Studies on thymus products. IV. Absence of serum `thymic activity'' in adult NZB and (NZB × NZW) F1 mice

New Zealand Black (NZB) mice and (B/W) F1 hybrids have a normal level of serum `thymic activity' (TA) at birth but this level decreases prematurely between the third and sixth weeks of life. At 2 months, NZB and NZ (B/W) F1 mice have no significant TA, whereas TA is still at birth's level in six control mouse strains and remains at this level until the fourth to the sixth month. Six weeks after the decline of serum TA, NZB mice show disappearance of theta-positive lymph node rosette-forming cells (RFC) and 2 weeks later, progressive decrease in spleen RFC sensitivity to anti-theta serum (AΘS) and azathioprine (AZ) as in neonatally thymectomized CBA mice. Normal AZ and AΘS sensitivity of spleen and lymph node RFC is reconstituted by in vitro or in vivo treatment by thymic extracts. The early thymic abnormalities found in NZB mice are in keeping with the thymic medullar epithelium atrophy reported in newborn NZB mice.     

Studies on thymus products: II. Demonstration and characterization of a circulating thymic hormone

Normal mouse serum confers on bone marrow rosette-forming cells (RFC) from normal mice a high sensitivity to anti-theta serum (AθS) and azathioprine (AZ) which they otherwise lack. Such an effect can also be demonstrated on spleen cells from adult thymectomized mice, `thymus-deprived' and nude mice, but the amount of serum required is higher in the latter mice. This activity of serum on RFC disappears after thymectomy of the serum donor with a half-life of 2.5 hours and reappears in thymectomized mice within 4 days after grafting of a thymus. Serum thymic activity (TA) is present in different amounts in different mouse strains and in ageing mice it progressively disappears. No TA is found in the serum of 4-week-old nude mice. TA is stable after lyophilization but is thermolabile in solution. It passes through UM 10 Amicon membranes, which suggests that its molecular weight (mol. wt) is probably < 10,000. TA is reversibly suppressed in the presence of a serum inhibitor with a mol. wt between 100,000 and 300,000. This inhibitor is not detectable in the serum of thymectomized mice unless the serum is incubated with TA containing serum for 60 minutes at 37°. The biological significance of TA is still a matter of speculation but its role in maturation or expansion of T-cells is suspected.     

Studies on thymus products. VI. The effects of cyclic nucleotides and prostaglandins on rosette-forming cells. Interactions with thymic factor

Cyclic AMP, its dibutyryl derivative and prostaglandins, PGE₁ and PGE₂, do not modify the numbers of rosette-forming cells (RFC), but they do alter these cells' sensitivity to in vitro inhibition by azathioprine (AZ) and anti-theta serum (ATS). In thymectomized mice, cyclic nucleotides and prostaglandins mimic the action of the thymic factor contained in thymic extracts and in normal serum, correcting the low spleen RFC sensitivity to AZ and ATS induced by thymectomy. Moreover, the mixture of infraliminal active doses of cyclic AMP and of thymic extracts (or normal mouse serum) also gives this effect. In normal mice, the nucleotides (but not prostaglandins) lower the AZ and ATS sensitivity of RFC, which remains insensitive to thymic extracts. Theophylline and isoproterenol have effects similar to those of cyclic AMP, a result in accord with their postulated interactions with the adenyl-cyclase system. Noncyclic AMP and cyclic GMP show no effect in the RFC assay system at doses 10–100 times higher than active doses of cyclic AMP. Other vasoactive compounds, including epinephrine, phentolamine, propanolol, and angiotensin, do not show any effect on RFC.     

Demonstration and characterization of a serum factor produced by activated T cells.

Spleen rosette forming cells (RFC) from adult thymectomized mice have a low sensitivity to inhibition by anitheta serum (AOS) and azathioprine (AZ) in comparison with normal spleen or thymus RFC. Thymus extracts and normal mouse serum (but not spleen extracts or thymectomized mouse serum) correct this abnormality after a 30 min in vitro incubation with spleen cells. We report here the existence of a serum factor produced in allogeneic reactions with the same activity on rosettes as thymic factor (TF). This 'allogeneic' factor (AF) is detectable in mice undergoing a graft versus host reaction (GVHR), rejecting skin allografts or allogeneic cells or responding to thymus-dependent antigens such as heterologous red blood cells or BSA. The T-cell origin of AF is indicated by AF presence in nude mice submitted to the same allogeneic stimuli as listed above and in normal mice injected with PVP or LPS. AF is distinct from the thymic factor as shown by differences in electric charge. Moreover, in contrast with TF there is no specific high molecular weight inhibitor of AF. Preliminary biochemical studies indicate that AF is probably a peptide of low molecular weight (greater than 5000 daltons). Its target cell is probably a T-cell precursor.     

Studies on thymus products: III. Epithelial origin of the serum thymic factor

Serum thymic factor (TF) has been tested by its action on spleen rosette-forming cells from adult thymectomized mice. It has been confirmed in a blind study using coded serum samples that TF disappeared early after adult thymectomy and reappeared after grafting a thymus, either as a free graft or enclosed in cell impermeable diffusion chambers. Similar reconstitution was also obtained by grafting a non-lymphoid epithelial thymoma or pure epithelial thymus, obtained by in vivo incubation of a thymus within a diffusion chamber in an intermediate host. Conversely, TF levels were not restored in thymectomized animals treated with dispersed spleen cells or with dispersed thymic lymphocytes.     

Induction of PHA response in mouse bone marrow cells by thymic extracts as studied by changes in the structuredness of cytoplasmic matrix.

The specificity and cross-species activity of thymic extracts and some non-specific agents on some alleged properties of T cells and their precursors in the bone marrow cells of normal, nude and thymectomized mice was studied in vitro. The phenomenon of changes in the structuredness of cytoplasmic matrix (SCM) was used to assess the maturation and responses of cells treated with the extracts to mitogens, i.e. phytohaemagglutinin and concanavalin A. We found that thymic extracts of calf and mouse origin induce functional 'maturation' which can be abrogated by cycloheximide, an inhibitor of protein synthesis. The nonspecific agents used (spleen extracts, PPD, dBcAMP and endotoxin) had no effect on bone marrow cells, as measured by changes in the SCM. The method of measuring SCM using the technique of fluorescence polarization is described.     

Theta-bearing cells in the bone marrow of thymus-deprived mice. Numbers and nature.

Theta-bearing cells in lymphomyeloid tissues of thymus-deprived and normal mice have been studied by the use of anti-theta antiserum and cytotoxicity tests in addition to functional tests. In contrast to the findings in peripheral lymphoid tissues, increased percentages and numbers of theta-bearing cells were found in the bone marrow of neonatally and nude mice as compared with normal and sham-thymectomized mice. In adult thymectomized mice, percentages comparable to those in sham-operated littermates were found. The findings were not due to irrelevant antibodies in the anti-theta antiserum, and neonatally thymectomized mice grafted with a thymic lobe showed percentages of theta-positive cells in the bone marrow comparable to those of sham-operated animals. Adrenalectomy did not lead to diminished percentages of theta-positive cells in the bone marrow of neonatally thymectomized mice, and the serum levels of hydrocortisone and corticosterone were within normal ranges in thymus-deprived mice. The mitogen responses and graft-versus-host activity of bone marrow cells from neonatally thymectomized mice suggest that most theta-positive cells in the bone marrow of these mice are functionally immature cells.     

Lymphocyte activation: I. Response of T and B lymphocytes to phytomitogens

The selectivity of phytomitogens for T (thymus derived/dependent) and B (`bursa-equivalent' dependent/derived) lymphocytes from the mouse spleen has been investigated. Responses of normal spleen cell cultures were compared with those of cultures of selected B cells. The latter were obtained from three sources (1) spleen cells of mice that had been thymectomized as adults, lethally irradiated and reconstituted with syngeneic bone marrow cells pretreated with anti-θ serum (2) spleen cells from congenitally athymic (`nude') mice and (3) spleen cells from normal mice treated with anti-θ serum plus guinea-pig complement prior to culture.

Using a variety of different culture conditions it was shown that B cells respond well to pokeweed mitogen, and poorly if at all to phytohaemagglutinin. Responsiveness to the latter mitogen in normal spleen cell cultures appears to be a property of T cells.

     

Thymus dependence of θ-bearing cells in the peripheral lymphoid tissues of mice

In mice thymectomized as newborns, thymectomized as adults and irradiated, and in nude mice with congenital absence of the thymus, there is a marked reduction in the number of θ-bearing lymphocytes in the lymph nodes and spleen. This suggests that θ is present on those lymphocytes which are thymus-derived. Using ⁵¹Cr and dye exclusion cytotoxic testing, approximately 65–85 per cent of lymph node and thoracic duct lymphocytes and 30–50 per cent of spleen lymphocytes were found to have θ on their surface.     

Theta-Bearing Cells in the Bone Marrow of Thymus-Deprived Mice

Theta-bearing cells in lymphomyeloid tissues of thymus-deprived and normal mice have been studied by the use of anti-theta. antisenun and cytotoxicity tests in addition to functional tests, In contrast to the findings in peripheral lymphoid tissues, increased percentages and numbers of theta-bearing cells were found in the bone marrow of neonatally thymectomized and nude mice as compared with normal and sham-thymectomized mice. In adult thymectomized mice, percentages comparable to those in sham-perated littermates were found. The findings were not due to irrelevant antibodies in the anti-theta antiserum, and neonatally thymectomized mice grafted with a thymic lobe showed percentages of theta-positive cells in the bone marrow comparable to those of sham-operated animals. Adrenalectomy did not lead to diminished percentages of theta-positive cells in the bone marrow of neonatally thymectomized mice, and the serum levels of hydrocortisone and corticosterone were within normal ranges in thymus-deprived mice. The mitogen responses and graft-versus-host activity of bone marrow cells from neonatally thymectomized mice suggest that most theta-positive cells in the bone marrow of these mice are functionally immature cells.     

c-Met signalling is required for efficient postnatal thymic regeneration and repair

We have reported that in vivo administration of the hybrid cytokine rIL-7/HGFβ or rIL-7/HGFα, which contains interleukin-7 (IL-7) and the β- or α-chain of hepatocyte growth factor (HGF), significantly enhances thymopoiesis in mice after bone marrow transplantation. We have shown that the HGF receptor, c-Met, is involved in the effect of the hybrid cytokines. To address the role of c-Met signalling in thymocyte development and recovery, we generated conditional knockout (cKO) mice in which c-Met was specifically deleted in T cells by crossing c-Met^ft/ft mice with CD4-Cre transgenic mice. We show here that although the number of total thymocytes and thymocyte subsets in young c-Met cKO mice is comparable to age-matched control (Ctrl) mice, the cKO mice were more susceptible to sub-lethal irradiation and dexamethasone treatment. This was demonstrated by low recovery in thymic cellularity in c-Met cKO mice after insult. Furthermore, the number of total thymocytes and thymocyte subsets was markedly reduced in 6- to 12-month-old cKO mice compared with age-matched Ctrl mice, and the thymic architecture of 12-month-old cKO mice was similar to that of 20-month-old wild-type mice. In addition, c-Met deficiency reduced cell survival and the expression of Bcl-xL in double-positive thymocytes, and decreased cell proliferation and the expression of cyclin E and cyclin-dependent kinase 5 in single-positive thymocytes. Our data indicate that c-Met signalling plays an important role in thymic regeneration after thymic insult. In addition, T-cell-specific inactivation of c-Met accelerates age-related thymic involution.     

Cell participation in immune response by immune ribonucleic acid. I. The role of T lymphocytes in immune response by immune RNA against T-dependent antigens

T- and B-cell participation in the immune response induced by immune ribonucleic acid (iRNA) preparations against T-dependent antigens was studied using athymic nude, neonatally thymectomized (NT) and cyclophosphamide-treated (CY) mice. The iRNA(T + B) preparations were made from the spleen of BALB/c mice immunized with these antigens. Injection of the iRNA into nude or NT mice caused an increase in the number of specific rosette-forming cells (RFC) and of memory cells capable of responding to secondary stimulus with a small dose of the corresponding antigen. Injection with T-dependent antigens or with iRNA(T + B) did not cause any immune response in CY mice, suggesting depletion of the B-cell function. The iRNA(T) and iRNA(B) were prepared, respectively, from the thymuses of BALB/c mice and from the spleens of nude mice which had been immunized with T-dependent antigens. Injection of nude mice with both iRNA(T) and iRNA(B) caused an increase in the number of specific RFC and the secondary antibody formation response after boosting with a small dose of the corresponding antigen. Injection of iRNA(T) preparation into nude mice could induce the anamnestic response after boosting. However, neither of the iRNA(T) or iRNA(B) preparation could induce in nude mice the proliferation of the number of specific RFC. These results indicate the presence of at least two kinds of iRNA preparations against T-dependent antigens and that the cooperation of iRNA(T) and iRNA(B) was required for the induction of immune response against T-dependent antigens.     

In vitro culture of functional human thymic epithelium.

Cultures of pure epithelial cells have been obtained from human thymus. Incubation of spleen cells from adult thymectomized mice on these cultures rendered RFC sensitive to inhibition by anti-theta serum, whereas no effect was obtained with fibroblasts or spleen cell cultures. The fact that theta conversion was obtained when spleen cells were incubated in the thymic culture placed within a Millipore chamber indicates that theta conversion was due to the effect of a humoral factor.     

The suppression of sheep rosette-forming cells and the inability of mouse bone marrow cells to reconstitute competence after infection with the nematode Trichinella spiralis

Mice infected with the nematode Trichinella spiralis or normal mice treated either with the serum of infected animals or with a parasite extract contained fewer spleen rosette-forming cells (RFC) to sheep erythrocytes than the controls. Bone marrow cells from infected mice were considerably less efficient than normal cells in reconstituting thymectomized, irradiated animals.     

Response of lymphocytes to plant lectins: I. A thymic-dependent lymphoid population responsive to Pokeweed mitogen

The response of lymph node, bone marrow, splenic and thymic lymphocytes to phytohaemagglutinin and Pokeweed mitogen has been studied. Depletion of theta positive or recirculating lymphocytes abolished the response to phytohaemagglutinin but did not impair the response to Pokeweed mitogen. Lymphocytes from thymectomized or athymic `nude' mice respond poorly or not at all to phytohaemagglutinin or Pokeweed mitogen.

We conclude that the normal response to Pokeweed mitogen depends upon an intact thymus gland. This despite the fact that one population of Pokeweed mitogen-responsive cells are theta-negative bone marrow-derived cells. This conclusion is based upon: (1) the equal reactivity of theta-positive and theta-negative lymphocytes to Pokeweed mitogen as shown by the normal response to Pokeweed mitogen of anti-theta serum treated lymphoid populations and (2) the impaired response of lymphocytes from nude mice and of lymphocytes from thymectomized, irradiated and bone marrow reconstituted mice to Pokeweed mitogen.

     

Suppressed Accumulation of Cerebral Amyloid β Peptides in Aged Transgenic Alzheimer’s Disease Mice by Transplantation with Wild-Type or Prostaglandin E2 Receptor Subtype 2-Null Bone Marrow

A complex therapeutic challenge for Alzheimer’s disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid β (Aβ) peptides. One potential target is selective suppression of microglial prostaglandin E₂ receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2^−/−) into lethally irradiated 5-month-old wild-type or APPswe-PS1ΔE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2^−/− microglia was more efficient in APPswe-PS1ΔE9 than in wild-type mice, and APPswe-PS1ΔE9 mice that received EP2^−/− BMT had increased cortical microglia compared with APPswe-PS1ΔE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Aβ plaques and potentially more neurotoxic soluble Aβ species. An additional 25% reduction in cerebral cortical Aβ burden was achieved in mice that received EP2^−/− BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Aβ peptide and plaque accumulation in the cerebrum of patients with AD.Alzheimer’s disease (AD), the most common dementing neurodegenerative disease,¹ is a major public health burden for older Americans.² Amyloid β (Aβ) peptides are pleotropic molecules that are directly neurotoxic and stimulate liberation of cytotoxic cytokines through activation of microglia innate immune response.³ However, activated microglia phagocytosis and degradation of Aβ species is key to cerebral Aβ homeostasis.⁴ Thus, an important but complex therapeutic challenge is balancing deleterious and beneficial aspects of microglial activation in AD.⁵ One proposed mechanism of microglial modulation is prostaglandin E₂ signaling, especially through activation of the E prostanoid receptor subtype 2 (EP2).⁶ Cultured microglia lacking EP2 (EP2^−/−) show enhanced phagocytosis of Aβ from human brain explants and reduced paracrine neurotoxicity.⁷ In vivo experiments with EP2^−/− mice have shown reduced accumulation of cerebral Aβ in a transgenic mouse model of AD,^7,8,9 as well as suppressed oxidative damage to neurons following innate immune activation.^7,10,11,12 However, because EP2 is expressed by several cell types in brain, including microglia and neurons, the importance of microglial-specific EP2 has not been established. To address this gap in our knowledge, bone marrow cells from EP2^−/− mice were transplanted into APPswe-PS1ΔE9 mice.Circulating bone marrow transplant (BMT)-derived cells can selectively replace resident microglia,¹³ and up to 30% of microglia can be derived from donor marrow in wild-type mice recipients up to a year after transplantation.^14,15 Moreover, engraftment of brain appears qualitatively more efficient in recipient AD mice than in wild-type controls.^16,17 The reasons for the apparent higher engraftment are not clear, but may be in response to chronic low level immune activation in AD mouse brains.^16,17 Some investigators have shown BMT-derived microglia associated with Aβ deposits in vivo, and that transgenic AD mouse BMT recipients have reduced Aβ plaque burden.¹⁷ Although previous data addressed potential mechanisms by which BMT-derived microglia might promote clearance of Aβ peptides,¹⁸ the results of these studies were confounded by the effects of preconditioning brain irradiation; it is possible that the reduced Aβ plaque burden was caused by irradiation-induced alteration of Aβ production or clearance rather than BMT-derived microglia. In the current studies, we robustly quantify microglial engraftment in brains of APPswe-PS1ΔE9 mice. In addition, we control for the potential confounder of irradiation-mediated Aβ peptide suppression by evaluating Aβ in mice that received cranial-specific irradiation with or without BMT. Finally, we test the hypothesis that BMT with cells from EP2^−/− mice would enhance cerebral bone marrow derived microglia engraftment and clearance of Aβ peptides from cerebrum of APPswe-PS1ΔE9 mice.     

Deficient antibody formation in the bone marrow of nude mice

The bone marrow of young adult nude mice was investigated as a site of antibody formation after intravenous immunization with the thymus-independent antigen Escherichia coli lipopolysaccharide (LPS). Mice heterozygous for the nu-gene were found to be capable of a plaque-forming cell (PFC) response in both spleen and bone marrow after primary and secondary immunization with LPS. Primary immunization of nude mice with LPS induced a normal PFC response in the spleen, but did not evoke the appearance of PFC in the bone marrow. During the secondary response the nude mice did show PFC activity in the bone marrow, but at a much lower level than their heterozygous littermates. At all time points after secondary immunization the number of splenic PFC was higher in nude mice than in the control mice.

Determination by immunofluorescence of cells containing cytoplasmic immunoglobulin (C-Ig cells) in the bone marrow of young adult nonimmune nude and heterozygous mice, revealed a three times higher number of C-IgM cells in the bone marrow of the heterozygous thymus-bearing mice. On the other hand, the number of splenic C-IgM cells was higher in the nude mice than in their heterozygous littermates. These results suggest that the presence of the thymus facilitates the appearance of mature antibody-forming cells in the bone marrow of young adult mice, irrespective of whether the generation of these cells is initiated by so called thymus-dependent or thymus-independent antigens.

     

Stimulation of DNA synthesis in mouse lymphoid cells by polyanions in vitro. I. Target cells and possible mode of action

The effect of dextran sulfate on [³H]dThd incorporation in lymphoid cells was investigated. The polyanion activated DNA synthesis in spleen and bone marrow cells of normal mice. The highest rate of activation was detected in spleen cells of athymic (nude) mice; the ratio of [³H]dThd incorporation was higher in TxBM spleen cells of thymectomized, lethally irradiated and bone marrow protected mice than in spleen cells of normal donors. Thymus cells of normal mice could not be stimulated, but a slight response was obtained in cortisone-resistant thymus cells. A synergetic effect was found in normal thymus cells using a combination of dextran sulfate and phytohemagglutinin (PHA). In contrast, lipopolysaccharide and PHA showed no synergetic effect. The possible mode of action of polyanions as de-repressors of DNA synthesis is discussed.     

Effects of levamisole on spontaneous rosette-forming cells in murine spleen.

Levamisole, an anti-anergic chemotherapeutic agent, is shown to depress the azathioprine sensitivity of rosette-forming cells (RFC) in the spleen of normal mice, and to restore the azathioprine sensitivity of RFC in the spleen of adult thymectomized mice. Using the fully active dose of 1.25 mg/kg i.v., the restoration effect was present 15 h after treatment with the drug and almost disappeared after 48 h. Levamisole was ineffective on RFC in vitro. These results suggest that Levamisole might interfere with the reactivity of T lymphocytes as has been described for drugs interfering with cyclic nucleotide metabolism.     

Graft-versus-host-disease-associated donor cell engraftment in an F1 hybrid model is dependent upon the Fas pathway

The graft‐versus‐host disease (GVHD) generated in BDF₁ mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell‐mediated attack on host lymphohaematopoietic populations, resulting in the reconstitution of the host with donor cells. We examined Fas–Fas ligand (FasL) interactions in donor and host haematopoietic cells over a prolonged period of parental‐induced GVHD. Fas expression on bone marrow cells of both donor and host origin increased at 2 weeks. Host cell incubation with anti‐Fas antibody induced apoptosis, and the number of haematopoietic progenitor cells decreased. Fas‐induced apoptosis by the repopulating donor cells, however, did not increase until 12 weeks, when more than 90% of the cells were donor cells. The expression of various cytokines, such as interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), and FasL gene expression in the bone marrow increased concomitantly. To examine directly whether FasL has a major role in the development of donor cell engraftment, FasL‐deficient (gld) mice were used as donors. Injection of B6/gld spleen cells induced significantly less host lymphohaematopoietic depletion, resulting in a failure of donor cell engraftment. Furthermore, injection of IFN‐γ gene knockout (gko) B6 spleen cells failed to augment Fas and FasL expression in recipient mice, resulting in a failure of donor cell engraftment. This suggests that the induction of apoptosis by Fas–FasL interactions in host cells may contribute to a reconstitution of the host with donor cells and that donor‐derived IFN‐γ plays a significant role for Fas–FasL interactions in host cells during parental‐induced GVHD.