Kinetics of Lymphocytes in Chronic Lymphocytic Leukemia: Studies Using Continuous 3H-Thymidine Infusion in Two Patients

Two patients with typical chronic lymphocytic leukemia received a continuous infusion of ³H-thymidine for 7 days. Theproduction rate of lymphocytes was lowin the lymph nodes, the marrow, and theblood. The fraction of newly formedlymphocytes appearing in the blood per daywas only about 0.5%. The absolute production rate of blood lymphocytes howeverwas very high, 690 cells/cu mm blood/dayin one patient and 725 cells/cu mm/dayin the other. The daily accumulation ofnewly produced lymphocytes in the circulating blood was estimated to be 3.0 x10⁹ cells. Most of these newly formedcells were lost from the blood lymphocytecompartment within 3 wk. They are considered to be short-lived lymphocytes withhalf-lives of 3-8 days. About 90% of theblood lymphocytes were long-lived with aturnover time in excess of 1 yr in theblood lymphocyte compartment. The short-lived cells were both large and smalllymphocytes, the long-lived cells werealmost exclusively small lymphocytes.Lymphoid cell proliferation was virtuallyabsent in the blood, very low in the marrow, and marked in the enlarged lymphnodes. The exchange of lymphocytesseemed to be partially intact betweenlymph nodes and blood but more or lesscompletely blocked between marrow andblood.

Submitted on October 16, 1972 Revised on February 22, 1973 Accepted on April 10, 1973     

Radioautographic Study of Cellular Migration Using Parabiotic Rats

Leukocyte exchange between thehemopoietic tissues of parabiotic ratswas studied subsequent to giving multiple injections of ³H-thymidine to onemember of each pair while arrestingthe cross-circulation. Cell types thatmigrated from one parabiont to theother were segmented granulocytes,small, medium and large lymphocytes,immunoblasts, monocytoid cells, macrophages or their immediate precursors, and plasma cells. Evidence for thetransformation of circulating cells toother cell types was rarely seen. Thelong-lived small lymphocytes wereequilibrated between parabionts, suggesting that this is a single pool ofcells with respect to kinetic behaviorand recirculation. There was no evidence for a trephocytic function oflymphocytes. A small number of bonemarrow lymphocytes coursed directlyto lymph nodes and spleen. Evidenceis given for a limited recirculation ofshort-lived lymphocytes of thoracicduct lymph (TDL), as well as for long-lived cells. Only a few immunoblasts ofTDL recirculated. The majority of cellsthat entered the white pulp of thespleen were long-lived small lymphocytes, while the majority of immigrantcells to the red pulp were monocytoidcells and granulocytes. Many smalllymphocytes originated in splenic redpulp and entered the blood. No immigrant cells to the thymic cortex werenoted, although some small lymphocytes and monocytoid cells enteredthe medullary areas. Immigrant cells tothe bone marrow (less than 2% of thecells in marrow) included monocytoidcells, small lymphocytes, and plasmacells. Evidence for the direct transformation of a circulating cell into a committed blast, based on reduction ingrain count, was noted only in bonemarrow.

Submitted on April 14, 1971 Revised on August 9, 1971 Accepted on August 17, 1971     

Kinetics of Small Lymphocytes in Normal and Nude Mice after Splenectomy

Autoradiography and various quantitations on lymphoid tissues have been used to evaluate the kinetics of small lymphocytes in normal (+/nu or +/+) and congenitally athymic nude (nu/nu) NMRI mice 1 month after splenectomy or sham-splenectomy. The results indicate that splenectomy causes depressed thymic activity and diminished numbers of T lymphocytes in peripheral lymphoid tissues. The total number of cells in these tissues as well as the blast cell activity, were within normal limits. Bone marrow lymphocyte numbers and kinetics as well as blood lymphocyte levels in splenectomized and sham-splenectomized normal animals were comparable. Blood lymphocyte numbers were at normal levels in splenectomized nude mice, in spite of reduced numbers of bone marrow and thoracic duct lymphocytes. It is suggested that increased number of newly-formed lymphocytes, found in lymph nodes and blood of splenectomized mice, are released from the lympho-myeloid organs in compensation for the loss of long-lived, thymus-derived cells.     

Circulation and migration of small blood lymphocytes in the rat: II. Study of the lymphocyte selective migration by light and electron microscopic autoradiography

Fourteen male Wistar rats were the recipients of labeled small lymphocytes (1.5 x 10(7) each) collected from the peripheral blood of syngeneic donors. The migrating labeled lymphocytes were traced in the various organs from one to 60 minutes following their transfusion. Sections from the lymph nodes, bone marrow, spleen, thymus, ileum, liver, lung, and kidney were analyzed morphologically by autoradiographic studies. The results showed that some of the labeled small blood lymphocytes migrate to the lymph node and bone marrow as early as one minute following transfusion; their exodus from these two organs occurs within three minutes. In the case of the spleen, the lymphocytes did not migrate selectively to the marginal zone of the lymphoid follicles until ten minutes following transfusion. The electron microscopic study of the spleen and lymph node showed that the labeled lymphocytes selectively migrate to certain areas which consist of reticulum cells, macrophages, unlabeled lymphocytes, and plasma cells. The term "immunocompetent zones" is proposed for these areas because of the biological significance of this selective migration with reference to immunity.     

A Light and Electron Microscopic Study of the Lymphomyeloid Complex in Hypervitaminosis A

Prolonged administration of vitamin Ato guinea pigs caused lymphomonocytosis and marked changes in organsof the lymphomyeloid complex, withproliferation of atypical mononuclearcells in the spleen, lymph nodes, andliver. In the bone marrow, atypicalmononuclear cell proliferation was alsoencountered and represented 13% ofthe entire marrow cell population insome animals. Electron microscopicexamination of the marrow revealedmany lymphocytes and plasma cellswith a well-developed rough endoplasmic reticulum (RER), suggesting thatthese cells are active in protein synthesis and antibody production. Theatypical mononuclear cells describedappeared to belong to the monocyticseries and contained a variety ofdense bodies, and a well-developedRER and Golgi apparatus. Vitamin Ais mitogenic, labilizes lysosomal andcell membranes, and can act as anadjuvant enhancing immune responses. It has been suggested thatvitamin A alters lymphoid cell membranes and enhances the degree ofimmune response by stimulation ofcell division in immunologically competent cells. The lesions describedhere may well be the morphologiccounterpart of an enhanced cellularimmune response.

Submitted on January 3, 1972 Revised on May 2, 1972 Accepted on May 5, 1972     

Leukocyte Labeling With 51Chromium. IV. The Kinetics of Chronic Lymphocytic Leukemic Lymphocytes

The kinetics of radiochromate-labeledautologous blood lymphocytes werestudied in ten cases of chronic lymphocytic leukemia (CLL). The labeledcells equilibrated with an enlarged recirculating lymphocyte pool (RLP) distributed between the blood, the spleen,and the bone marrow. The size of theRLP, estimated from dilution of thelabeled cells, was better correlatedwith the degree of splenomegaly andmarrow infiltration than was the bloodlymphocyte count. The marrow fraction of the RLP turned over moreslowly than the splenic pool. The half-life of the labeled cells in the RLPvaried in individual cases from one to11 days. Lymphocyte life span was notrelated either to the blood lymphocytecount or to the size of the RLP. Themean half-life of 3.8 days was significantly shorter than the normal lymphocyte half-life of 18 days. We conclude that CLL is characterized byoverproduction of a strain of abnormally short-lived lymphocytes.

Submitted on June 12, 1972 Revised on August 28, 1972 Accepted on August 30, 1972     

Cell Migration from the Thymus to Other Lymphoid Organs in Hamsters of Different Ages

Hamsters of different ages were labeled intra thymus and intravenously with³H-thymidine. After extraction of nucleic acids, measurements of DNA content and tritium activity, the specific activity of different organs was calculated.By comparing the pattern of label of different organs after these quantitativemeasurements, it could be concluded that a good local labeling was obtainedin all intra thymus injected animals, and a quantitatively significant transportof label could be shown to occur from the thymus to the mesenteric lymphnodes and Peyer’s patches in animals of both sexes, and also to the spleen ofmale animals. This transport was shown in 23 day old hamsters in which thelymphatic organs except the thymus still are immature. No such transportcould be demonstrated in adult animals. Autoradiographic examinations alsoshowed a good local labeling in the intra thymus injected animals and amigration of heavily labeled cells to mesenteric lymph nodes in 7 days oldanimals, and to mesenteric lymph nodes, Peyer’s patches and the white pulpof the spleen in 23 days old animals. Only single heavily labeled cells werefound in the "peripheral" lymphatic organs of adult animals.

Submitted on March 14, 1967 Accepted on November 6, 1967     

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.     

Leukocyte Labeling With 51Chromium. III. The Kinetics of Normal Lymphocytes

The kinetics of radiochromate-labeledautologous blood lymphocytes werestudied in normal subjects. The labeledcells equilibrated within hours with arecirculating lymphocyte pool (RLP)distributed between the blood, thespleen, and the bone marrow. The RLPwas found to be about twice the sizeof the blood lymphocyte pool contained within it and turned overthrough the blood about 12 times daily.The labeled cells had an average halflife in the RLP of 18 days.

Submitted on October 13, 1971 Revised on January 24, 1972 Accepted on January 26, 1972     

Lymphocyte Production and Life-span in the Bone Marrow of the Guinea Pig

Guinea pigs were given 14 daily injections of ³H-thymidine to label a proportion of cells with a slow rate of turnoverin addition to rapidly proliferating cells.In the bone marrow the only unlabeledcells were some reticular, endothelial, andplasma cells, damaged cells, and 14.1%of small lymphocytes. Six weeks afterdiscontinuation of ³H-thymidine 7% ofthe marrow lymphocytes remained labeled. In guinea pigs injected every 4 hrwith ³H-thymidine for 4 days to label allcells entering DNA synthesis, 14.4% ofsmall lymphocytes remained unlabeledalong with some reticular, endothelial,phagocytic, monocytoid, damaged, andplasma cells. The pattern of appearanceof labeled lymphocytes was consistentwith the kinetics of transitional cells thatfunction as their precursors. Thus, in thebone marrow of the guinea pig the majority of lymphocytes have a short lifespan and a rapid turnover, whereas about14% turn over more slowly and 7% havea life-span exceeding 4 wk. In this respect the kinetics of marrow lymphocyteproduction differs from that of the rat.

Submitted on March 11, 1971 Revised on April 9, 1971 Accepted on April 14, 1971     

Regeneration of Marrow Tissue in Chronic Iron Deficiency in Postweanling Rats

The regenerative potential of chronically iron-deficient rat marrow wasstudied in extramedullary marrowautotransplants and intramedullarycavity after ablation. Subcutaneousimplantation of normal marrow resultsconsistently in establishment of a marrow nodule with a mean weight ratioof 30.7% of the implanted tissue. Iniron deficiency, the mean weight ratiowas 7.1%, and the take was 11%.After ablation of femoral marrow, theregenerative process proceeds distallyfrom the uninjured marrow at thefemoral head. The process of regeneration reached the midshaft after 11days in iron-deficient animals but tookonly 5 days for normal animals. Ironrepletion accelerated the process toward normal. These results are consistent with biochemical data fromiron-deficient marrow (low nucleic acidcontent, decreased incorporation of³H-thymidine into DNA, and decreasedutilization of ⁵⁹Fe and ¹⁴C-glycine forheme synthesis) and suggest that thereported hypercellularity of the marrowin iron deficiency may reflect sequestration of erythroid precursors, ratherthan the compensatory mechanism ofincreased cell proliferation.

Submitted on July 28, 1971 Revised on January 24, 1972 Accepted on January 29, 1972     

Oral administration of cyclosporin does not prevent expansion of antigen-specific,gut-associated,and spleen lymphocyte populations duringChlamydia trachomatis proctitis in nonhuman primates

To study the effects of oral cyclosporin (CsA) administration on immune responses in the gastrointestinal tract, humoral and cellular immune responses were studied in CsA-treated nonhuman primates having Chlamydia trachomatis proctitis (lymphogranuloma venereum, LGV). There was no apparent effect of CsA treatment on the gross or microscopic appearance of LGV proctitis, but CsA-treated animals, with or without LGV infection, had lymphoid hyperplasia of spleen and mesenteric lymph nodes. CsA treatment inhibited the primary antibody response to LGV, inhibited peripheral blood lymphocyte mitogen-induced proliferation and IL-2 production, and inhibited LGV-specific proliferation of peripheral blood lymphocytes. In contrast, mitogen-stimulated proliferation of spleen, mesenteric lymph node, and lamina propria lymphocytes was not significantly inhibited in CsA-treated animals. In addition, LGV-specific proliferation of spleen and mesenteric lymph node lymphocytes was not inhibited. High mitogen-stimulated IL-2 production of lamina propria lymphocytes was only partially inhibited in CsA-treated animals. In vitroCsA treatment had the expected inhibitory effects on mitogen- and antigen-induced proliferation of spleen and mesenteric lymph node lymphocytes. Thus, although oral cyclosporin inhibits the antibody and proliferative responses of peripheral blood lymphocytes to antigens and mitogens in animals having Chlamydia trachomatis proctitis, it does not prevent the expansion of antigen-specific, gut-associated, and spleen lymphocyte populations.Part of this work has been published as a preliminary report in abstract form (Clinical Research 34:446A, 1986).     

Differences of immunoglobulin secreting cells in bone marrow from those in circulation: relevance to their development

The surface markers of immunoglobulin secreting cells (ISC) in bone marrow and peripheral blood were analysed. Circulating ISC bear surface Ig and Ia-like antigens. However, these markers were not detectable on ISC in bone marrow. Fc and complement receptors were not present on circulating ISC. The areas of plaques corresponding to Ig secretion by bone marrow cells were always larger than those of peripheral blood cells. Although the majority of ISC were typical plasma cells, plasmacytoid lymphocytes were observed in peripheral blood. These findings seem to indicate that ISC in the peripheral blood are less advanced in their differentiation and maturation pathway from B lymphocytes to plasma cells than those in bone marrow. ISC in mesenteric lymph nodes exhibited nearly the same phenotype as peripheral cells.     

Distribution of B, T, and O lymphocytes in blood and tissues of normal humans reflecting a kinetic model.

The occurrence of the lymphocyte subpopulations TE, BFc, BC3, BFc + C3, BIg, and O-, percentage as well as absolute, was studied in lymphocyte suspensions from tonsils, abdominal lymph nodes, spleens, bone-marrow aspirates, and at the same time in venous blood. The absolute lymphocyte content (number of lymphocytes per g tissue) was highest in the abdominal lymph nodes, lower in the spleens and tonsils, and lowest in the blood. TE lymphocytes were found in the significantly highest percentage, 60%, in the blood. B lymphocytes, comprising BFc + C3 plus BIg, were present in the highest percentage in the bone marrow: 74%. Tonsils, spleens, and abdominal lymph nodes contained fewer B lymphocytes, and the blood fewest: 39%. A significant correlation was found only between the absolute numbers of T and B lymphocytes. A relationship between the absolute number of T lymphocytes and the total number of B lymphocytes as well as fractions thereof was thus demonstrated in the various tissues and in the blood and also between the blood and the tissue. O- lymphocytes were found in bone marrow, lymph nodes, and spleens, apparently as markerless precursors of other subpopulations. The main conclusion of the study is: In the lymphocytokinetic system the T lymphocytes must play a guiding role as an afferent vector, trigging the B fractions which thus constitute the efferent vector of the system.     

Junin virus infection of guinea pigs: immunohistochemical and ultrastructural studies of hemopoietic tissue

An association between viral antigens, cytopathic effect (CPE) and viral titers in blood and lymphoid tissues suggests a direct CPE of Junin virus on the lymphopoietic organs of guinea pigs infected with 10(3) 50% lethal doses of the XJ prototype strain. After seven days of infection, all lymphoreticular organs had infectivity titers higher than those for blood. Virus was recovered from bone marrow and lymph nodes at day 5 after infection; peak titers were obtained from bone marrow, spleen, and lymph nodes after day 10. Granular specific fluorescence was detected in the cytoplasm of reticular monocytes after day 7; megakaryocytes showed positive fluorescence, but specific staining of other lymphoid cells was not observed. Necrosis of bone marrow, lymph nodes, and spleen was observed after day 9. CPE consisted of overdevelopment of reticuloendoplasmic cisterne of reticulomonocytes and myeloblasts. Typical Junin virus particles were observed. Reticular cells were gradually destroyed, and simultaneous necrosis of surrounding lymphoid cells was observed.     

Relationships of adrenals,gonads and thyroid to thymus and lymph nodes,and to blood and thoracic duct leukocytes

The effects of gonadectomy, adrenalectomy, and thyroidectomy, and ofthyroxine administration on the weight of lymphatic tissues and on the numbersof blood and thoracic duct leukocytes have been studied over a 40 day period inyoung male rats. The results of the present experiment are interpreted to indicate that:

1. Removal of the gonads caused a marked increase in the weight of thethymus, whereas but slight effect was noted on the weights of the lymph nodes.Removal of the adrenal glands, however, resulted in a marked increase in theweight of all lymphatic tissues.

2. Thyroxine administration was demonstrated to exert a hyperplastic effecton the lymph nodes, an effect which was accentuated by the removal of theadrenals and gonads. The lymph nodes were more sensitive than the thymus tothe stimulating influence of thyroxine administration in the intact animal.

3. A comparison of the weights of the thymus and lymph nodes to controlvalues was more indicative of the hypoactivity or hyperactivity of the thyroid,adrenals and gonads, than were the weights of the thymus or lymph nodes considered separately.

4. The increase in the level of blood lymphocytes following removal of theadrenals appeared to result from a failure of normal removal of the lymphocytesfrom the bloodstream, rather than from an increased delivery of the lymphocytesto the bloodstream via the thoracic duct. Administration of thyroxine to normalanimals did not produce a blood lymphocytosis, lending further weight to thesuggestion of Marine and others that the blood changes seen its toxic hyperthyroidism are the result of adrenocortical insufficiency.

5. Changes in the weight of the lymphatic tissues were not necessarily reflectedby a proportional change in the number of thoracic duct lymphocytes, nor didthe number of blood lymphocytes necessarily reflect the number of lymphocytesdelivered to the bloodstream via the thoracic duct lymph.

6. The marked increase in the volume and cell content of thoracic duct lymphnoted its thyroxine treated adrenalectomized-gonadectomized animals appearsto be directly related to an increased sensitivity to the effects of thyroxine administration in the absence of these glands.

7. Removal of the thyroid gland vitiated the increases in weight of thymusand lymph nodes produced after castration and/or adrenalectomy.

8. The eosinophilic leukocytosis consequent on adrenalectomy was markedlyaugmented by removal of the thyroid and gonads.

Submitted on August 13, 1953 Accepted on January 1, 1955     

Marrow Transplantation and Iron Therapy in Mouse Hereditary Microcytic Anemia

The defective tissues causing themicrocytic anemia in mk/mk micehave been studied by transplantationof marrow erythrocyte precursors intolethally irradiated recipients. Microcytic (mk/mk) marrow in irradiatednormal mice produced erythrocyteparameters, blood values, spleen/bodyweight ratios, and reticulocyte percentages characteristic of the microcyticanemia, but splenic iron stores remained high. Normal marrow in irradiated microcytic mice producederythrocyte parameters intermediatebetween microcytic and normal and,combined with iron injections, produced normal erythrocyte parameters,blood values, spleen/body weightratios, and reticulocyte percentages;long-term splenic iron stores remainedlow. Iron injections did not improveblood values of either irradiated normal mice implanted with microcyticmarrow or untreated microcytic mice.Microcytic mice injected with iron recovered more rapidly from repeatedsevere bleedings than did uninjectedmicrocytic mice, but iron injectionsmade no difference in final bloodvalues reached. Only slightly less ⁵⁹Feremained in mk/mk than in normalmice 22 days after injection. These results indicate that not only the erythrocyte precursor cells but also iron supplies to these cells and splenic ironstores are defective in microcytic miceand that iron loss is not responsiblefor the iron storage defect.

Submitted on March 10, 1972 Revised on June 7, 1972 Accepted on July 5, 1972     

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.     

The Migratory Properties of Indium-111 Oxine Labelled Lymphocytes in Patients with Chronic Lymphocytic Leukaemia

S ummary . These studies describe the application of a new method for following the migration of autologous lymphocytes in normal subjects and patients with chronic lymphocytic leukaemia (CLL). There is evidence that Indium 111 oxine is a reliable radioactive cell label for in vivo studies of lymphocyte traffic in man. In normal subjects, where 60-70% of the lymphocytes labelled are T cells, the results are different from those of previous workers. The lymphocytes leave the blood initially but later return to it. It is believed that this is due to the reappearance of cells which at first entered the spleen. It is suggested that the difference between these data and those of Hersey (1971) is due to the failure of lymphocytes in the latter studies to return to the blood after primary migration. In CLL no such reappearance in the blood is seen and the lymphocytes do not leave the spleen in significant numbers over 48 h. This suggests that CLL B cells either do not recirculate through spleen or that their transit time is greater than 48 h. Bone marrow localization in CLL patients is greater than in the normal subjects, suggesting that either a larger proportion of the neoplastic B lympliocytes enter this compartment or their transit time through it is longer than the transit time of a normal T cell predominant population. Localization in normal-sized lymph nodes in patients with CLL is less than that in the lymph nodes of normal subjects. This may possibly be explained by the greater propensity of T lymphocytes to enter lymph nodes than B lymphocytes, or by the altered migratory properties of CLL B lymphocytes as compared with normal B cells.     

Thymus-Derived Lymphocytes in Blood,Lymph and Lymphoid Organs after Intrathymic Labelling with 3H-Thymidine

Guinea-pigs were investigated 2, 4 and 8 days after intrathymic or intraperitoneal labelling with tritiated thymidine. By the comparison between the content of lymphocytes in afferent and efferent thymic blood, an export of heavily labelled lymphocytes from the thymus was demonstrated in the intrathymically labelled animals after 2 and 4 days but not after 8 days. By use of liquid scintillation tritiated thymidine was localized to the spleen in a significantly higher amount 2 days after intrathymic than after intraperitoneal labelling. By autoradiography heavily labelled lymphocytes derived from the thymus were found in the spleen after 2 days and also to a lesser extent after 4 and 8 days, and in the mesenteric lymph nodes at all times after labelling. In the thoracic duct lymph the frequency of heavily labelled lymphocytes was the same as in the blood after intrathymic labelling, but less than in the blood after intraperitoneal labelling. The result indicates that thymus-derived lymphocytes are recirculating between blood and lymph.