after the series of T T H injections reveals differences in rates, which are believed to reflect differences in sites of origin as well as in routes of entry into these tissues. For example, labeled small lymphocytes appeared in blood at a faster rate and reached a higher level than in TDL. Moreover, the disappearance curves show that the percentage of labeled cells decreased more rapidly in blood than in T D L during the first 2 weeks and was lower in blood than in T D L from 4-8 weeks postinjection. This difference in percentage of labeled cells in these compartments we have attributed to direct-entry lymphocytes (lymphocytes which enter the bloodstream directly without first entering the lymph). (Direct-entry lymphocytes have been considered in some detail by Yoffey. 2, 16) The most likely sources for the direct-entry, short-lived lymphocytes would seem to be thymus, bone marrow, and spleen. There

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is no convincing evidence, however, that significant numbers of thymocytes enter the blood and, in fact, the recent labeling experiments of Rieke and Schwarz iv using thymectomized rats indicate that the thymus is not a primary source of direct-entry cells. The spleen is, no doubt, a major contributor of the direct-entry cells because approximately one third of its small lymphocytes are of the short-lived variety. Moreover, it is not common for lymphatic vessels of the spleen to be well developed. It is probable, however, that the bone marrow serves as the major source of the direct-entry, short-lived lymphocytes in the rat. Studies have shown that the population of bone marrow lymphocytes in the guinea pig and rat is approximately 20 times the number in blood. 2, 18 Furthermore, recent studies indicate that in the guinea pig and rat the great majority, at least, of bone marrow lymphocytes arise in situ. 3, 4, 8 The latter studies also provided evidence that significant numbers of bone marrow small lymphocytes of the rat enter the bloodstream. Localization and origin o f long- and short-lived small lymphocytes As indicated above, studies from this laboratory have demonstrated that approximately 90 per cent of the small lymphocytes in T D L of the rat are of the long-lived variety. 8 Thus, it is clear that long-lived lymphocytes comprise the major population of cells which have been shown to enter the blood and recirculate repeatedly from blood via the lymph nodes and Peyer's patches. 19 This recirculating pool has been estimated to contain approximately 7.8 ;< 10~ small lymphocytes per gram of body weight for rats, 7 which is in accord with studies by Gowans and Knight. 19 Small lymphocytes of this recirculating pool may be localized in or course through specific areas of the hemopoietic tissues. Tissue sections from rats which received multiple injections of T T H 2 weeks or more prior to sacrifice often evidenced numerous labeled lymphocytes in the white pulp of the spleen, in Peyer's patches, and in the peripheral zone of germinal centers of lymph nodes. No labeled small lymphocytes were present in the bone marrow or thymus at these postinjection intervals, and they were seldom observed in the red pulp of the spleen or within the germinal centers of the lymph node or spleen. These tissues, or areas thereof, which were essentially free of labeled cells are sites in which approximately 100 per cent of the lymphocytes were labeled after 5 days of continuous T T H injections and are thus considered to be areas of short-lived lymphocyte formation. Experiments have shown that, after transfusion of labeled, long-lived small lymphocytes from TDL, cells were localized in lymph nodes, splenic white pulp, and Peyer's patches.8, 13, 19, 20 Bone marrow and thymus, however, have not been observed as sites of localization for transfused long-lived cells, s , 2o In contrast, when cells of labeled bone marrow or thymus were transfused, considerable numbers of labeled small lymphocytes appeared in bone marrow and splenic red pulp. s, 21 Animals may be depleted of the long-lived small lymphocytes by chronic drainage of the TDL. 8, 2z, 23 The tissues of these animals show a depletion of lymphocytes in the areas normally occupied by the long-lived cells. Immediately after chronic drainage of TDL, the thymus and bone marrow also show a depletion of lymphocytes. Our unpublished observations indicate, however, that the loss of cells from thymus and bone marrow results from operational stress and not from drainage of short-lived cells through the fistula. In light of the above, it is appropriate to emphasize that the ceils of T D L are essentially a select group of long-lived recirculating cells, unlike

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the average small lymphocytes of the thymus and bone marrow. This consideration has important functional implications. Although the origin of the long-lived small lymphocytes has not been definitely established, indirect evidence indicates that they actually originate in the thymus. The origin of the short-lived lymphocytes appears to be at lymphopoietic sites which have been shown to have a high mitotic r a t e - f o r example, bone marrow, thymus, and splenic red pulpy Functional considerations of small lymphocytes The view of many hematologists that lymphocytes may serve as stem cells for a variety of other cell lines, and more particularly as granulopoietic and erythropoietic precursors, has been seriously challenged by experiments using T T H and radioautography. Application of these techniques, however, has shown that the small lymphocyte is not necessarily an end cell. It is now well established, both from i, citro and in vivo studies, that small lymphocytes when appropriately stimulated may transform into large "blast-like" cells which have immunologic and mitotic potential. The recently developed monolayer culture system of Ginsburg and Sachs, 24 which has been used for culturing lymphocytes, offers considerable promise for a functional analysis of the cells which arise from stimulated small lymphocytes. The system has, in fact, permitted obtaining an effective graft-versus-host reaction. Stated simply, the method as applied by Ginsburg and Sachs involves culturing rat lymphocytes on monolayers of growing mouse embryo cells. On the second or third day of culture, large lymphoid cells appear, some of which have a morphology similar to the pyroninophilic cells of Gowans and to phytohemagglutinin (PHA) cells (Fig. 4). Clones of enlarged cells are produced, apparently from single cells. These initiate a destruction of the mouse monolayer at the areas of contact, and dissolution spreads until complete destruction of the monolayer occurs, usually within 6-9 days of culture. The transfer of the large lymphoid cells to a second plate of the same type of monolayer produces a destructive effect within a few hours. The transfer of the supernate fails to produce any destruction, indicating a cell-associated antibody rather than a humoral one. This culture system, coupled with T T H and radioautography, has recently been used in this laboratory in collaboration with Dr. Ginsburg in studying small lymphocytes, z5 The cells cultured were from rat T D L in which only the long-lived cells were labeled (the lymph was collected 4 weeks after the last multiple T T H injections). After 2 days of culture, labeled large blast cells appeared with the same degree of label as the control of small lymphocytes (Fig. 5). Moreover, the initial percentage of blasts labeled was the same as for the small lymphocytes of the original sample. The percentage of labeled blasts decreased with time, which reflected a dilution of label through cell division. Destruction of the monolayer was essentially complete by day 6 and at this time approximately 40 per cent of the lymphoid cells were large blast forms. None of these were labeled. There was no change in the percentage of labeled small lymphocytes during the culture period. It was estimated that less than 3 per cent of the small lymphocytes enlarged to give rise to the original blasts which served as stem cells for the clones of enlarged cells. The majority of small lympbocytes in the system either remained unchanged or died during the culture time. These results may be compared with the in viro transfusion experiments of Gesner and Gowans 6 in that the majority of the small lymphocytes transfused remained un-

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changed, and only a small percentage gave rise to large pyroninophilic cells. In accord with these observations are those of Astaldi and Airo 26 who reported that, when human lymphocytes were cultured with PHA, the blasts arose from a small percentage of the lymphocytes which underwent continued proliferation to give rise to more blasts with time. There is considerable variation in the morphology of cells which develop in the monolayer cultures as well as in the PHA culture system. Variation in the latter has recently been discussed and well illustrated in color. 27 It is possible that the various morphological forms which appear in lymphocyte cultures arise from small lymphocytes with different developmental potentials. Another possibility is that variations in morphology may reflect differences in reaction or response to different stimuli. In this connection it may be noted that, in addition to the blast-like forms which developed in monolayer cultures of long-lived lymphocytes of TDL, cells arose which resembled the so-called "activated" small lymphocytes of irradiated or pertussis vaccine-treated animals. 28, 29 The monolayer destruction, however, appeared to be more closely associated with the large blast forms. Electron microscope studies have been made of the blast forms from the monolayer system (H. Ginsburg and D. Lagunoff, unpublished data) as well as from PHA cultures 3° (M. R. Schwarz, unpublished data) and in neither case has a rough endoplasmic reticulum been found. Moreover, an analysis of the supernatant fluid from the monolayer system, by immunophoresis with anti-rat-serum antibody, showed no 7-globulin production, although a-2- and r-globulins were present in some of the cultures. 25 It may be noted, however, that pokeweed mitogen-stimulated cells have been reported to have a well-developed endoplasmic reticulum) 0 Evidence for 7-globulin production by these cells is inconclusive. Thus, it may be said that even among the long-lived small-lymphocyte population there appears to be a functional heterogeneity in which only a small portion of the cells respond to any one antigen and that the transformed cells may vary greatly in their morphological appearance. With regard to the relationship between the lymphocytic and plasmacytic series, it has been established that some plasma cell precursors are found in TDL. The electron microscopic studies by Zucker-Franklin 31 show that some of the large and medium-size cells in T D L have rough endoplasmic reticulum. It is reasonable to assume that these would give rise to mature plasma cells. Cell cultures of T D L in our own laboratory have yielded small numbers of mature plasma cells, and labeling experiments show that they are derived from the large and medium cells of T D L which incorporate T T H rather than from the long-lived small lymphocyte. Additionally, Gowans 32 observed that some T D L small lymphocytes of parental strains transformed into large pyroninophilic cells when injected in F1 hybrid rats. The most convincing evidence showing that small lymphocytes are directly involved in circulating antibody synthesis comes from the single-cell-culture experiments of Attardi e t al. ~3 These investigators showed that small lymphocytes contained antibody capable of inactivating bacteriophage, although only a small percentage of the cells appeared to be active. Whether or not the short-lived small lymphocyte is capable of hypertrophy and division has not been definitely established. It has not been possible to obtain T D L or lymph node cells in which the label is retricted to the short-lived variety because the large and medium lymphocytes and some long-lived cells also are labeled under

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similar conditions. Recently, however, Schwarz and Rieke :~4 reported that a small percentage of rat small thymocytes transformed into blast cells when cultured with PHA. Since the number of thymus cells required in culture was four times that of T D L lymphocytes, it is possible that the cells which enlarged represented the small percentage of long-lived cells in the thymus. The recent preliminary report by Osmond 35 would suggest that bone marrow small lymphocytes of the guinea pig may enlarge and undergo D N A synthesis when stimulated in t,itro with PHA. The definitive progeny of the blasts which arise from small lymphocytes thus far appear to be only small lymphocytes. The morphological similarity of the enlarged cells to other blast forms such as erythroblasts, myeloblasts, plasmoblasts, and hemoblasts, and hemocytoblasts only illustrates the inadequacy of purely morphological criteria for characterizing these cells. That lymphocytes are involved in a variety of immune or "immune-like" responses is no longer questioned even though there are many uncertainties relative to the extent of their involvement as well as to the precise way in which they may function in response to certain immunological stimuli. Much of the confusion relative to lymphocyte function as indicated in the above account relates to our failure to appreciate the heterogeneity of the lymphocyte population. Moreover, we have lacked adequate techniques to establish clearly the interrelations and developmental potentials of the various mononuclear cells which are generally placed in the lymphocyte category. We can, however, be optimistic that continued application of the techniques now at our disposal will provide answers to such questions as the nature and origin of the mononuclear cells from blood which appear in sites of inflammation and give rise to the macrophages of inflammatory exudates. Source o f mononuclear cells in inflammatory exudates

Strong support for the view that small lymphocytes are the source of these macrophages has been provided by Rebuck and his associates. 36, 37 This conclusion has been reached by following the time sequence of cells which appear on coverslips that have been applied to human skin from which the epithelium has been scraped away. Lymphocytes were reported to appear within the first few hours and with increasing frequency with time so that at 12 hr they constituted the majority of the exudate cells. These evidenced a progressive hypertrophy of nucleus and cytoplasm, definite by 16 hr, and by 24 hr could not be distinguished from tissue macrophages. Evidence for the transformation of lymphocytes into macrophages has also been provided by Rebuck and colleagues 37 from use of the blister technique which has allowed for continuous and direct observation of exudate cells in the aspirated blister fluid. Braunsteiner et al. 3s applied the skin window technique to rats in combination with T T H and also concluded that lymphocytes serve as precursors of macrophages. They reported that macrophages which appeared on the coverslips were labeled after multiple injections of T T H had been given. Volkman and Gowansp 9 in a more recent application of the skin window technique as well as from use of s.c. coverslips in rats, provided evidence for excluding small lymphocytes of blood as precursors of the macrophages of exudate. These investigators showed that up to 57 per cent of the macrophages on skin windows removed after 24 hr were labeled by a single injection of T T H given 1 day before

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application of the coverslip. In contrast, labeled small lymphocytes of blood did not exceed 2 per cent after a single T T H injection. In an extension of these studies, Volkman and Gowans 40 found that lymphocyte depletion by either chronic thoracic duct drainage or x-irradiation failed to suppress the appearance of labeled macrophages on coverslips after an injection of TTH. Moreover, after the injection of labeled cell suspensions from TDL, lymph nodes, thymus, spleen, and bone marrow into syngeneic rats, labeled macrophages appeared on coverslips only in the recipients of bone marrow and spleen cells. It was also noted that labeled monocytes appeared in the blood of animals which received injections of labeled bone marrow. In accord with the observations of Volkman and Gowans and with earlier studies by Ebert and Florey 41 are those of Spector and colleagues 42, 43 who found that essentially all of the mononuclear cells which appeared in inflammatory exudates were derived from circulating monocytes. These investigators used T T H and colloidal carbon as markers for the mononuclear cells which appeared in cutaneous exudates induced by administering fibrinogen or paraffin oil. In accord with these observations are those of Trepel and Begemann 44 who combined the s.c. coverslip method with the use of thymidine-H ~ or India ink for following the labeling pattern of cells which appeared in inflammatory exudates of rats. More recently, by using labeling techniques, Spector 45 has shown that the mononuclear cells of tuberculin reaction sites in rats are derived primarily from blood monocytes. Earlier, Kosunen e t al. 46 found that, when T T H was given before a challenging dose of tuberculin, up to 50 per cent of the mononuclear cells in the resulting exudates were labeled. This implicated cells with a high turnover rate and it was suggested that large or medium lymphocytes were primarily involved. In view of these conflicting reports relative to the identity and source ofmononuclear cells in inflammatory exudates, we have recently approached the problem, using the rat as the experimental animal. In these studies it seemed desirable to use procedures that would allow for preparing tissue smears much the same way as we have in our rather extensive studies of lymphocytes during the past several years. It was found that insertion of coverslips s.c. as described by Volkman and Gowans39,40 stimulated the accumulation of considerable amounts of tissue fluid and cells which could be withdrawn by pipette. Accordingly, conventional type smears were made of this fluid from a series of young adult rats at intervals from 1 to 24 hr after applying several coverslips to each animal. Low-speed centrifugation was used in some instances to increase the cell concentration before smearing. The smears were stained routinely with MacNeal's tetrachrome. By 3 hr after insertion of the coverslips, considerable amounts of tissue fluid and ceils had accumulated in the tissue spaces. At this time, neutrophils were the predominant cell type although there were numerous mononuclear cells and many of these were monocytic or monocytoid in appearance (Figs. 6 A and B). Typical small lymphocytes were rarely seen at this or at any of the other intervals sampled. By 6 hr the percentage of mononuclear cells had increased and, on the average, these were larger than at the 3-hr interval (Fig. 6C). It is to be emphasized, however, that there was considerable variation in cell size. In samples from some of the animals at this time, there were a few cells which evidenced characteristics of early macrophages. By 12 hr there was a further increase in the number of mononuclear cells. Moreover in some of the exudates there was a further increase in mononuclear cell size as well

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as an increase in the number of cells which were clearly identifiable as macrophages. Neutrophils were still numerous at 12 hr. In the 24-hr exudates, large mononuclear cells were obviously the predominant cell type and a high percentage of these evidenced vacuolar cytoplasm typical of phagocytic cells (Fig. 6D). Results of studies in progress support the findings of Volkman and Gowans 40 in showing that bone marrow is a source of the mononuclear cells which appear in inflammatory exudates. This confirmatory evidence derives from observation of heavily labeled mononuclear cells in radioautographs of the subcutaneous exudates of animals in which localized T T H injections were made into the tibial marrow as described previously. 4 Three 20-/xc injections of T T H were made into the tibial marrow parenchyma over a period of 48 hr before insertion of the subcutaneous coverslips. (As in earlier experiments, the escape of T T H into other body tissues was prevented by a rubber compression bandage placed around the thigh. Moreover, as a further control, excess amounts of unlabeled thymidine were administered into the general circulation while the hind limb circulation was occluded.) Radioautographs of the tissue exudates made at 6, 12, and 24 hr after insertion of the coverslips revealed considerable numbers of distinctly labeled mononuclear cells of the same morphology as described above for the nonradioautographic preparations. Considerable numbers of labeled neutrophils were also present. No other labeled cell types were noted. In assessing these results we can only conclude that the mononuclear cells which first appeared in the inflammatory exudates and served as macrophage precursors were not small lymphocytes. They have the morphology of cells for which we have used the term "monocytoid" and were shown to include monocytes as well as cells responsible for the recovery of bone marrow in rats which received lethal doses of irradiation. 47 The term "monocytoid" was used because the cells with stem cell capacity could not be distinguished morphologically from cells in the monocytic series. It is to be emphasized, however, that the term was not meant to be synonymous with the term "monocyte." Our earlier description 47 of rat monocytoid cells is repeated here because it is applicable to the mononuclear cells which first appeared in the inflammatory exudates of the present experiments and served as macrophage precursors: The cells which were identified in the present study as monocytoid cells were approximately the size of a medium lymphocyte (8-10~ in nuclear diameter). The majority have an irregular shaped nucleus with many invaginations and folds, while the nucleus of the lymphocytic series tends to be round. The nucleus is usually located centrally while the nucleus of the lymphocytic series is more eccentric. The chromatin does not form dense masses as in the small lymphocytes. The cytoplasm is not as basophilic as in the lymphocytic series and it is much more abundant than that seen in the majority of medium to large lymphocytes. In conclusion, the results of our preliminary studies are in line with those suggesting that the mononuclear cells of inflammatory exudates which serve as macrophage precursors are derived from circulating monocytes and not fi'om small lymphocytes. Moreover, the results are in accord with those of Volkman and Gowans relative to an origin from bone marrow.

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It is r e c o g n i z e d t h a t t h e differences in r e p o r t e d results r e l a t i v e to the c e l l u l a r o r i g i n o f i n f l a m m a t o r y m a c r o p h a g e s are, in p a r t , a t t r i b u t a b l e to t h e i n a d e q u a c y o f m o r p h o l o g i c a l c r i t e r i a a l o n e f o r d i s t i n g u i s h i n g b e t w e e n c e r t a i n cells. M o r e o v e r , it is a p p r e c i a t e d t h a t s o m e i n v e s t i g a t o r s use the t e r m " l y m p h o c y t e , " o r " l y m p h o i d , " m o r e b r o a d l y t h a n d o others, w h i c h f u r t h e r p o i n t s to t h e n e e d f o r c o n s i d e r i n g a d d i t i o n a l c r i t e r i a s u c h as f u n c t i o n a n d o r i g i n in c h a r a c t e r i z i n g c e r t a i n cells. REFERENCES 1. J. M. YOFFEYand F. C. COORT~CE,in Lymphatics, Lymph and Lymphoid Tissue. Edward Arnold, London (1956). 2. J. M. YOFFEY, Quantitative Cellular Haematology. Charles C. Thomas, Springfield (1960). 3. D. G. OSMONDand N. B. EVERETT,Blood 23, 1 (1964). 4. N. B. EVERETT and R. W. CAFEREY, in The Lymphocyte in Immunology and Haemopoiesis (Ed. J. M. YOFFEY), p. 108. Edward Arnold, London (1967). 5. W. O. RIEKE, R. W. CAFFREYand N. B. EVEREaT, Blood 22, 674 (1963). 6. B. M. GESNER and J. L. GOWANS, BE. J. exp. Path. 43, 431 (1962). 7. R. W. CAFrREY, W. O. RIEKE and N. B. EVERETT,Acta haemat. 28, 145 (1962). 8. N. B. EVERETT, R. W. CAErREYand W. O. RIEKE, Ann. N. Y. Acad. Sci. 113, 887 (1964). 9. N. B. EVERETTand R. W. TYLER, in International Review Cytology (Ed. J. F. DANNI~LU), VOI. 22, in press. 10. D. G. OSMONDand N. B. EVERETT,Nature, Lond. 196, 488 (1962), 11. S. H. ROBINSON,G. BRECHER,S. I. LOURIEand J. E. HALEY, Blood26, 281 (1965). 12. R. W. CAFrREY, N. B. EVERETTand W. O. 1OEKE, Anat. Rec. 155, 41 (1966). 13. N. B. EVERETT, R. W. CAFFREYand W. O. RIEKE, Proc. IXth Congr. Int. Soc. Hemat., 3, 345 (1963). 14. N. B. EVEREYTand R. W. CAFFREY,Anat. Rec. 148, 279 (1964). 15. J. E. KINDRED, Am. J. Anat. 71,207 (1942). 16. J. M. YOFFEY, in The Lymphocyte in Immunology and Haemopoiesis (Ed. J. M. YOErEY), p. 1. Edward Arnold, London (1967). 17. W. O. RaEKE and M. R. SCI-tWARZ, in The Lymphocyte in Immunology and Haemopoiesis (Ed. J. M. YOFFEY), p. 224. Edward Arnold, London (1967). 18. T. G. RAMSELLand J. M. YOFFEV, Acta anat. 47, 55 (1961). 19. J. L. GOWANS and E. J. KNIGHT, Proc. R. Soc. B159, 257 (1964). 20. D. O. ANDERSONand D. M. WHITELAW,Am. J. Physiol. 199, 824 (1960). 21. C. A. MIMS, BE. J. exp. Path. 43, 639 (1962). 22. J. L. GOWANS,J. PhysioL, Lond. 146, 54 (1959). 23. D. D. McGREGOR and J. L. GOWANS,J. exp. Med. 117, 303 (1963). 24. H. GINSBURGand L. SACHS,J. cell comp. PhysioL 66, 199 (1965). 25. l-l-. GINSBURG,R. W. TYLER and N. B. EVERETT,Anat. Ree. 157, 247 (1967). 26. G. ASTALDIand R. AIRO,in The Lymphocyte in Immunology and Haemopoiesis (Ed. J. M. YOFFEY), p. 73. Edward Arnold, London (1967). 27. J. M. YOFEEY,G. C. B. WINTER,D. G. OSMONDand E. S. MEEK, Br. J. Haemat. 11,488 (1965). 28. N. B. EVERE2WF,R. W. CAFFREYand W. O. RIEKE, Radiat. Re~. 21,383 (1964). 29. N. B. EVERETT,R. W. CAFFREY,W. O. RIEKE and M. R. SCHWARZ,in Use o f Radioautography Invest(~ation in Protein Synthesis, p. 143. Academic Press, New York. 30. S. D. DOUGLAS,P. F. HOFEMAN,J. BORHESONand L. N. CHESSIN,J. Immun. 98, 17 (1967). 31. D. ZUCKER-FRANKLIN,Or. Ultrastruct. Res. 9, 325 (1963). 32. J. L. GOWANS, Ann. N. Y. Acad. Sci. 99, 432 (1962). 33. G. ATTARDI, M. COHN, K. HORIBATAand E. S. LENNOX,J. Immun. 92, 346 (1964). 34. M. R. SCHWARZand W. O. RIEKE,Anat. Rec. 155, 493 (1966). 35. D. G. OSMOND, Anat. Rec. 157, 295 (1967). 36. J. W. REBUCK and J. H. CROWLEY, Ann. N. Y. Acad. Sci. 59, 757 (1955). 37. J. C. SIERACKIand J. W. REBUCK,in The Lymphocyte andLymphocytic Tissue (Ed. J. W. REBUCK), p. 71. Paul B. Hoeber, New York (1960).

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COMMENTS In answer to a question of Dr. Diener concerning the possible lymphocytic origin of plasma cells, Everett replied that work in his own laboratory led him to believe that typical lymphocytes, certainly not the small ones, did not give rise to plasma cells. Rather, it was the occasional large, very distinctly basophilic cells sometimes found in thoracic duct lymph wbich had the potential to differentiate into the plasma cell precursor and thence the plasma cell. In answer to questions by both Dr. Diener and Dr. Cline concerning the possibility that the labeled small lymphocytes three weeks after injection of tritiated thymidine were so labeled because of reutilization, Dr. Everett replied that this was of insufficient magnitude to cause confusion. Furthermore as controls, testes, small intestine and several compartments in which thele was rapid proliferation were sampled to be sure that there was no significant labeling following a localized injection. Dr. Weissmann then also pointed to the work of Nowell utilizing entirely different methodology that in the human and in most mamn-tals the small lymphocyte lives for one to three years in and out of the circulation without dividing, but inquired as to how the activated, transformed blastoid lymphocytes could attack or bother other cells. To this, Dr. Everett responded, citing also the work e f D r . Ginsburg that such clones of cells began their destruction of monolayer cultures about the fifth or sixth day not through humoral antibody intermediation but by a cell-contact phenomenon. In this connection, these cells or their progeny also gave rise to other small lymphocytes but there was no evidence that they formed plasma cells.