Effects of heterologous anti-lymphocyte serum on the distribution of 51Cr-labelled lymph node cells in mice

The effects of heterologous anti-lymphocyte serum (ALS) were studied in a syngeneic cell transfer system, in which lymph node cells from donor CBA mice were labelled in vitro with ⁵¹Cr and transferred intravenously into syngeneic recipients. Labelled cells treated in vitro with ALS were unable to migrate to lymph nodes or spleens of recipients, as did normal cells, but instead distributed themselves very similarly to cells which had been killed by exposure to heat. It is thus likely that cells treated in vitro with ALS are killed after transfer by the cytotoxic action of ALS mediated by the complement of the recipient.

ALS administered directly to the recipients of labelled lymphocytes could also reduce their uptake into lymphoid tissue; however, the magnitude of this effect appeared to be critically dependent upon the timing of the antiserum dose with respect to the labelled cell dose. ALS given immediately prior to labelled cells showed the greatest effect, while treatment given either 24 hours before or after the labelled cells was much less effective. While with prior treatment the reduced effect could be due to a fall off in antibody titre during the interval between the dose of antiserum and cells, in the latter situation no drop in titre would have occurred. It thus seems that lymphocytes that have already established themselves in lymphoid tissue may be less susceptible to the action of ALS.

ALS given chronically to lymphoid cell donors resulted in a population of cells which upon transfer to normal recipients were distributed differently from either normal or NRS-treated donor cells.

These data support the hypothesis that the effects of ALS may be exerted preferentially on circulating lymphocytes, and that ALS may act to selectively reduce the representation of this cell type in lymphoid tissue.

     

Radioautographic study of cellular mechanisms in delayed hypersensitivity: IV. Distribution of injected lymph node, spleen, thymus and bone marrow cells

Mononuclear cells from various organs of sensitized donor rats were labelled in vitro with tritiated leucine and injected intravenously into sensitized, syngeneic recipients. The localization of injected cells was studied in radioautographs.

Bone marrow cells were the most frequent of the labelled cells in skin reactions. Equal numbers of bone marrow cells were found in specific and non-specific delayed reactions and irritation reactions induced with turpentine.

Lymph node cells were found in delayed but not in turpentine reactions. Spleen cells were less frequent than lymph node cells in delayed skin reactions; small numbers were found in the turpentine reactions. Lymph node and spleen cells did not show detectable antigenic specificity.

Thymus cells were not found in the skin sites, even when they were allowed to circulate 2–3 days in the recipients before biopsies were taken.

Irrespective of the source of the cells injected few or no labelled cells were found in the recipient thymuses. Lymph node and spleen cells migrated equally into lymph nodes and spleen, but were infrequent in bone marrow. Only bone marrow cells had a decided preference for their organ of origin.

     

A Histological Study of the Early Stages of the Development of the Tuberculin Reaction after Passive Transfer of Cells Labelled with [3H]Thymidine

A comparison has been made of the histology of the lesions developing as a result of the injection of PPD (protein purified derivative of tuberculin) or HGG (human γ globulin) into the skin of normal guinea-pigs and those passively sensitized with lymphoid cells to PPD or actively sensitized with antigen—antibody precipitates to HGG. Little significant difference was found until 12 hours after skin test.

[³H]thymidine was injected into tuberculin-sensitized donor guinea-pigs. 24 hours later spleen cells from these donors were injected into recipient guinea-pigs. These recipients had been previously actively sensitized so as to show delayed-type hypersensitivity to HGG. The arrival of the labelled cells in lesions produced in the recipient by tuberculin (passively transferred delayed-type hypersensitivity) and by HGG (actively induced delayed-type hypersensitivity) was compared at intervals up to 12 hours. No significant difference was found between the arrival of labelled cells in passively induced tuberculin lesions and actively induced HGG lesions.

     

Inflammatory lymphoid cells: Cells in immunized lymph nodes that move to sites of inflammation

The arrival of cells from normal and immunized lymph node cells at sites of inflammation was studied. Mice were immunized with `oxazolone' or picryl chloride and 3–4 days later the draining lymph node cells were dissociated, labelled in vitro with ⁵¹Cr and injected intravenously into recipients.

Sites of inflammation were produced in the recipients by painting the ear with chemically reactive contact sensitizing agents or croton oil. The net arrival of normal lymph node cells at sites of inflammation was 0.1 per cent. In contrast the arrival of cells from immunized lymph nodes was 4–8 times greater. The peak number of cells that moved to sites of inflammation occurred 4 days after immunization with `oxazolone'.

An increase in the percentage of cells that moved to sites of inflammation occurred in lymph nodes after immunization with a number of agents including contact sensitizing agents, skin grafts and Freund's complete adjuvant. There was little or no increase in mice rendered unresponsive to picryl chloride and then immunized with picryl chloride or in mice injected with aluminium hydroxide, alum precipitated mouse serum or pneumococcal polysaccharide. This suggested that immunization and possibly the induction of delayed hypersensitivity was necessary for the generation of these cells.

The arrival of the cells did not depend on antigenic similarity between the agent used to immunize the lymph node and the agent used to produce inflammation. For example increased arrival of cells from immunized lymph nodes occurred at sites of inflammation produced in the ear by croton oil or in the peritoneal cavity by paraffin oil.

     

Cytotoxic T cell response to lymphocytic choriomeningitis virus: Properties of precursors of effector T cells, primary effector T cells and memory T cells in vitro and in vivo

Spleen cells from CBA/H mice pre-primed intravenously at various intervals with lymphocytic choriomeningitis (LCM) virus were tested for their capacity to respond and generate cytotoxic effector T cells on secondary stimulation both in vitro and in vivo. In vitro secondary stimulation was performed by culturing preprimed spleen cells with infected, syngeneic, peritoneal `stimulator' cells for periods of up to 7 days at 37°. Controls were incubated with uninfected `stimulators'. In vivo secondary stimulation was obtained by intravenous transfer of pre-primed spleen cells into irradiated, heavily infected CBA/H recipients at various times prior to assay of recipients' spleens. Controls were uninfected, irradiated recipients. Effectors were assayed against LCM virus-infected or uninfected, H-2 compatible L929 target cells in a ⁵¹Cr release assay.

Spleen cells gave three different patterns of cytotoxic T cell response following in vitro or in vivo stimulation, depending upon the interval between priming and secondary stimulation. Populations taken from mice approximately days 2–5 post-infection (PI) developed increasing cytotoxic activity against virus-infected targets in vitro when cultured with infected or uninfected peritoneal cells, or in vivo when transferred into irradiated, uninfected recipients. However, these early populations did not generate cytotoxic activity when transferred into irradiated, heavily infected recipients. Established primary effector populations (i.e. approx. days 7–11 PI) exhibited continuing effector activity when maintained in vitro or in vivo, more so when peritoneal `stimulator' cells or irradiated recipients were infected. Memory populations (i.e. more than approx. day 13 PI) developed highly potent secondary effectors when cultured with infected peritoneal cells, or on transfer to irradiated, infected recipients.

The three different patterns of cytotoxic T cell response of the different preprimed spleen cell populations can be interpreted as indicating three different phases of functional activity of the same population of antigen-reactive T cells from LCM virus-primed mice.

     

Factors affecting transfer of experimental autoimmune thyroiditis in rats

Actively induced experimental autoimmune thyroiditis in inbred Lewis rats was comparable using standard Freund's complete adjuvant (FCA) and Perrin's modification of FCA. However, adoptively transferred disease using lymph node cells from rats immunized with Perrin's FCA was significantly more severe. With this adjuvant, and pertussis vaccine as co-adjuvant, transfer was uniformly successful when at least 480 × 10⁶ lymph node cells were taken 10 days after immunization and recipients were killed 3 days after transfer. Lymphocytic infiltrates were seen in recipient thyroids as early as 18 hours after transfer. Whole body irradiation of the recipients at 550 r reduced the severity of transferred disease. The frequency and severity of lesions were higher when the lymph node cells were first incubated with low doses of antigen. Thymectomy of the recipients decreased the severity of transferred disease. Under the conditions tested, transfer of disease could not be accomplished by antiserum alone, even using thyroidectomized donors. Administration of an early immune serum with sensitized lymph node cells significantly depressed the severity of transferred disease, while a late antiserum increased it.     

The localization of antigen in relation to specific antibody-producing cells: I. Use of a synthetic polypeptide [(T,G)-A--L] labelled with iodine-125

A synthetic multichain polypeptide, (T,G,)-A--L 509, was trace labelled with ¹²⁵I in the tyrosine end groups, composing the main antigenic determinants, and used to study the distribution of antigen in the draining lymph nodes after injection into the footpads of mice. The polypeptide was administered: (a) in saline solution into unprimed mice—in which form it is not demonstrably immunogenic; (b) in Freund's adjuvant into unprimed mice—in which form it is immunogenic; and (c) in saline solution to primed mice, so as to give a booster response. Experiments comparable to (c) were also done with [¹²⁵I]haemocyanin.

At various time intervals from 12 hours to 21 days later sections of the draining nodes were examined by radioautography and methylgreen—pyronine staining, or by a combination of immunofluorescent staining for antibody containing cells with radioautography. In unprimed mice, irrespective of whether they made antibody, labelling was found predominantly in the phagocytic cells of the medulla and the cortical sinus, but definite weaker labelling was also seen in the germinal centres. The label persisted throughout the period of observation, but tended to become more prominent with time in the germinal centres of mice making antibody in response to antigen in Freund's adjuvant. In primed mice the label was rapidly and predominantly concentrated in germinal centres, where it persisted throughout the period of observation while its intensity gradually diminished in other sites. The distribution of label in germinal centres was in a lace-like or dendritic pattern, similar to that described by Nossal and co-workers, and did not correspond to the lymphocytes or pyroninophilic cells in the centres.

Grain counts were made over specific antibody containing cells in both primary and booster responses to (T,G)-A--L. Such cells sometimes lay close to and sometimes many cell diameters distant from phagocytic cells containing concentrations of the antigen, but the cells themselves did not have grain counts significantly different from the background count. If the antigen remained undegraded, under the conditions of the experiment it would have been possible to detect the presence of fifteen molecules or less in a cell. Reasons are given for supposing that ¹²⁵I constituted a valid label of the main antigenic determinant groups, of which there were a maximum of 100 per molecule.

     

The cellular transfer of immunity to Trichostrongylus colubriformis in an isogenic strain of guinea-pig: III. The localization and functional activity of immune lymph node cells following syngeneic and allogeneic transfer

Mesenteric lymph node cells obtained from guinea-pigs immunized with Trichostrongylus colubriformis have been transferred allogeneically (McMaster outbred to Heston inbred) and syngeneically (Heston to Heston inbred) by intravenous injection. The immune cells were transferred on days 6, 7 and 8 of infection when the parasite was in the susceptible fourth stage of development. The syngeneically transferred cells caused rejection of the parasite but the allogeneically transferred cells were not effective.

Immune mesenteric lymph node cells were labelled in vitro with ⁵¹Cr before transfer to infected recipients. The percentage of the intravenously injected cells which localized in infected small intestine has been calculated from γ-counts recorded at 1, 6, 16, 24 and 48 hours after injection of the labelled cells. The mean percentage of cell dose per 12 in. of small intestine recorded following syngeneic transfer was 1·29 and following allogeneic transfer 0·68. A significant difference in the localization of allogeneic and syngeneic cells in the small intestine was already apparent at 6 hours following intravenous injection.

     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1^lv1) rats received orthotopic corneal isografts or Wistar-Furth (RT1^u) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8⁺ than CD4⁺ cells was found at days 1–3 following rejection, whereas there was a higher proportion of CD4⁺ cells at days 5–8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1–2, 4 and 7–10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4⁺ > CD8⁺ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1–2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes—from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4⁺ cells was found in both aqueous and graft at later times.     

Amyloidosis in neonatally thymectomized and anti-lymphocyte serum treated mice

A marked lymphocyte depletion and a striking impairment of their capacity to form antibodies against sheep erythrocytes was shown by outbred Swiss albino mice thymectomized or sham-thymectomized at birth and later treated with anti-lymphocyte serum.

Ninety-four per cent of allogeneic and 53% of heterogeneic skin grafts applied to the former, and 63% and 0% of those applied to the latter, survived up to the time of killing, i.e. 41 days after transplantation.

The remaining allogeneic and heterogeneic skin grafts were rejected by mice belonging to both experimental groups in a minimum of 18 days and a maximum of 35 days, which is much longer than is usually required by normal recipients (allogeneic grafts = about 10–11 days; heterogeneic grafts = about 7–8 days).

Despite the severe immunological depression caused by anti-lymphocyte serum treatment, either associated or not with neonaial thymectomy, all the mice chronically exposed to casein developed amyloidosis.

The present results are in accordance with previous findings indicating that mechanisms other than immunity may be involved in the pathogenesis of amyloidosis.

     

The fate of proliferating cells in skeletal muscle after denervation or tenotomy: an autoradiographic study

During the first 7 days after denervation or tenotomy of skeletal muscle there is increased cell proliferation, as measured autoradiographically by [3H]thymidine incorporation. By 21 days most of the labelled cells have disappeared. The present experiment was designed to measure residual levels of labelling in skeletal muscles at 21 days after denervation or tenotomy, when [3H]thymidine had been available for the first 7 days. This was to determine the fate of cells labelled at 7 days and to detect whether continued cell proliferation had occurred during the intervening period, as evidenced by the dilution of nuclear label. Tibialis anterior muscles of 16 mice were denervated, tenotomized or sham-operated, and the animals injected with a single daily pulse of [3H]thymidine for the first 7 days only. In denervated muscle the 11% of muscle nuclei and 41% of connective tissue nuclei labelled at 7 days had decreased to 2 and 13%, respectively, by 21 days. These values were still significantly higher than in 21 day controls (0.2 and 1.9%, respectively). In muscle nuclei (myonuclei and satellite cell nuclei) there was a significant decrease in the amount of label per nucleus. This was interpreted as evidence of continued cell proliferation. However, this did not produce an increase in numbers of muscle nuclei. It was concluded that: there was turnover of satellite cells and possibly myonuclei, the latter probably through continued division and incorporation of satellite cells; and the extra satellite cells and myonuclei produced were probably extruded from the fibre to the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)     

The intragraft vascularized bone marrow component plays a critical role in tolerance induction after reconstructive transplantation

The role of the vascularized bone marrow component as a continuous source of donor-derived hematopoietic stem cells that facilitate tolerance induction of vascularized composite allografts is not completely understood. In this study, vascularized composite tissue allograft transplantation outcomes between recipients receiving either conventional bone marrow transplantation (CBMT) or vascularized bone marrow (VBM) transplantation from Balb/c (H2d) to C57BL/6 (H2b) mice were compared. Either high- or low-dose CBMT (1.5 × 10⁸ or 3 × 10⁷ bone marrow cells, respectively) was applied. In addition, recipients were treated with costimulation blockade (1 mg anti-CD154 and 0.5 mg CTLA4Ig on postoperative days 0 and 2, respectively) and short-term rapamycin (3 mg/kg/day for the first posttransplant week and then every other day for another 3 weeks). Similar to high-dose conventional bone marrow transplantation, 5/6 animals in the vascularized bone marrow group demonstrated long-term allograft survival (>120 days). In contrast, significantly shorter median survival was noted in the low-dose CBMT group (~64 days). Consistently high chimerism levels were observed in the VBM transplantation group. Notably, low levels of circulating CD4⁺ and CD8⁺ T cells and a higher ratio of Treg to Teff cells were maintained in VBM transplantation and high-dose CBMT recipients (>30 days) but not in low-dose VBM transplant recipients. Donor-specific hyporesponsiveness was shown in tolerant recipients in vitro. Removal of the vascularized bone marrow component after secondary donor-specific skin transplantation did not affect either primary allograft or secondary skin graft survival.     

Depression of delayed hypersensitivity by pretreatment with Freund-type adjuvants. III. Depressed arrival of lymphoid cells at recently immunized lymph nodes in mice pretreated with adjuvants

Pretreatment of mice with Freund's complete adjuvant (FCA) or Corynebacterium parvum depresses the contact sensitivity which otherwise follows immunization with picryl chloride in FCA (PCL/FCA). In contrast, Bordetella pertussis is inactive. The hypothesis that this depression was partly due to the failure of lymph nodes immunized with PCL/FCA to collect lymphoid cells was tested. Mice were pretreated with FCA, C. parvum or B. pertussis, subsequently immunized with PCL/FCA, and injected with ⁵¹Cr-labelled normal lymph node cells.

Pretreatment with FCA 10 days before immunization depressed the arrival of labelled cells at draining nodes immunized with PCL/FCA by 10–19%.

Pretreatment intravenously with C. parvum 4 days before immunization virtually abolished the increased arrival at draining lymph nodes which is otherwise seen 2 and 4 days after immunization, but had little effect on the increased arrival seen 1 day after immunization. In contrast, B. pertussis caused a slight but significant depression at day 1, but had no effect 2 and 4 days after immunization.

The depression of contact sensitivity by these three bacterial adjuvants was paralleled by their depression of the arrival of labelled lymph node cells at recently immunized nodes, and supported the hypothesis that failure of recently immunized lymph nodes to collect lymphoid cells is part of the mechanism of depression of delayed hypersensitivity by bacterial adjuvants, and in particular, by C. parvum.

     

Experimental immune inflammation in the synovial membrane: II. The origin and local activity of inflammatory cells

Synovitis was produced in guinea-pigs previously immunized with bovine γ-globulin and Freund's complete adjuvant, by injection of bovine γ-globulin and tuberculin PPD into the knee joints. In animals given prior intravenous injections of tritiated thymidine and colloidal carbon, up to 40 per cent of the local accumulations of mononuclear cells 48 hours after antigen challenge were tritium-labelled as seen by autoradiography. These inflammatory cells are considered to be of haematogenous origin. A smaller percentage of cells was carbon-labelled. The percentage of tritium-labelled cells decreased with duration of the granuloma following antigen challenge. In other animals, synovial inflammatory cells were labelled by intra-articular injection of tritiated thymidine at different times following the establishment of synovial inflammation by injection of antigen. The highest proportion of cells labelled was 17 per cent, but when label was administered as late as 60 days after the inflammatory stimulus, only about 3 per cent of cells were labelled. Grain counts of cells labelled by local tritium administration or by short-term incubation of slices in vitro showed approximately 30–100 grains per nucleus. Sequential study of locally labelled synovia showed that such high-grain cells were still present 12 days after tritium administration, suggesting that at least some of the mononuclear cells were relatively long-lived.     

Prolonged survival of H-2 incompatible skin allografts on F1 animals treated with parental lymphoid cells

The survival times of H-2 incompatible skin allografts were studied in F₁ hybrids treated with parental strain lymphoid cells. The injection of 50×10⁶ A/Sn lymphoid cells into A/Sn×CBA F₁ hybrids greatly prolonged the survival of 5M skin allografts applied 7 days later. Suppression of allograft rejection was observed also in F₁ recipients that were actively or adoptively immunized against the skin graft donor.

The immunocompetence of the sensitized F₁ hosts was supressed to a greater degree than that of normal non-sensitized recipients. It is unlikely that immuno-incompetence is caused by basic effects of the graft-versus-host reaction or by antigenic competition since overt signs of a graft-versus-host reaction were not a necessary prerequisite for immunosuppression. It is suggested that a general inhibition of expression of immunity develops as a consequence of the immune reaction, possibly mediated by humoral factors.

     

Selective migration of lymphocytes within the mouse small intestine

The factors which determine the migration of lymphoid cells to lamina propria or Peyer's patches of mouse small intestine have been investigated by autoradiographic tracing of intravenously injected spleen, thymus and lymph node cells. The numbers of labelled cells found in antigen-free grafts of foetal small intestine were compared with the numbers in normally sited gut. Thymus, normal spleen and B spleen lymphocytes, labelled with [³H]adenosine or [5-³H]uridine, were confined to Peyer's patches in normal and grafted gut. [³H]Thymidine-labelled lymphoblasts from the mesenteric nodes of young (19–22 days) mice and mice infected with Nippostrongylus brasiliensis were found in the lamina propria of both graft and normal small intestine, but [³H]thymidine-labelled lymphoblasts from oxazolone-primed lymph nodes did not migrate to the villi. The possible roles of intraluminal antigens, source of cells and changes in cell surface receptors during differentiation, in determining the selective migration of cells to the lamina propria and Peyer's patches, are discussed.     

Secondary cytotoxic cell response to lymphocytic choriomeningitis virus: III. In vivo protective activity of effector cells generated in vitro

Secondary cytotoxic T cells were generated in vitro by culturing WE3-LCM virus-specific memory CBA/H spleen cells with WE3-LCM virus-infected syngeneic peritoneal cells at 37° for 5 days, and were found to be highly potent in reducing virus titres in the visceral organs of syngeneic recipients when transferred 24 h before intravenous virus challenge (e.g. 3.1×10⁶ cells transferred gave approximately 3 logs reduction of virus titre). Protection was measured by titrating spleens for virus in a plaque assay, usually 2 days post virus-challenge. Intravenously administered secondary effectors did not, however, elicit a reduction in brain virus titres by 3 days after intracerebral inoculation of virus. Cells mediating protection were sensitive to treatment with anti-θ ascitic fluid plus complement. Protection occurred only when donors of secondary cells, infected stimulator cells and recipients shared H-2K or H-2D, and there was a similar genetic restriction for these effectors to efficiently lyse virus-infected targets in vitro.

Spleen cells from ectromelia virus-memory mice were cultured with ectromelia virus-infected syngeneic peritoneal `stimulator' cells and generated secondary effector cells which protected syngeneic recipients from subsequent challenge with ectromelia but not LCM virus. However, secondary effector cells derived from in vitro stimulation of spleen cells from WE3-LCM virus-memory mice with WE3-LCM virus-infected syngeneic stimulator cells caused a significant reduction in spleen virus titres in syngeneic recipients challenged with either ectromelia or LCM virus. If WE3-LCM virus-infected stimulator cells were fixed and responder spleen cells were either from very old (6–12 months post-priming) WE3-LCM virus-memory mice, or from ARM-LCM virus-memory mice, this unidirectional cross-protection of ectromelia virus-challenged mice was less pronounced. One explanation for the unidirectional cross-protection was that the transferred secondary effectors contained and released infectious LCM virus (or defective virions) which adsorbed to recipients' spleen cells; these latter cells displayed both LCM and ectromelia virus-specific antigenic patterns after ectromelia virus challenge, rendering them susceptible to specific T-cell mediated lysis. Clear specificity of effector cells was demonstrated at the effector: target level in vitro between LCM virus and ectromelia virus. (Experiments to compare the in vivo activity of secondary effectors with primary effector T cells raised in vivo or in vitro were hampered because of the large amounts of infectious virus in the latter two populations.)

Secondary effectors reduced spleen, lung and liver virus titres when transferred 24 h after virus challenge, but were less efficient in protecting recipients than when given before virus.

     

Origin and kinetics of Kupffer cells during an acute inflammatory response

The course of the increased number of liver macrophages and the origin of these cells were studied after intravenous stimulation by zymosan, stilboestrol, or corynebacterium. The macrophages were isolated by digestion of the liver with pronase and DNAase.

Zymosan doubled the number of liver macrophages per gram of liver and led to a four- to five-fold increase in the number of blood monocytes, whereas stilboestrol induced a four-fold increase of liver macrophages and a two-fold increase of blood monocytes. Corynebacterium administration gave a two-fold rise in the number of liver macrophages and a six-fold increase of blood monocytes.

The in vitro labelling index of the liver macrophages showed a transient but marked increase after the administration of zymosan or stilboestrol, but returned to approximately normal values 4 days after the stimulus. Hydrocortisone given 48 h before zymosan prevented this increase in the in vitro labelling index of liver macrophages, thus demonstrating that the mononuclear phagocytes labelled in vitro had recently been recruited from the bone marrow to the liver.

Stimulation by stilboestrol or zymosan of mice labelled with [³H]-thymidine caused an increase in the number of labelled liver macrophages and blood monocytes as compared with the numerical course of the labelled Kupffer cells and monocytes in untreated mice. It may be concluded that this increase is attributable to the increased influx into the circulation of labelled monocytes from the bone marrow, which in turn migrate to the liver in larger numbers than are seen in the normal steady state. Local proliferation could not have been responsible for the increased number of labelled liver macrophages, because free [³H]-thymidine was no longer available when the stimulus was applied. Evidence supporting the bone marrow origin of the increased number of monocytes and liver macrophages after intravenous stimulation was provided by the course of the number of monocytes and liver macrophages in hydrocortisone-treated mice given zymosan as stimulus.

     

The different migratory characteristics of lymphocyte populations from a whole spleen transplant.

Spleens from AS x BN donor rats labelled in vivo by multiple doses of [3H]thymidine were transplanted into syngeneic recipients by anastomosis to the abdominal great vessels. The recipients were killed 1-5 days after receiving the whole spleen transplants and the numbers and location of the [3H]thymidine-labelled cells which had migrated from the labelled donor spleen traced by means of autoradiographs of sections, imprints and smears of various recipient lymphoid tissues. These results were compared with the migration pattern of labelled dissociated spleen cell suspensions injected intravenously. The latter consists almost entirely of small lymphocytes which migrate to T or B areas of recipient spleen, lymph nodes and Peyer's patches. The labelled whole spleens also contained cells which migrated to the T and B areas of recipient lymphoid tissues, but in addition contained many lymphoid cells which migrated to the red pulp of the recipient spleen and to the lamina propria of the gut. These experiments showed, therefore, that the spleen contains mobile elements which have not been detected by transfer of spleen cell suspensions.     

Malignant changes in New Zealand black mice

Ageing, Coombs positive, NZB mice, may spontaneously develop a neoplasia of the reticulum cell type which can be transferred by serial passage of their lymphoid tissues in young intact or neonatally thymectomized syngeneic recipients. The recipients (103/117) of cell suspensions prepared from the enlarged spleens and/or lymph nodes of four such donors developed an extensive and lethal reticulum cell neoplasia affecting the spleen, lymph nodes, lungs and liver but the bone marrow, thymus and kidneys were seldom involved. The recipients (16/17) of spleen cells from a fifth donor showed massive proliferations of eosinophils in all the organs examined.

Prematurely positive antiglobulin (Coombs) reactions were detected in only two recipients. Although there was an indication that the IgM content of the sera decreased as one of the passages progressed, the levels of IgG and IgA were not seriously distorted.

Particles resembling murine leukaemia virus were identified by electron microscopy in the spleen and in plasma or serum pellets of passage recipients. However, similar particles were also seen in the thymus and/or spleen or bone marrow of untreated NZB mice including an 18-day embryo and animals aged 1–56 weeks, although no particles were found in plasma and serum pellets of mice aged 6–70 weeks.

The theory that autoimmunity, malignancy and virus infection are directly related is discussed.