Studies of intestinal lymphoid tissue. II. Aspects of proliferation and migration of epithelial lymphocytes in the small intestine of mice.

Mice were given either intraperitoneal tritiated thymidine (3H-Tdr) or colchicine to study proliferation and migration of intestinal epithelial lymphocytes. Both labelled medium and large lymphocytes ('immunoblasts') were observed throughout the epithelium, crossing the basement membrane and within villous lymphatics for at least seven days after 3H-Tdr administration. Epithelial lymphocytes are predominantly young cells, actively dividing at the rate of 1% per hour. They do not migrate along the villi, unlike epithelial cells, but circulate rapidly through the epithelium, returning to the lamina propria at the rate of approximately 3 epithelial lymphocytes/1000 epithelial cell nuclei/hour. The labelling pattern of epithelial lymphocytes and intralymphatic cells with time was very similar suggesting that epithelial lymphocytes therefore may directly enter adjacent lymphatics and hence gain access to thoracic duct lymph.     

Migration pattern of splenic lymphocytes after local labelling of the pig spleen with 3H-cytidine

In normal young pigs splenic lymphocytes were selectively labelled by injecting tritiated cytidine into a splenic artery. 10 h later several lymphoid and non-lymphoid organs were investigated for spleen-derived lymphocytes by autoradiography. The relative and absolute organ distribution of the labelled cells was determined. Labelled lymphocytes appeared rapidly in the peripheral blood reaching a mean labelling index of 4.8%. More splenic lymphocytes were found in thymus dependent areas than in thymus independent areas of lymphoid organs. Nearly 40% of all emigrated lymphocytes homed to lymph nodes and only about 1% in the thymus. A surprisingly high number of splenic lymphocytes were located in the bone marrow and the lung.     

In situ labelling of bone marrow lymphocytes with fluorescein isothiocyanate for lymphocyte migration studies in pigs

In normal young pigs, the femoral artery and vein were cannulated and after occluding other vessels to one hind leg they were connected to an extracorporeal perfusion system. Fluorescein isothiocyanate (FITC) was added to the perfusate to selectively label cells in the bone marrow. Large numbers (approximately 0.9 X 10(9] of labelled lymphocytes left the bone marrow of one leg within 1 d and migrated via the blood to the bone marrow in other bones, lymph nodes, spleen, Peyer's patches and even into the thymus. On average 1.7% of the lymphocytes in the blood and about 1% within the spleen were labelled. Peyer's patches and the thymus showed very low indices. Thus the bone marrow is an integral part of the migratory route of lymphocytes. Selective labelling of bone marrow cells in their normal microenvironment with FITC is a suitable method for studies of cell migration from the bone marrow.     

Evidence for cell division in synoviocytes in acutely inflamed rabbit joints.

DNA synthesis has been measured both by Feulgen cytophotometry, quantified by the DNA synthesis index, and by tritiated thymidine autoradiography, quantified by the labelling index. In the early acute inflammation resulting from the intra-articular challenge of ovalbumin in sensitised rabbits both indices rose considerably, so that at least 1 in 10 synoviocytes was heavily labelled 3 days after challenge. The results are compatible with the concept that even such apparently differentiated synoviocytes are capable of cell division.     

Cell proliferation of pericryptal fibroblasts in the rat colon mucosa.

The turnover of pericryptal fibroblasts in the rat colon mucosa was analysed after in vivo incorporation of tritiated thymidine. Thirty-six rats were serially killed one hour to 21 days after intraperitoneal injection of the radionuclide. At one hour, the labelling index of pericryptal fibroblasts was only 2.44%; labelled fibroblasts were slightly predominant along the lower two-thirds of the crypts. Within 24 hours, most underwent at least one cell division. No migration was observed and a significant proportion of labelled fibroblasts was still present after three weeks. It is concluded that those fibroblasts constitute a slowly renewing cell population. The data failed to confirm the hypothesis of an 'en bloc' migration of fibroblasts in synchrony with the epithelial cells.     

Proliferative Activity of Leukaemic Cells at Various Stages of Acute Leukaemia of Childhood

The DNA synthesis and content of individual leukaemic cells have been measured respectively by in-vitro labelling with tritiated thymidine and micro-densitometry of Feulgen-stained cells. These investigations have been carried out on bone-marrow and blood samples obtained from children at the time of diagnosis of acute leukaemia, and during subsequent remissions and relapses.
At diagnosis, the labelling index of bone-marrow blasts was low (6.8 ± 3.51 per cent), and that of blasts in the peripheral blood was usually still lower; most cells were at the 2n mode. There was no correlation between the labelling index and either the numbers of blasts in bone marrow or blood, or the duration of symptoms. During induction of remission, labelled cells disappeared more rapidly from the blood than unlabelled cells.
In relapse, the labelling index was significantly higher than at diagnosis, and there was evidence of aneuploidy in most patients, as shown by the distribution of DNA content. The mean labelling index of the small proportion of blasts in the marrow of five patients during remissions was intermediate between those found at diagnosis and during relapse.
The labelling index of leukaemic cells with tritiated uridine was high both at diagnosis and during relapse, the most heavily labelled cells usually being amongst the largest members of the population.
The possible significance of these findings is discussed in relation to the natural history of the disease.     

Lymphocyte Number and Response to Phytohaemagglutinin in Chronic Lymphocytic Leukaemia

The mitotic index and uptake of tritiated thymidine after 3 days culture with phytohaemagglutinin was markedly lower in lymphocytes from patients with chronic lymphocytic leukaemia than in lymphocytes from normal persons. The number of lymphocytes in leukaemic blood showed a strong negative correlation with the percentage of tritium labelled cells in culture, and a similar but nonsignificant correlation with the mitotic index. The reduced response of leukaemic cells was not due to cell damage caused by treatment.     

Angiostrongylus cantonensis: lymphoid cell responsiveness and antibody production in rats.

Angiostrongylus cantonensis-infected rats were examined for the presence of antigen sensitive lymphocytes, as assessed by the in vitro uptake of tritiated thymidine by cells of various lymphoid organs (cervical, mediastinal and mesenteric lymph nodes, spleen and periphereal blood), following stimulation by adult worm antigen. The lymphoid cell response of rats to A. cantonensis appeared to be local in nature in that significant responses were noted only in the cervical lymph node cells during the first 4 weeks of infection. The responses of spleen cells to phytohemagglutinin gradually declined as the infection progressed and this reduced responsiveness was statistically significant during the period of 5 to 10 weeks of infection. Homocytotropic antibody, demonstrated by 72-hour homologous passive cutaneous anaphylaxis, was detected throughout 2 to 17 weeks postinfection with a peak response at the 5th week of infection. The antibody was heat labile and sensitive to reduction by 2-mercaptoethanol and alkylation. Hemagglutinating antibody was first observed 5 weeks after infection and high titers occurred throughout 6 to 17 weeks postinfection.     

Studies of Lymphocyte Kinetics in Man

Studies of certain aspects of lymphocyte kinetics were performed in ninepatients with malignancies but who were hematologically normal.

Following the administration of tritiated thymidine, well-labeled large lymphocytes appeared very promptly in thoracic duct lymph along with somelightly labeled small lymphocytes. Specific activity was higher in the thoracicduct lymph lymphocytes as compared to the peripheral blood leukocytes forat least the first 50 hours.

When male patients were transfused with thoracic duct lymphocyte obtained from female donors, lymphocytes with a female karyotype were observed as early as 10 hours in the thoracic duct lymph and as early as one hourin the peripheral blood.

The evidence presented in these studies confirms data previously obtainedonly in animal experiments and indicates that homologous lymphocytes maycirculate as long as 9 days in appropriate recipients.

Submitted on April 4, 1966 Accepted on May 20, 1966     

The Appearance of Labeled Cells in the Thoracic Duct Lymph of the Guinea Pig after the Administration of Tritiated Thymidine

Tritium-labeled thymidine was given by either intraperitoneal or intravenousinjection to 13 male guinea pigs of approximately 400 Gm. weight.

At times varying from 1 hour to 30 days after the administration of thymidine, thoracic duct lymph was obtained and examined for the presence oflabeled cells.

After a single dose of thymidine, a steady stream of labeled lymphocytes,ranging from 2 to 7 per cent of the total cells, enters the blood over theperiod studied. The intensity of the labeling appears to diminish gradually.

Labeled large and medium lymphocytes were found in the lymph duringthe first hour. Labeled small lymphocytes began to appear in the fourth hour,in small numbers, and thereafter increased, whereas the proportion of labeledlarge and medium lymphocytes steadily diminished.

This sequential appearance of large, medium and small lymphocytes is interpreted as indicating the pattern of development of the cell series. The labeledsmall lymphocytes appearing in the lymph are considered to be newly formedfrom precursor cells located in the various lymphatic tissues.

Submitted on January 26, 1959 Accepted on July 13, 1959     

Cell renewal in non-specific duodenitis, ulcer-related duodenitis and duodenal ulcer. Experiments in Mastomys (Praomys natalensis)

Cell renewal in the duodenal mucosa of Mastomys was studied by autoradiography 1 and 24 h after intraperitoneal injection of tritiated thymidine. Non-specific duodenitis and duodenal ulceration were produced with a continuous infusion of histamine. Mucosal renewal in the duodenum of 30 control Mastomys was compared with 30 which received histamine dihydrochloride for 5 days. No abnormality developed in the controls, but 11 of the experimental group developed non-specific duodenitis and 12 duodenal ulcers. The size of the proliferative zone was increased in the Mastomys sacrificed 1 h after injection of tritiated thymidine which received histamine, compared with controls (p = 0.004). The number of labelled nuclei (p = 0.0003) and the size of the columns of labelled nuclei (p = 0.001) were increased in the Mastomys receiving histamine and sacrificed 24 h after injection of tritiated thymidine, compared with controls. The number of labelled nuclei (p = 0.004) and the size of the columns of labelled nuclei (p = 0.01) were increased in the Mastomys with ulcers compared with the other Mastomys which had received histamine. Cimetidine prevented duodenitis and ulceration, normalising the pattern of cell renewal. There was correlation between the severity of non-specific and ulcer-related duodenitis as judged by Lance's system and the number of labelled nuclei (Spearman rank correlation coefficient 0.771, p less than 0.002). Cell renewal increased as duodenitis became more severe and duodenal ulcers were found in duodenal mucosa where cell renewal was fastest.     

51Cr Labelling of Normal Human T and B Lymphocytes for Kinetic Studies in Vivo

Lymphocyte traffic between blood and tissues was assessed by ⁵¹Cr labelling of lymphocytes and subsequent autologous reinfusion in 10 normal elderly persons. The technique for isolation and platelet depletion of lymphocyte suspensions is described. By the labelling procedure used about 70 μCi ⁵¹Cr may be incubated in about 100 million lymphocytes. This permits measurement of lymphocyte-bound radioactivity on the T and B fractions separately. The blood disappearance curves for labelled lymphocytes indicate the existence of exchangeable pools in the tissues of T as well as of B lymphocytes, that of the T lymphocytes being apparently larger. A characteristic finding in the blood disappearance curves for total lymphocytes is an increase in lymphocyte-bound radioactivity in the blood 4–6 h after reinfusion, designated reappearance. The disappearance curve of the B lymphocytes shows reappearance 4–10 h after reinfusion, whereas that of the T lymphocytes falls exponentially without any recordable reappearance. On the basis of the disappearance curves and a knowledge of the topographic distribution of T and B lymphocytes in the lymphoid tissues, a model of T and B lymphocyte traffic in the lymph nodes is discussed. This model operates with T and B lymphocyte passage by way of postcapillary venules and describes the migration in and around the germinal centres. The T lymphocytes in the periphery of the germinal centres are assumed to derive mainly from the afferent lymph, whereas the B lymphocytes in the centres are exchanged with lymphocytes in the blood in an exchangeable pool. The functional implications are discussed.     

On the Origin of Foà-Kurloff Cells

The thymic and splenic release of Foà-Kurloff cells into the blood was studied in estradiol-treated male guinea pigs by comparison between the cellular content in afferent and efferent blood. The amount and distribution of such cells in thymus, spleen, lymph nodes, and bone marrow was investigated. The treatment with estradiol caused involution of the thymus and splenomegaly. An abundance of Foà-Kurloff cells was found in the red splenic pulp and a considerable release of such cells from the spleen into the blood was demonstrated. At the same time the output of lymphocytes from the spleen was reduced, suggesting that the Foà-Kurloff cells are transformed lymphocytes. The spleen contained an increased amount of erythroblasts, indicating a stimulation of splenic erythropoiesis by estradiol. In the bone marrow and the thymus the number of Foà-Kurloff cells was much smaller than in the spleen and no emigration of such cells from the thymus into the blood was demonstrated. A very small amount of Foà-Kurloff cells was found in the lymph nodes and very few occurred in the thoracic duct lymph. Thus, the Foà-Kurloff cells of the blood do not originate in the lymph nodes and do not recirculate between blood and lymph. It is concluded that the spleen is the major producer of Foà-Kurloff cells and that they are released from the spleen into the blood.     

Spontaneous Lymphocyte Transformation in vitro: Negative Evidence from a Study of Thymidine Uptake

Studies of the uptake of tritiated thymidine by human peripheral lymphocytes cultured in autologous plasma or, after washing, in foetal calf serum have failed to provide evidence for the occurrence of spontaneous blastoid transformation during 1–7 days in vitro.
Repeated washing of peripheral lymphocytes caused an inconstant reduction in their ability to respond to stimulation with PHA.
The accuracy and sensitivity of the technique of measuring the degree of in vitro transformation activity of lymphocyte cultures by their uptake of radioactively labelled thymidine is increased by partially removing polymorphonuclear leucocytes from the leucocyte suspensions and by restricting exposure of the cultures to the radioactive thymidine to the final 1 hour of culture.     

Studies on T and B lymphocytes in rats bearing methylcholanthrene-induced tumor.

The authors have studied the influence of primary, methylcholanthrene-induced tumor on T and B lymphocytes of spleen, thymus, draining lymph nodes and peripheral blood of rats. Differences in weight of tumors were found to correlate with changes in proportionality of T and B lymphocytes of followed organs. Small tumors induced but insignificant changes. There was increased trapping of T lymphocytes in spleen and lymph nodes with simultaneous decrease in peripheral blood. The authors noted a high percentage of blasts. B lymphocytes showed a tendency to compensate for the loss of T cells. Large, progressively growing tumors caused evident exhaustion in the number of both cell types in lymph nodes and peripheral blood. In the spleen there was slower exhaustion. Reduction in the number of B lymphocytes correlated with the size of tumor. Blasts disappeared. Proportionality of T and B lymphocytes in thymus did not appear to be influenced by the size of tumor.     

Neonatal administration of prolactin antiserum alters the developmental pattern of T- and B-lymphocytes in the thymus and spleen of BALB/c female mice.

We have evaluated the effect of neonatal administration of mouse prolactin (PRL) antiserum on the developmental expression of T- and B-lymphocytes in the thymus and spleen of female BALB/c mice. Newborn female mice were injected subcutaneously with a 50-microliters aliquot of PRL antiserum or normal rabbit serum on days 1, 2, and 3. On neonatal day 5, the PRL antiserum-treated group had a significantly (P less than 0.05) increased population of cells in the thymus and the spleen that were positive for Thy-1.2 and for L3T4. Increases in Thy-1.2- and L3T4-positive cells in the thymus were detectable also on days 8 and 14 in mice that received the PRL antiserum and in mice injected with bromocriptine, a dopamine agonist that inhibits PRL release from the anterior pituitary. On neonatal days 21, 28, and 32, there were no significant differences in the percentage of cells positive for Thy-1.2, Ly-2 (formerly Lyt-2), or L3T4 antigens in the thymus. However, there were significant increases in the percentage of Thy-1.2- and L3T4-positive spleen cells in the bromocriptine-treated group at all times monitored and in the PRL antiserum-treated group except on day 14. In addition, the percentage of splenocytes that were positive for IgG was significantly increased in the PRL antiserum-treatment group on days 8-28, although not on neonatal day 32. Of tissues known to contain PRL receptors, neonatal administration of PRL antiserum or bromocriptine resulted in a significant alteration in the wet weight of spleen and liver, with no significant effect in thymus, heart, and kidney. Pituitary implants also resulted in a significant increase in both concanavalin A- and lipopolysaccharide-stimulated thymidine incorporation into murine splenic lymphocytes prepared from 45-day-old female mice. These data extend the role of PRL as an immunomodulator of adult lymphocyte function to a role in the developmental expression of T- and B-lymphocyte populations in the thymus and spleen of mice.     

Immunomodulatory role of thyroid hormones: in vivo effect of thyroid hormones on the blastogenic response of lymphoid tissues

In an attempt to find out the mechanism of immunomodulation by thyroid hormones (T3 and T4), their in vivo effect on the blastogenic response of lymphocytes from various lymphoid tissues of hormone-treated and thyroidectomized rats were studied. The blastogenic response of lymphocytes from thymus, peripheral blood and mesenteric lymph nodes to pokeweed mitogen (PWM) was found to be increased significantly following T3 or T4 administration for 15 days or 30 days. However, the response to phytohaemagglutinin (PHA) increased only after 1 month of T3 or T4 administration. The blastogenic response of spleen cells to both PHA and PWM was, on the other hand, found to be depressed following 15 days of hormone administration. Thyroidectomy invariably induced significant depression in the blastogenic response to both PHA and PWM in lymphocytes of all the lymphoid tissues. Thyroid hormone (T3) administration was found to restore the blastogenic response of the lymphocytes of thyroidectomized animals.     

Activated lymphocytes in the peripheral blood of patients with rheumatoid arthritis.

The peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA), when compared to controls, has a higher proportion of mononuclear cells actively synthesizing DNA (S-phase cells). Such cells can be marked by radioautography after incubation with tritiated thymidine and can also be quantitated by the 4-h spontaneous in vitro uptake of tritiated thymidine. As our previous studies showed a marked and rapid decrease in this latter measure, in response to in vivo methotrexate, we suggested that the responsible cells may have a direct role in pathogenesis rather than merely reflecting the inflammation that is present. We have therefore tried to characterize them further. A mean of 46% of these autoradiography positive cells from the peripheral blood of patients with active RA bear CD3 markers and 61% Ia. On some occasions up to 25% stain for Leu M3 and 25% for Leu 7. None were esterase positive. Cell separation studies confirm that such cells were found in both T cell and non-T cell enriched populations. These cells, therefore, appear to be heterogeneous. A decreased number was seen in peripheral blood lymphocytes from patients who had received remittive therapy for over 4 months when compared to other patients with RA and this was associated with a lower 4-h spontaneous uptake of labelled thymidine.     

Development of a new Leydig cell population after the destruction of existing Leydig cells by ethane dimethane sulphonate in rats: an autoradiographic study

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [3H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [3H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [3H]thymidine at days 2, 3 or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered. In a second series of experiments, [3H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)