The predominant role of the spleen in lymphocyte recirculation. II. Pre- and postsplenectomy retransfusion studies in young pigs.

Autologous blood lymphocytes from three normal pigs were labelled with 3H-uridine and retransfused before and after splenectomy. Frequent samples for up to 150 min after retransfusion were evaluated autoradiographically to determine the rate of disappearance of labelled lymphocytes from the blood. In one pig retransfusion was performed before and after sham-splenectomy. In all preoperative experiments the pattern of disappearance of labelled lymphocytes was very similar. After a first rapid decline (halving time on average 8 min) a short rise of the labelling index was observed from 10 to 15 min after retransfusion. Then a second more gradual decrease of labelled lymphocytes followed. The mean halving time during this period was less than 32 min. From 60 min onwards the labelling index remained nearly constant. Retransfusions performed 3 days after splenectomy revealed only one nearly constant decline of the labelling index (halving time on average 129 min). After sham-splenectomy the pattern of disappearance was similar to the preoperative experiment. One hour after the end of retransfusion the labelling index had decreased by three-quarters of the initial value in normal pigs and by only one-third in the splenectomized ones. These results indicate that in the pig the total rate of recirculation is at least 4 times faster with the spleen in situ than without the spleen.     

THE PREDOMINANT ROLE OF THE SPLEEN IN LYMPHOCYTE RECIRCULATION

Lymphocyte recirculation through the isolated pig spleen was studied by means of a perfusion system which kept the organ alive for a prolonged period of time. By changing the perfusate to a leucocyte-enriched or cell-free perfusate and taking serial arterial and venous samples, the numbers of lymphocytes which homed to or were released from the spleen were measured. In all experiments more lymphocytes homed than were released per minute. There was no apparent difference when autologous or allogeneic cells were used. The number of lymphocytes released depended on the number of lymphocytes homed previously. During the phase of constant release up to 3-3 × 10⁶ lymphocytes were released per gram spleen per minute. From these values it can be extrapolated that up to 270 × 10⁹ lymphocytes recirculate through the isolated pig spleen per day. Based on kinetic data from other species it is estimated that in the entire pig a total number of 300–400 × 10⁹ lymphocytes recirculate per day. Thus, it can be concluded that the spleen is the most important organ for lymphocyte recirculation in the pig.     

Epidermal cell kinetics of the pig: a review

Age-related changes in cell kinetic parameters for the epidermis of pigs have been shown to be small, indicating that young pigs may be used for experimental studies. It was not possible to draw any firm conclusions about any strain-related differences in the cell kinetics of the epidermis of the pig. Lower LI values have been quoted for the miniature pig and the Yorkshire pig than for the Large White pig. However, these differences may be related to variations in experimental technique. The cell kinetic data for the Yorkshire pig are not consistent. Very high values for the mitotic index suggested a high rate of cell turnover, whilst data from single pulse labelling and grain count halving studies indicate a relatively low rate of cell turnover. The results from continuous labelling studies on the epidermis of the Yorkshire pig suggest that the basal cell turnover time (TT) is a factor of two or more shorter (136 h) than the estimates obtained using other methods. In the Large White pig estimates of TT were similar using a variety of techniques and were comparable with the TT estimate for the Yorkshire pig obtained using the continuous labelling method. There is some degree of inconsistency in the literature with regard to possible diurnal variations in the cell kinetic parameters for the epidermis. In the study of Archambeau & Bennet (1984) distinct diurnal variations were found in the LI, although the reliability of this finding is questionable due to the small number of animals used. Later studies by Morris et al. (1987) have suggested that diurnal variations are negligible in the epidermis of the pig. The majority of labelled cells (80%) in pig epidermis are located in the basal layer, although a significant proportion (20%) occurs suprabasally, in the cell layer immediately above the basal layer. Therefore, the epidermis can be regarded as having a bilayered proliferative cell compartment. The results from studies on irradiated pig skin (Morris & Hopewell, 1986, 1988, 1989) are not consistent with the presence of a homogeneous proliferative compartment in the epidermis, and are best explained by the occurrence of an heterogeneous proliferative compartment consisting of a stem cell subpopulation and a much larger population of transit proliferative cells.     

Splenic lymphocytopoiesis and migration pattern of splenic lymphocytes

The spleens of young pigs were selectively labeled with tritiated thymidine ([³H]-TdR) and the relative and absolute numbers of labeled lymphocytes found 24 hr later in different lymphoid and nonlymphoid organs were determined autoradiographically. It was deduced that about 4.6 × 10⁹ lymphocytes (that is, about 15% of all splenic lymphocytes) are produced by the spleen per day and about 17% of the newly formed lymphocytes leave the spleen within the first day of labeling. Spleen-derived lymphocytes could be found in relatively high numbers in the lymph nodes, blood, gut-associated lymphoid tissues, and, surprisingly, in the bone marrow, whereas the concentration in the thymus was very low. In a second series, pigs were labeled with [³H]TdR and only the spleen was excluded from labeling. The labeling index of splenic small lymphocytes was about 10% 1 day later, indicating a high rate of influx of newly formed lymphocytes into the pig spleen. The spleen of the young pig is an important lymphocytopoietic organ and exports and imports newly formed lymphocytes at high rates.     

AUTORADIOGRAPHIC CONFIRMATION OF THE MITOTIC DIVISION OF EVERY MOUSE JEJUNAL CRYPT CELL LABELLED WITH 3H-THYMIDINE

The question was investigated of whether for crypt epithelia of the jejunum of the mouse all cells labelled after a single injection of ³H-TdR subsequently divide or whether cells exist in the crypt which synthesize metabolic DNA and, therefore, do not undergo division after labelling.
A double labelling experiment was performed with a first injection of ³H-TdR followed 1 hr later by an injection of ¹⁴C-TdR. Then from double emulsion autoradiographs of isolated squashed crypts the number of ³H-only, ¹⁴C-only and double labelled cells and mitoses were counted.
The double labelling produced a narrow, 1 hr wide sub-population of ³H-only labelled cells. This subpopulation of S cells completed its division before labelled cells were lost from the crypts by migration onto the villi. The results showed that this subpopulation of ³H-only cells completely doubled within 3 hr and then remained constant through 6 hr. From this result it was concluded that every cell labelled after a single injection of ³H-TdR divides.
From the same autoradiographs the flow rate through the end of mitosis was measured. From the flow rate and the mitotic index a mitotic duration of 0·5 hr was determined. The agreement of this measured mitotic time with the value calculated from the labelling index, mitotic index and S duration is also strong evidence that every labelled cell divides.
Both experiments show that the intestinal crypt does not contain cells synthesizing metabolic DNA.     

THE KINETICS OF LYMPHOCYTE RECIRCULATION WITHIN THE RAT SPLEEN

The migration of lymphocytes from the blood into the splenic pulp and the release of lymphocytes from the spleen into the blood was studied by isolating the rat spleen and perfusing it with 15 ml of recirculating, oxygenated blood. When thoracic duct lymphocytes labelled with tritiated uridine were added to the initial perfusate the concentration of these cells fell exponentially for 2–3 hr and then rose to a flat secondary peak. From this pattern it was inferred that small lymphocytes entered the spleen at a rate proportional to their instantaneous concentration in the perfusate, traversed the splenic pulp and re-entered the perfusate with a minimum transit time of 2–3 hr. The rate of release of small lymphocytes from the spleen was not influenced by the prevailing concentration of small lymphocytes in the perfusate but probably reflected the rate of migration into the spleen over a period earlier than 2 hr before. The rate of exchange of small lymphocytes between the blood and the intact spleen in vivo was estimated to be about 84 × 10⁶ cells/hr. The size of the intrasplenic pool of recirculating small lymphocytes was probably 400–500 × 10⁶ cells. The rate of migration of small lymphocytes into the spleen was not affected by prior irradiation of the spleen donor. When either of two antigenic materials were added to the perfusate no inhibition of lymphocyte migration into the spleen was noted although the release of lymphocytes from the spleen was diminished by the addition of a large dose of sheep erythrocytes.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

Delayed labelling of brain glutamate after an intra-arterial [13C]glucose bolus: evidence for aerobic metabolism of guinea pig brain glycogen store.

Glycogen in glial cells is the largest store of glucose equivalents in the brain. Here we describe evidence that brain glycogen contributes to aerobic energy metabolism of the guinea pig brain in vivo. Five min after an intra-arterial bolus injection of d-[U-14C]glucose, 28+/-11% of the radioactivity in brain tissue was associated with the glycogen fraction, indicating that a significant proportion of labelled glucose taken up by the brain is converted to glycogen shortly after bolus infusion. Incorporation of 13C-label into lactate generated by brains made ischaemic after d-[1-13C]glucose injection confirms that these glucose equivalents can be mobilised for anaerobic glucose metabolism. Aerobic metabolism was monitored by following the time course of 13C-incorporation into glutamate in guinea pig cortex and cerebellum in vivo. After an intra-arterial bolus injection of d-[1-13C]glucose, glutamate labelling reached a maximum 40-60 min after injection, suggesting that a slowly metabolised pool of labelled glucose equivalents was present. As the concentration of 13C-labelled glucose in blood was shown to decrease below detectable levels within 5 min of bolus injection, this late phase of glutamate labelling must occur with mobilisation of a brain storage pool of labelled glucose equivalents. We interpret this as evidence that glucose equivalents in glycogen may contribute to energy metabolism in the aerobic guinea pig brain.     

THE TEMPO OF LYMPHOCYTE RECIRCULATION FROM BLOOD TO LYMPH IN THE RAT

Radioactively labelled thoracic duct lymphocytes were obtained either by incubation in vitro with ³H-uridine or ¹⁴C-uridine or by giving potential donors repeated injections of ³H-thymidine finishing 17 days before thoracic duct cannulation. These labelled TDL were injected i.v. into syngeneic recipients which had been subjected to splenectomy and thoracic duct cannulation on the previous day. The tempo of lymphocyte recirculation from blood to lymph was reflected by the time at which radioactivity was recovered in the thoracic duct lymphocyte output of the recipient. This was measured by scintillation counting of 2-hourly fractional collections for 36 hr after the injection. Two lines of evidence showed that the majority of small lymphocytes which label intensely with radioactive uridine in vitro were uniform in their 'migration potential'with a modal blood to lymph transit time of 14–18 hr. By contrast the cells which were labelled in vivo with ³H-thymidine included a slower population with a modal transit time of 24–28 hr. These conclusions can be more fully interpreted in the light of recent evidence on thymic-independent ('B') lymphocytes.     

Are all lymphoid blasts in the cell cycle?

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with mediumsized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no G_o cells. Supported by the Deutsche Forschungsgemeinschaft DFG Grant SFB 112     

An Autoradiographic Study of the Developing Parietal Cell Population In Neonatal Pigs

Abstract. Autoradiographic labelling using tritiated thymidine ([³H]TdR) was used to examine the pattern of development of gastric parietal cells in newborn pigs. Specific objectives were to establish sites in the gland where cells with a characteristic parietal cell morphology first appear, the extent of their migration or displacement, and the kinetics of any development and migration that occurs. Five newly-born littermate piglets were given a virtually continuous label of [³H]TdR over 24 hr, sacrificed at 1, 3, 5, 7 and 10 days thereafter, and samples of the gastric mucosa taken. the percentage of labelled parietal cells as a function of position in the oxyntic gland was measured for each pig. A generalized log linear model was fitted to the data using the statistical package GLIM, confirming a significant trend for labelled cells to occupy higher sites in the oxyntic gland as the time since labelling of cells increased. Goodness of fit tests showed that the trend effect was highly unlikely to be due to the variability of cell distribution from animal to animal. the dynamics of the parietal cell population and the strengths of GLIM for analysing cell labelling data are discussed.     

An autoradiographic study of the developing parietal cell population in neonatal pigs

Autoradiographic labelling using tritiated thymidine ([3H]TdR) was used to examine the pattern of development of gastric parietal cells in newborn pigs. Specific objectives were to establish sites in the gland where cells with a characteristic parietal cell morphology first appear, the extent of their migration or displacement, and the kinetics of any development and migration that occurs. Five newly-born littermate piglets were given a virtually continuous label of [3H]TdR over 24 hr, sacrificed at 1, 3, 5, 7 and 10 days thereafter, and samples of the gastric mucosa taken. The percentage of labelled parietal cells as a function of position in the oxyntic gland was measured for each pig. A generalized log linear model was fitted to the data using the statistical package GLIM, confirming a significant trend for labelled cells to occupy higher sites in the oxyntic gland as the time since labelling of cells increased. Goodness of fit tests showed that the trend effect was highly unlikely to be due to the variability of cell distribution from animal to animal. The dynamics of the parietal cell population and the strengths of GLIM for analysing cell labelling data are discussed.     

RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm.     

Are all lymphoid blasts in the cell cycle? DNA synthesis in the lymphoid tissues of the dog

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with medium-sized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no Go cells.     

3H-URIDINE INCORPORATION IN THE PREMITOTIC STAGE OF RHIZOID CELL DIFFERENTIATION IN PTERIS VITTATA L.

The premitotic rhizoid stage (corona stage) of P. vittata gametophytes was pulsed in radioactive uridine for 5, 15, or 30 min and the data analyzed quantitatively by autoradiography. After 5 min, only the nucleoplasmic compartment is labelled significantly, suggesting that this short pulse is insufficient time for labelled precursor to be fixed in the nucleolus or to be transported from the nucleus to the cytoplasm. Since after 15 and 30 min, all compartments are labelled, with the greatest proportional increase over the nucleolus, it is concluded that cytoplasmic labelling is nuclear in origin and that nucleolar RNA activity is relatively high in this stage.     

EPIDERMAL REGENERATION AFTER CELLOPHANE TAPE STRIPPING OF HAIRLESS MOUSE SKIN

After repeated applications of cellophane tape to the dorsal skin of hairless mice, the proliferative response in the treated epidermis was estimated by three different methods. The mitotic rate was determined in the interfollicular epidermis using the Colcemid technique, and the DNA synthetic activity was estimated after ³H-thymidine injection by counting labelled interfollicular cells in autoradiographs and by determining the specific activity of epidermal DNA. An initial 40–50% inhibition of DNA synthesis and mitosis was followed by an increase in the labelling index and the mitotic rate 8–10 hr after tape stripping. By 24 hr, peak values 5–6 times the controls were attained for both parameters. The labelling index and the mitotic rate were nearly normal at 3–4 days, but a second small peak was seen on day 5. Normal values were found on days 6 and 8. A similar pattern of response was found biochemically, but the peak of DNA specific activity was much broader and the extent of the increase was only about half as great as the increase in the labelling index. Possible reasons for these differences are discussed.     

Blood volume determinations in sheep before and after splenectomy

Using 51Cr labelled RBCs, total blood volume, red cell volume and plasma volume were measured in fifteen adult, female, domestic sheep both before and after splenectomy. Eight of the fifteen animals studied were anemic. Statistical analyses revealed no significant differences in blood volume parameters whether animals were grouped together or separated into normal and anemic groups. We observed: (a) splenectomy produced modest reductions in blood volume parameters in 12 of 15 animals, (b) preoperative variability in blood volume parameters caused by release of sequestered RBCs from the spleen was eliminated after splenectomy, and (c) equilibration of 51Cr required at least 30 minutes in intact animals, but only 10 minutes in splenectomized animals. After volume parameters were normalized to body weight, they were found to agree closely with values reported previously. This study demonstrates the dynamic function of the sheep spleen in the regulation of blood volume.     

The predominant role of the spleen in lymphocyte recirculation. I. Homing of lymphocytes to and release from the isolated perfused pig spleen.

Lymphocyte recirculation through the isolated pig spleen was studied by means of a perfusion system which kept the organ alive for a prolonged period of time. By changing the perfusate to a leucocyte-enriched or cell-free perfusate and taking serial arterial and venous samples, the numbers of lymphocytes which homed to or were released from the spleen were measured. In all experiments more lymphocytes homed than were released per minute. There was no apparent difference when autologous or allogeneic cells were used. The number of lymphocytes released depended on the number of lymphocytes homed previously. During the phase of constant release up to 3-3 X 10(6) lymphocytes were released per gram spleen per minute. From these values it can be extrapolated that up to 270 X 19(9) lymphocytes recirculate through the isolated pig spleen per day. Based on kinetic data from other species it is estimated that in the entire pig a total number of 300-400 X 10(9) lymphocytes recirculate per day. Thus, it can be concluded that the spleen is the most important organ for lymphocyte recirculation in the pig.     

AUTORADIOGRAPHIC INVESTIGATION ON THE CELL KINETICS OF CRYPT EPITHELIA IN THE JEJUNUM OF THE MOUSE °CONFIRMATION OF STEADY STATE GROWTH WITH CONSTANT FREQUENCY DISTRIBUTION OF CELLS THROUGHOUT THE CYCLE

Cell kinetic parameters of cells in the crypt of the jejunum of the mouse were obtained autoradiographically. A number of different methods used in cell proliferation studies were applied to the same animal strain kept under constant conditions. In order to avoid effects of geometrical factors, squashes of isolated crypts were used.
The generation time was determined by the per cent labelled mitoses method of Quastler, modified by double labelling with ³H- and ¹⁴C-TdR. This modified method permits a more exact determination of the generation time. The duration of the cycle was 14 hr.
Double labelling experiments in which an injection of ³H-TdR was followed by an injection of ¹⁴C-TdR after 1 hr showed that the cell flux was 7.0%/hr at the beginning of the S-phase and 7.68%/hr at the end. Assuming steady state growth a constant cell flux of 7.15%/hr within the whole cycle can be derived from the measured generation time of 14 hr. These results clearly show that the crypt epithelia constitute a steady state system with constant frequency distribution of the cells throughout the cycle.
The per cent labelled mitoses method after a single injection of ³H-TdR as well as double labelling experiments with ³H- and ¹⁴C-TdR give an estimate of the S-phase of 8.0 or 7.4 hr respectively. Double determinations lead to a value of 0.54 or 0.52 hr respectively for the duration of mitosis and to values of 77% and 72%     

Studies on control of granulopoiesis in man II. Influence of circulating neutrophil count on release of labelled bone marrow cells

Leukocyte kinetic studies were performed by labelling neutrophilic leukocytes in vivo with radioactive diisopropylfluorophosphate and measuring the time taken for the label to traverse the myelocyte compartment, marrow granulocyte reservoir and blood (myelocyte-to-tissue transit time). Leukocyte specific activity (LSA) reached background levels within 17 to 22 days in normal subjects and more rapidly than normal in patients with neutropenia due to hypersplenism or marrow aplasia. When neutropenia was partially or completely corrected by splenectomy the label disappeared more slowly. In patients with stable neutrophilia the label disappeared slowly and a terminal plateau in the LSA curve was encountered. Analysis of LSA curves suggests that disappearance of labelled neutrophil cohorts is influenced by circulating neutrophil levels, not only by an effect on release of cells from the marrow granulocyte reservoir but also, directly or indirectly, by an effect on myelocyte proliferation.