Interleukin 2 regulation following semi-allogeneic bone marrow transplantation in mice

Success of allogeneic and autologous bone marrow transplantation (BMT) is hampered by susceptibility to infection during the first two post-treatment years. Further, in treating malignant diseases, impaired anti-host reactivity for donor cells may contribute to a high rate of relapse. Both complications are a consequence of immune deficiency involving B and T lymphocytes. The present study evaluates several key parameters of the immunologic reconstitution mechanism in mice subjected to myeloablative total body irradiation following semi-allogeneic (parental) BMT. This resulted in a gradual reduction of splenic CD3, CD4 and CD8 cells until day 45 post-BMT. Concomitantly, there was an increase in monocytes and CD4⁺/CD8⁺ (double positive) cells, accompanied by a persistent elevation in the percentage of B lymphocytes. The total thymic and splenic T cell populations were reduced until day +30. The cellular reduction correlated with the poor proliferative response of the thymic and splenic cells. A decrease occurred in IL-2 mRNA expression in thymic cells during days 15–20 post-transplant, corresponding with the low level of IL-2 secretion in the spleen and thymus of the transplanted mice. In conclusion, following semi-allogeneic BMT, there was an overall immune down-regulation in the cells, gene and protein levels. Reduced immunological responsiveness following BMT reinforces the need for improving the immune dysfunction by immunotherapy post-BMT.     

Immune recovery following bone marrow transplantation

83 patients undergoing allogeneic or autologous BMT because of haematologic malignancies have been studied before and after transplantation at different intervals. The determinations consisted of lymphocyte counts, E-rosetting, lymphoblastic response, evaluation of serum immunoglobulin levels, skin testing, and in a smaller part of the patients surface marker studies using monoclonal antibodies of the BL-series. At first after BMT the lymphocyte and T cell counts went to normal between 4-18 weeks post transplant, about 4 weeks earlier in autologous than in allogeneic BMT. T suppressor cells showed an early increase compared to T helper cells which normalized much slower about 6 months after BMT. Lymphoblastic responses, however, tended to normal not before the second half of the first year both in autologous and allogeneic transplantation. Skin test reactivity became normal during the 2nd and 3rd year posttransplant, which was more complete in autologous than in allogeneic BMT. The IgG and IgM levels were depressed for half a year and IgA levels for 2 years. The most striking aspect was the multiphase course of lymphoblastic response in every individual patient. We suggest this to be the expression of sequential differentiation of donor lymphocytes.     

Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E(2) (PGE(2)) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF-/- donors (GM-CSF-/- BMT) with untransplanted mice. GM-CSF-/- BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF-/- BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF-/- BMT mice overproduced PGE(2), but expression of the inhibitory EP(2) receptor was diminished. As a consequence of decreased EP(2) receptor expression, we found diminished accumulation of cAMP in response to PGE(2) stimulation in GM-CSF-/- BMT AMs compared with control BMT AMs. In addition, GM-CSF-/- BMT AMs retained cysteinyl leukotriene production and normal TNF-alpha response compared with AMs from control BMT mice. GM-CSF-/- BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF-/- recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.     

The role of busulfan in bone marrow transplantation

High-dose busulfan is an important component in many conditioning protocols for hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation (BMT) in both adults and children. During the past 12y several studies have reported the wide inter-invidual variability in busulfan disposition. Age, disease status, hepatic function, circadian rhythmicity, drug interactions and bioavailability, were identified as factors contributing to the high inter-individual variability found in busulfan disposition. Traditionally, a standard busulfan dose of 4mg/kg/d for four days is used in most BMT/HSCT protocols. Many investigations have pointed out the pharmacodynamic relationship between a high busulfan systemic exposure and the occurrence of BMT related toxicity including hepatic veno-occlusive disease (VOD), interstitial pneumonia and alopecia in adult patients. However, studies in young patients have shown a high rate of graft failure and subsequently relapse which most probably is due to the low systemic exposure despite the standard dose schedule. In children and infants VOD was not observed with the standard doses. Increasing interest for the drug and new modification strategies for children led to higher rate of VOD and CNS toxicity when busulfans was administered according to the body surface area. More pharmacodynamic studies are required to establish the relation between the systemic exposure to busulfan and the therapeutic efficacy, especially in young children undergoing BMT or HSCT. In the present time an accurate and effective busulfan plasma level monitoring combined with dose adjustment based on the known pharmacological parameters may improve the clinical outcome for patients undergoing BMT.     

Linomide administration following bone marrow transplantation in mice

The effect of linomide, an immunomodulatory drug, on natural killer (NK) cells and T cell-dependent immune responses following syngeneic or allogeneic bone marrow transplantation (BMT) was investigated in BALB/c mice inoculated with B-cell leukemia (BCL1). Linomide given in the drinking water had no impact on graft survival or graft versus leukemia (GVL) effects. Although linomide regulates anti-self reactivity in mice with experimental and spontaneous autoimmune disorders, the anti-tumor effects induced by allogeneic donor lymphocytes were not affected. This indicates that different mechanisms regulate anti-self and anti-leukemia effects. Alternatively, linomide might affect the homing of self-reactive lymphocytes to specific target organs in autoimmune disorders, although the homing process may not be relevant to the control of leukemia by alloreactive lymphocytes.     

The regulation of hematopoiesis following bone marrow transplantation

Allogeneic bone marrow transplantation requires that donor stem cells home to the recipient bone marrow, proliferate and differentiate under normal physiologic regulatory mechanisms. Recent observations that T cell depletion of donor bone marrow leads to a greatly increased incidence of graft failure mandate a detailed understanding of the engraftment process. Post-transplant hematopoietic deficiencies appear to be related to several sources: decreased number of stem cells, activation of donor hematopoietic suppressor cells, rejection of donor stem cells by residual recipient lymphocytes and abnormal function of accessory cells that produce hematopoietic growth factors. A better understanding of the relative roles of these factors should lead to a better understanding of engraftment as well as graft failure and its prevention.     

Serum immunoglobulin levels following allogeneic bone marrow transplantation

In order to study the posttransplant evolution of serum immunoglobulin levels, we measured serum IgG, IgA and IgM levels in 50 recipients of allogeneic bone marrow before transplantation and at different intervals thereafter (days 39, 120, 365 and 730). IgG and IgM levels were depressed for 1 year and IgA levels for 2 years posttransplant. Immunoglobulin deficiency was more severe and prolonged in patients with graft versus-host-disease. Hypogammaglobulinemia may contribute to the frequent infections observed in these patients, especially those with chronic graft-versus-host disease.     

Impaired neonatal macrophage phagocytosis is not explained by overproduction of prostaglandin E2

Background

Neonates and young infants manifest increased susceptibility to bacterial, viral and fungal lung infections. Previous work has identified a role for eicosanoids in mediating host defense functions of macrophages. This study examines the relationship between alveolar macrophage (AM) host defense and production of lipid mediators during the neonatal period compared to adult AMs.

Methods

AMs were harvested from young (day 7 and day 14) and adult (~10 week) rats. The functionality of these cells was assessed by examining their ability to phagocytose opsonized targets, produce cytokines, eicosanoids and intracellular cAMP measured by enzyme immunoassays, and gene expression of proteins, enzymes and receptors essential for eicosanoid generation and phagocytosis measured by real time RT-PCR.

Results

AMs from young animals (day 7 and 14) were defective in their ability to phagocytose opsonized targets and produce tumor necrosis factor (TNF)- α. In addition, young AMs produce more prostaglandin (PG) E₂, a suppressor of host defense, and less leukotriene (LT) B₄, a promoter of host defense. Young AMs express higher levels of enzymes responsible for the production of PGE₂ and LTB₄; however, there was no change in the expression of E prostanoid (EP) receptors or LT receptors. Despite the similar EP profiles, young AMs are more responsive to PGE₂ as evidenced by their increased production of the important second messenger, cyclic AMP. In addition, young AMs express higher levels of PDE3B and lower levels of PDE4C compared to adult AMs. However, even though the young AMs produced a skewed eicosanoid profile, neither the inhibition of PGE₂ by aspirin nor the addition of exogenous LTB₄ rescued the defective opsonized phagocytosis. Examination of a receptor responsible for mediating opsonized phagocytosis showed a significant decrease in the gene expression levels of the Fcgamma receptor in young (day 7) AMs compared to adult AMs.

Conclusion

These results suggest that elevated production of PGE₂ and decreased production of LTB₄ do not contribute to impaired opsonized macrophage phagocytosis and highlight an important difference between young and adult AMs.     

Whole bone marrow transplantation induces angiogenesis following acute ischemia

Several recent studies have shown that purified subsets of bone marrow (BM) cells can differentiate into endothelial, cardiac, and other cell types. During coronary artery bypass graft (CABG) surgery, sternal BM is routinely discarded. To determine if this BM can be used to induce angiogenesis and augment perfusion of the cardiac tissues after CABG, a simplified and more practical approach of using whole BM extract was tried to determine whether it would be adequate for the induction of BM-derived angiogenesis in experimental acute limb ischemia. BM was prepared from FVB/N-TgN(TIE2 lacZ)182 Sato (Tie2-lacZ) or B6.129S7-Gtrosa 26 (Rosa 26) mice that express beta-galactosidase (beta-gal) in endothelial cells and most adult tissues, respectively. Acute limb ischemia was induced in either C57BL6/J or FVB/N mice by double ligation of the left femoral artery just distal to the profunda femoral artery branch. Occlusion of the ligated artery was verified by angiography. The study group (n = 31) received an intramuscular injection of 50 micro l containing 1 x 10(6) BM cells, 5 mm proximal to the site of ligation. Experimental controls (n = 21) had an intramuscular injection of 50 micro l of saline. Angiogenesis in the mice was assessed by histological analysis. BM-derived beta-gal(+) cells were observed to aggregate in the vicinity of the ligated artery and not in the injected musculature BM-derived endothelial cells were incorporated within capillaries and small size blood vessels near the site of ligation. Generation of BM-derived blood vessels in experimental acute limb ischemia does not require purification of specific subset of cells. The elimination of cell purification will enhance the ease of using BM transplantation in generating blood vessels.     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E2 (PGE2), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [3H]Thymidine and [3H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h. PGE2 (10(-7) M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10(-7) M) inhibited DNA and collagen synthesis. The addition of PGE2 reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collagen synthesis in central bone to levels above control untreated cultures. We conclude that PGE2 has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Impaired bone resorption to prostaglandin E2 in prostaglandin E receptor EP4-knockout mice

Prostaglandin E(2) (PGE(2)) acts as a potent stimulator of bone resorption. In this study, we first clarified in normal ddy mice the involvement of protein kinase A and induction of matrix metalloproteinases (MMPs) in PGE(2)-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE(2) action using mice lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In calvarial culture obtained from normal ddy mice, both PGE(2) and dibutyryl cyclic AMP (Bt(2)cAMP) stimulated bone resorption and induced MMPs including MMP-2 and MMP-13. Addition of an inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94, significantly suppressed bone-resorbing activity induced by PGE(2.) In calvarial culture from EP1-, EP2-, and EP3-knockout mice, PGE(2) stimulated bone resorption to an extent similar to that found in calvaria from the wild-type mice. On the other hand, a marked reduction in bone resorption to PGE(2) was found in the calvarial culture from EP4-knockout mice. The impaired bone resorption to PGE(2) was also detected in long bone cultures from EP4-knockout mice. Bt(2)cAMP greatly stimulated bone resorption similarly in both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by PGE(2) was greatly impaired in calvarial culture from EP4-knockout mice, but Bt(2)cAMP stimulated MMPs induction similarly in the wild-type and EP4-knockout mice. These findings suggest that PGE(2) stimulates bone resorption by a cAMP-dependent mechanism via the EP4 receptor.     

Effect of selective phosphodiesterase inhibitors on response of ovine pulmonary arteries to prostaglandin E2

Gao, Yuansheng, Jean-François Tolsa, Hai Shen, and J. Usha Raj. Effect of selective phosphodiesteraseinhibitors on response of ovine pulmonary arteries to prostaglandinE₂. J. Appl. Physiol. 84(1): 13-18, 1998.Several adenosine3,5-cyclic monophosphate (cAMP)-hydrolyzingphosphodiesterase isozymes are present in the pulmonary vasculature.The present study was designed to determine the effect of selectiveinhibitors of phosphodiesterase subtypes on prostaglandinE₂(PGE₂)-induced relaxation ofisolated fourth- generation pulmonary arteries of newborn lambs.PGE₂ and forskolin causedpulmonary arteries to relax and induced an increase in theintracellular cAMP content in the vessels. The relaxation and change incAMP content were augmented by milrinone and rolipram, inhibitors ofphosphodiesterase type 3 (PDE3) and type 4 (PDE4), respectively. Theaugmentation in relaxation and the increase in cAMP content caused bymilrinone plus rolipram was greater than the sum of theresponses caused by either of the inhibitors alone.8-Methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine, an inhibitor of phosphodiesterase type 1, had no effect on relaxation andchange in cAMP induced by PGE₂ andforskolin. Acetylcholine alone had no effect on cAMP content in thevessels but augmented the relaxation and the increase in cAMP inducedby PGE₂ and forskolin in arterieswith endothelium. This effect was not observed in arteries withoutendothelium or in arteries with endothelium treated withN^G-nitro-L-arginine.These results suggest that PDE3 and PDE4 are the primary enzymeshydrolyzing cAMP of pulmonary arteries of newborn lambs and that aninhibition of both PDE3 and PDE4 would result in a greater effect thanthat caused by inhibition of either one of the subtype isozymes alone.Furthermore, endothelium-derived nitric oxide may enhance cAMP-mediatedrelaxation by inhibition of PDE3.

     

Membrane-bound prostaglandin E synthase-1-mediated prostaglandin E2 production by osteoblast plays a critical role in lipopolysaccharide-induced bone loss associated with inflammation

PGE(2) acts as a potent stimulator of bone resorption in several disorders including osteoarthritis and periodontitis. Three PGE synthases (PGES) were isolated for PGE(2) production, but which PGES has the major role in inflammatory bone resorption is still unclear. In this study, we examined the role of PGE(2) in LPS-induced bone resorption using membrane-bound PGES (mPGES)-1-deficient mice (mPges1(-/-)). In osteoblasts from wild-type mice, PGE(2) production was greatly stimulated by LPS following the expression of cyclooxygenase 2 and mPGES-1 mRNA, whereas no PGE(2) production was found in osteoblasts from mPges1(-/-). LPS administration reduced the bone volume in wild-type femur that was associated with an increased number of osteoclasts. In mPges1(-/-), however, LPS-induced bone loss was reduced. We next examined whether mPGES-1 deficiency could alter the alveolar bone loss in LPS-induced experimental periodontitis. LPS was injected into the lower gingiva and bone mineral density of alveolar bone was measured. LPS induced the loss of alveolar bone in wild-type, but not in mPges1(-/-) mice, suggesting an mPGES-1 deficiency resistant to LPS-induced periodontal bone resorption. To understand the pathway of LPS-induced PGE(2) production in osteoblast, we used C3H/HeJ mice with mutated tlr4. Osteoblasts from C3H/HeJ mice did not respond to LPS, and PGE(2) production was not altered at all. LPS-induced bone loss in the femur was also impaired in C3H/HeJ mice. Thus, LPS binds to TLR4 on osteoblasts that directly induce mPGES-1 expression for PGE(2) synthesis, leading to subsequent bone resorption. Therefore, mPGES-1 may provide a new target for the treatment of inflammatory bone disease.     

Lymphokines and the acute-phase response in clinical bone marrow transplantation.

This paper reviews the role of the acute phase response and of cytokines in clinical bone marrow transplantation. Data are discussed from the literature and from the authors experience which show that measurement of C-reactive protein is a rather non-specific marker of tissue injury, but that it is elevated in graft-versus-host disease, and especially in infection. Cytokines are clearly implicated in several aspects of transplantation, and tumour necrosis factor in particular may be important. Although there are some data which associate high TNF levels with severe graft-versus-host disease, this association may not hold true in individual patients.     

Frontiers in bone marrow transplantation

The early murine experiments and human studies that indicated the potential of marrow transplantation are reviewed. The results of marrow grafting for a variety of human diseases are summarized. Current directions of research are indicated.     

GM-CSF and the impaired pulmonary innate immune response following hyperoxic stress

We have previously demonstrated that mice exposed to sublethal hyperoxia (an atmosphere of >95% oxygen for 4 days, followed by return to room air) have significantly impaired pulmonary innate immune response. Alveolar macrophages (AM) from hyperoxia-exposed mice exhibit significantly diminished antimicrobial activity and markedly reduced production of inflammatory cytokines in response to stimulation with LPS compared with AM from control mice in normoxia. As a consequence of these defects, mice exposed to sublethal hyperoxia are more susceptible to lethal pneumonia with Klebsiella pneumoniae than control mice. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a growth factor produced by normal pulmonary alveolar epithelial cells that is critically involved in maintenance of normal AM function. We now report that sublethal hyperoxia in vivo leads to greatly reduced alveolar epithelial cell GM-CSF expression. Systemic treatment of mice with recombinant murine GM-CSF during hyperoxia exposure preserved AM function, as indicated by cell surface Toll-like receptor 4 expression and by inflammatory cytokine secretion following stimulation with LPS ex vivo. Treatment of hyperoxic mice with GM-CSF significantly reduced lung bacterial burden following intratracheal inoculation with K. pneumoniae, returning lung bacterial colony-forming units to the level of normoxic controls. These data point to a critical role for continuous GM-CSF activity in the lung in maintenance of normal AM function and demonstrate that lung injury due to hyperoxic stress results in significant impairment in pulmonary innate immunity through suppression of alveolar epithelial cell GM-CSF expression.     

Dynamics of lineage-restricted mixed chimerism following sex-mismatched allogeneic bone marrow transplantation

Scant knowledge is available about the dynamics of lineage-specific mixed chimerism (Ch) following bone marrow transplantation (BMT). This review is focused on findings derived from bone marrow (BM) biopsies in patients with chronic myeloid leukemia (CML) including a sex-mismatched host/donor constellation. Appropriate techniques involved immunophenotyping by monoclonal antibodies to identify the various cell lineages, dual color fluorescence in situ hybridization (FISH) with x- and y-chromosome-specific DNA-probes and a proper detection system for a simultaneous labeling of the bcr/abl locus. A significant degree of Ch with more than 20% host CD34+ progenitors was found in the early and late (up to 200 days after BMT) posttransplant period. However, only 10% of these cells harbored the bcr/abl translocation gene. This result fits well with corresponding molecular biological findings of so-called minimal residual disease. Conversion of Ch evolved during leukemic relapse with 90% host progenitors of which 50% revealed the bcr/abl locus. A Ch of nucleated erythroid percursors (5%) and CD68+ macrophages (8%) was expressed to a significantly lower degree. The slightly increased frequency found in CD61+ megakaryocytes (16%) was probably due to the polyploid state of these cells. Similar to the CD34+ progenitor cells abrupt changes from donor to host type was associated with an insidious transformation into recurrent leukemia. The CD34+ endothelial cells showed a minor degree of Ch, because donor-derived elements ranged from 18% to 25%. Leukemic relapse was characterized by an almost complete conversion of the endothelial cells to a host type. These findings point towards a CD34+ progenitor cell origin of the (leukemic) endothelial cell layer and suggests that their dysfunction may contribute to an expansion of the neoplastic clone.     

Immune complex glomerulonephritis following bone marrow transplantation in C3 deficient mice

Background

The role of circulating complement in host defense and immune disease is well established. Although a number of cells and tissues are capable of synthesizing complement components locally, the importance of such local synthesis in immune disease has been difficult to establish.

Methodology/Principal Findings

We used bone marrow transplantation (BMT) between C3 knockout (C3KO) and wild type (WT) mice to construct animals that were discordant for systemic (hepatic) and local (monocytic) C3 synthetic capacity. An immune complex glomerulonephritis (GN) was then induced using intraperitoneal injections of horse spleen apoferritin (HSA) with a lipopolysaccharide (LPS) adjuvant. All HSA/LPS animals developed a proliferative GN with glomerular infiltration by monocytes. By sensitive ELISA, monocyte C3 synthesis could be detected in C3KO animals transplanted with WT bone marrow cells. Despite this, there were no significant differences among groups of mice in measures of clinical (proteinuria, renal function) or histologic (glomerular cellularity, crescents) disease severity.

Conclusions/Significance

In this model of GN, local synthesis of C3 by infiltrating cells does not appear to be of pathologic importance.