Tolerant Animals Research Articles

Evidence for an important role of protein phosphatases in the mechanism of morphine tolerance

Acute morphine antinociception has been shown to be blocked by very low picogram doses of okadaic acid indicating that inhibition of protein phosphatase PP2A allows for increases in phosphorylation to inhibit antinociception. Comparative studies in morphine tolerant animals have not been reported. In the present study, we showed a significant increase in the total phosphatase activity in the periaqueductal gray matter (PAG) from morphine-pelleted versus placebo-pelleted mice, 72-h after pellet implantation. This supports our hypothesis that phosphatase activity is increased in tolerance as a compensatory mechanism for the increase in kinase activity during the development of tolerance. We also demonstrated that i.c.v. administration of the phosphatase inhibitor okadaic acid (3 pmol/mouse; a dose tested to be inert in placebo-pelleted mice) enhanced the level of morphine antinociceptive tolerance assessed by the tail immersion test, 72-h following pellet implantation. This was supported by the fact that the same treatment with okadaic acid blocked the increase in phosphatase activity in PAG of morphine tolerant mice indicating that selective inhibition of PP2A contributes to enhanced levels of morphine tolerance. We have previously reported that PKC or PKA inhibitors reversed morphine antinociceptive tolerance in mice. The current study shows that i.c.v. administration of the PKC inhibitors bisindolylmaleimide I or Go6976 reversed the enhanced level of morphine tolerance induced by okadaic acid treatment to the same level of tolerance observed in non-okadaic acid-treated tolerant mice. However, the PKA inhibitor PKI-(14-22)-amide only partially reversed the enhancement of morphine tolerance induced by okadaic acid. Our data suggest an important role for the balance between kinases and phosphatases in modulating tolerance levels. Further studies will be directed towards a better understanding of the role of different phosphatase isoforms in morphine tolerance.

Postconditioning in rabbits with haemodynamic nitrate tolerance

We tested whether postconditioning limited infarct size in rabbits with haemodynamic nitrate tolerance. Male rabbits made tolerant (tol.) to the hypotensive response to 30 μg/kg intravenous nitroglycerin (NG) by a preceding 1-week exposure to transdermal NG (0.07 mg/kg/h) were subjected to 35 min coronary occlusion (test ischaemia) followed by 3 h of reperfusion with the following additional interventions: no intervention (NI); postconditioning pacing (PPC): five cycles of 5-min periods of rapid ventricular pacing (500 b.p.m.) or postconditioning coronary occlusion (PCO): five cycles of 5-min coronary occlusion with 10-min interpacing/inter-occlusion intervals, applied after the end of the test ischemia. These protocols was applied in both nitrate tolerant and non-tolerant animals. Infarct size expressed as a percentage of area at risk (I/R) was determined by triphenyltetrazolium chloride staining, left ventricular cyclic nucleotides (pmol/mg ww) were determined by radioimmunoassay from samples out of the area at risk, 75 min after the test ischaemia. The results obtained are seen in the table:

Reduced central c-fos expression and febrile response to repeated LPS injection into periodontal tissue of rats

Systemic injection of repeated doses of Escherichia coli lipopolysaccharide (LPS) results in attenuation of the febrile response, i.e. endotoxin tolerance, which has been fairly well characterized in rats. In the present study, we tested the hypothesis that endotoxin tolerance also occurs after repeated local injection of LPS into periodontal protection tissue. Male Wistar rats were given a gingival intra-pouch injection of sterile saline or LPS at dose of 100 μg/kg on three consecutive days. Body core temperature (Tb) was measured with a miniature datalogger. Another group of animals were used for Fos immunohistochemistry 3 h after the injections in both non-tolerant and tolerant animals. On day one we observed a polyphasic febrile response after LPS injection. The increase in body temperature started about 2 h after LPS administration and lasted 5 h. On day two this response was sensitized and on day three the febrile response was completely abolished. These data suggest that rats develop endotoxin tolerance after repeated LPS administrations into tissues within the oral cavity. Moreover, immunohistochemistry detected a reduction in LPS-induced Fos-like immunoreactivity expression in the subnucleus caudalis of spinal trigeminal nucleus and in the preoptic area of the hypothalamus (POA) in tolerant rats compared with non-tolerant animals, indicating that the endotoxin tolerance may be locally mediated in the periodontal protection tissues of rats.

Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1(u) recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1(u) recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.

Differential gene expression profiles associated with heavy metal tolerance in the soil insect Orchesella cincta

Field-selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola, springtails) populations occurring at metal-contaminated mining sites. Metal tolerance in O. cincta is correlated with heritable increase of excretion efficiency, decrease in cadmium-induced growth reduction, and over-expression of the single copy metallothionein ( mt) gene. Although mt plays a major role in cadmium (Cd) detoxification an additional number of gene products may be altered in the tolerant phenotype that influence quantitative traits like excretion efficiency and growth. Using suppression subtractive hybridization analysis (SSH) genes were cloned that showed up- or down-regulation in response to Cd. This study reports about 192 SSH derived cDNA clones, of which 49 were confirmed to be differentially expressed after spot blot analysis. After sequence analysis 19 of the 49 differential clones could be annotated. Real-time RT-PCR of a reference culture and tolerant populations revealed significant gene responses upon Cd exposure. The reference culture showed stress induced gene expression, whereas the tolerant animals maintained normal gene expression or down regulation of cDNAs upon Cd exposure. Seven cDNAs with homology to genes involved in cell signaling, transport and programmed cell death showed an interaction between treatment and population.

T-Cell Depletion Eliminates the Development of Cardiac Allograft Vasculopathy in Mice Rendered Tolerant by the Induction of Mixed Chimerism

We previously demonstrated that cardiac allografts to fully tolerant chimeric mice developed cardiac allograft vasculopathy (CAV). Here we begin to examine which components of the immune system are responsible for the pathogenesis of CAV in such tolerant recipients. B10.A/B6 mixed chimeric mice were created by receiving injections of bone marrow cells from B10.A (H-2 k) mice given to C57BL/6 (B6; H-2 b) mice with some preparations. B10.A skin grafts were first placed onto B10.A/B6 mixed chimeric recipients. When the donor strain skin grafts had survived perfectly for at least 56 days, B10.A hearts were transplanted heterotopically into B10.A/B6 mixed chimeric recipients. Hearts were examined for the presence of CAV 56 days later. To determine the effector cells that contribute to the development of CAV, they were treated weekly with a combination of anti-CD4/CD8 monoclonal antibodies (mAbs) or anti-NK1.1 mAb continuing until 56 days. 14 B10.A cardiac transplants of 18 otherwise untreated B10.A/B6 chimeric recipients developed CAV; concurrent B6 isografts were unaffected (0/7). In chimeric recipients treated with anti-CD4/8 mAbs, the prevalence of CAV was greatly reduced (0/6, P < .01 compared to the untreated group). Anti-NK1.1 mAb was not effective in the prevention of CAV (4/5). These data suggest that T cells may contribute in some way to the development of CAV that occurs in those fully tolerant recipients. Host T cells that may still be responsive to non-major histocompatability complex antigens, including tissue-specific antigens presented not on skin but on heart, may also be responsible for the development of CAV in tolerant animals.

CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge

Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.

CTLA4-Ig-Based Conditioning Regimen to Induce Tolerance to Cardiac Allografts

Transplant rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem. Mixed hematopoietic chimerism has been shown to produce tolerance to solid organ transplants. However, currently available protocols to induce mixed hematopoietic chimerism invariably require toxic pre-conditioning. In this study, we investigated a non-toxic CTLA4-Ig-based protocol to induce donor-specific tolerance to cardiac allografts in rats. Fully mismatched, 4 to 6 week old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as cell/organ donors and recipients, respectively. Recipients were treated with CTLA4-Ig 2 mg/kg/day (on days 0, 2, 4, 6, 8), tacrolimus 1 mg/kg/day (daily, from days 0 to 9), and a single dose of anti-lymphocyte serum (10 mg) on day 10, soon after total body irradiation (300 cGy) and donor bone marrow (100 x 10(6) T-cell depleted cells) transplantation (BMT). Six weeks after BMT, chimeric animals received heterotopic heart transplants. Hematopoietic chimerism was 18.8 +/- 10.6% at day 30, and was stable (24 +/- 10%) at 1 year post-BMT; there was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>360 days) donor-specific (RT1.A(a); n = 6) hearts, yet rapidly rejected (<9 days) third-party hearts (RT1.A(l); n = 5). Graft (heart) tolerant (>100 days) recipients accepted donor-specific secondary skin grafts (>200 days) while rejected the third-party skin grafts (<9 days). Lymphocytes of graft tolerant animals demonstrated hyporesponsiveness in mixed lymphocyte cultures in a donor-specific manner. Tolerant graft histology showed no obliterative arteriopathy or chronic rejection. The CTLA4-Ig based conditioning regimen with donor BMT produced mixed chimerism and induced donor- specific tolerance to cardiac allografts.

Dynamic changes in CA1 dendritic spines associated with ischemic tolerance

Hippocampal CA1 neurons are particularly vulnerable to 5–10 min durations of global ischemia. These cells can develop tolerance to ischemia through prior exposure to brief episodes of ischemia (ischemic preconditioning, IP). Dendritic spines are implicated in various forms of neuroplasticity including memory and recovery of function. Here we characterized the changes in hippocampal CA1 dendritic spines during the development of ischemic tolerance and the subsequent postischemic recovery period. Gerbils received 5 min, bilateral carotid artery occlusions preceded by two 1.5 min occlusions each of which were 24 h apart (tolerance groups). Spine densities were calculated from CA1 apical and basilar dendrites in tolerant animals that survived 3 (IP3), 10 (IP10) or 30 (IP30) days as well as sham-operated animals and those that received only the two preconditioning episodes (PO). Habituation to a novel open-field was assessed 3, 7, 10 and 30 days after ischemia to gauge CA1 functional integrity. Dendritic spines were quantified from Golgi-Cox stained sections of the CA1 subfield. IP10, IP30 and PO animals had significantly higher CA1 basilar and apical spine densities than all other groups. Tolerant animals initially displayed open-field habituation impairments at a time when spine densities were reduced. Behavioral impairments gradually subsided over time in coincidence with an increase in CA1 spine densities. These findings suggest that dendritic spines may play a role in recovery of function associated with ischemic tolerance and stroke.

ENDOTOXIN TOLERANCE ENHANCES INTERLEUKIN-10 RENAL EXPRESSION AND DECREASES ISCHEMIA-REPERFUSION RENAL INJURY IN RATS

The potential implication of interleukin (IL) 6, tumor necrosis factor alpha (TNF-alpha), and IL-10 in the protective effect of low-dose lipopolysaccharide (LPS) administration against renal ischemia-reperfusion injury was evaluated in a rat model. Eighteen male Sprague-Dawley rats were injected intravenously with either 0.5 mg/kg of LPS (tolerant group) or saline (control group) 2 days before surgery. Ischemic renal injury was induced by clamping the left renal artery for 60 min on rats immediately after right-side nephrectomy. Reperfusion was obtained by clamp removal and was studied at R0 (no reperfusion), 2H (R2), and 24H (R24) by renal tubular disorder characterization and by plasma creatinine as well as renal cytokine (IL-6, IL-10, and TNF-alpha) studies. No differences were observed between the two groups as concerns the period immediately after renal ischemia (R0). The endotoxin-tolerant group was associated with a significantly lower creatinine level at R24 (231 +/- 28 vs 315 +/- 36 micromol/L; P = 0.007). Pretreatment with LPS significantly reduced the degree of proximal tubule necrosis and outer medulla congestion. In such tolerant animals, renal IL-6 production was decreased, whereas IL-10 production was significantly increased at R2 and R24. There were no differences in TNF-alpha renal production. In this study, we demonstrated that administration of low doses of LPS to rats had a protective effect from renal reperfusion injury, and our data suggest that IL-10 might play a role in this phenomenon.

Dominant Tolerance to Kidney Allografts Induced by Anti-Donor MHC Class II Antibodies: Cooperation between T and Non-T CD103+ Cells

Allograft acceptance can be induced in the rat by pretransplant infusion of donor blood or spleen cells. Although promoting long-term acceptance, this treatment is also associated with chronic rejection. In this study, we show that a single administration of anti-donor MHC class II alloimmune serum on the day of transplantation results in indefinite survival of a MHC-mismatched kidney graft. Long-term recipients accept a donor-type skin graft and display no histological evidence of chronic rejection. The kidney grafts of tolerant animals display an accumulation of TCR Cbeta, FoxP3, and IDO transcripts. Moreover, as compared with syngeneic recipients, tolerant recipients harbor a large infiltrate of MHC class II(+) cells and CD103(+) cells. In vitro, splenocytes from tolerant recipients exhibit decreased donor-specific proliferation, which is restored by depletion of non-T cells and partially restored by the blockade of IDO. Finally, splenocytes from tolerant recipients, but not purified T cell splenocytes, transfer donor-specific infectious tolerance without chronic rejection, after infusion into naive recipients, over two generations. However, splenocytes depleted of T cells or splenocytes depleted of CD103(+) cells fail to transfer tolerance. Collectively, these data show that a single administration of anti-donor MHC class II alloimmune serum induces a tolerant state characterized by an infiltration of the kidney graft by regulatory T cells and CD103(+) cells. These data also show that the transfer of tolerance requires the presence of both T cells and CD103(+) dendritic cells. The precise mechanism of cooperation of these two cell subsets remains to be defined.

Facilitation of spinal morphine analgesia in normal and morphine tolerant animals by neuropeptide SF and related peptides

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.

Systemic effects of oral tolerance on inflammation: mobilization of lymphocytes and bone marrow eosinopoiesis

Oral tolerance is a T-cell mediated phenomenon defined by inhibition of immune responsiveness to a protein previously contacted by the oral route. Oral tolerance may prevent autoimmune and allergic diseases that involve the recruitment and/or activation of different cell types including mast cells, neutrophils, eosinophils, monocytes and lymphocytes. The mechanisms by which oral tolerance avoids these immunological disorders are still controversial. Herein we used a murine model of ovalbumin (OVA)-induced peritonitis to investigate the effect of oral tolerance on allergic inflammation. Frequency of leucocyte subpopulations was evaluated by global and differential cell counts in peritoneal lavage fluid, peripheral blood, and bone marrow. Changes on lymphocyte subsets and adhesion molecules expression by these cells were analysed by flow cytometry. As compared with OVA-immune mice, intraperitoneal challenge of tolerant animals with OVA resulted in a significantly milder peritonitis, mostly affecting neutrophils and eosinophils; a concomitant reduction in total white blood cell counts was also observed, mainly because of lower neutrophil and eosinophil counts. Eosinophils, but not neutrophils, were also reduced in the bone-marrow of OVA-challenged tolerant mice. No changes occurred in total peritoneal lymphocyte counts in OVA-tolerant mice, however, there was a significant decrease in CD3+ CD8+ T cells and an increase in B cells (CD45R+) in these animals as compared to immune OVA-challenged animals. Altered expression of CD18 and CD54, respectively, in blood and peritoneal lymphocytes was also noted. These results suggest that, in addition to local specific effects, oral tolerance has systemic effects on the mobilization of leucocytes and bone-marrow eosinopoiesis.

Long-Term Acceptance of Fully Allogeneic Cardiac Grafts by Cotransplantation of Vascularized Thymus in Miniature Swine

We have previously reported the ability of both thymokidney and vascularized thymic lobe (VTL) allografts to induce transplantation tolerance to renal allografts across a full major histocompatibility complex (MHC) mismatch in thymectomized miniature swine. However, whether vascularized thymus is capable of inducing tolerance to less tolerogeneic organs when it is transplanted simultaneously is not yet known. The present study investigates cardiac allograft survival and the mechanism of long-term acceptance in recipient swine following cotransplantation of VTL and cardiac grafts from fully MHC-mismatched donors. Animals received a heart graft, a heart graft and a VTL, or a heart graft and a donor thymocyte infusion. Immunosuppressive regimens consisted of 12 or 28 days of tacrolimus. All animals that received a VTL maintained their grafts significantly longer than their counterparts that received only a heart graft, and those receiving 28 days of tacrolimus maintained their heart grafts long-term. Recipients of a donor thymocyte infusion demonstrated slightly prolonged cardiac graft survival but all rejected their grafts, highlighting the importance of thymic stroma. Cytotoxic T-lymphocyte responses against third-party antigens by cells from tolerant animals showed restriction by both self and donor MHC, whereas responses of controls were restricted to self MHC only. The presence of donor dendritic cells in the VTL grafts and results of co-culture assays suggest that both central and regulatory mechanisms were involved in achieving long-term acceptance. This is the first demonstration of the long-term acceptance of fully MHC-mismatched cardiac allografts in large animals.

The effect of systemic iNOS inhibition during human endotoxemia on the development of TLR-tolerance in vitro

The phenomenon of repeated exposure to endotoxin resulting in diminished release of proinflammatory cytokines is called endotoxin tolerance. After receiving a low-dose pretreatment with endotoxin, tolerant animals survive a challenge with a 'lethal dose' of endotoxin. Recent studies suggests a pivotal role for NO arising from iNOS in the development of tolerance. Toll-like receptors (TLRs) are receptors that recognize specific pathogen-associated molecular patterns. Ten members of the so-called TLR family have been identified in humans, and several of them appear to recognize specific microbial products, including LPS (TLR-4), peptidoglycan (TLR-2) and flagelline (TLR-5). Recognition leads to activation of intracellular signaling pathways, which upregulate a wide array of inflammatory modulators that contribute to the early host cell response. The aim of this study was to test whether administration of the selective iNOS inhibitor aminoguanidine during human experimental endotoxemia influences the development of tolerance to several TLR-agonists in vitro.

Inhibition of Phosphodiesterase 5 Selectively Reverses Nitrate Tolerance in the Venous Circulation

An important component of the antianginal efficacy of glyceryl trinitrate (GTN) is attributable to its selective venodilator effect, resulting in decreased cardiac preload and myocardial oxygen demand. Tolerance to nitrates occurs during chronic exposure, and the current study assessed whether this was due to increased phosphodiesterase (PDE) activity in the venous circulation. Tolerance was induced in rats by continuous exposure to 0.4 mg/h GTN for 48 h. Tension recordings of isolated femoral artery and vein indicated that tolerance was more pronounced in femoral vein. 4-[[3,4-(Methylenedioxy)benzyl]amino]-6-chloroquinazoline (MBCQ), a selective PDE5 inhibitor, significantly decreased the EC(50) values for GTN-induced relaxation in both tolerant and nontolerant tissues, but with the greatest relative shift occurring in tolerant veins. MBCQ also increased the vasodilator potency of 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), a nitric oxide donor; however, cross-tolerance between DEA/NO and GTN was not observed. A significant increase in cGMP PDE activity was observed in tolerant femoral vein, whereas PDE activity was unchanged in femoral artery. Conscious rats treated with hexamethonium (30 mg/kg) to induce ganglionic blockade exhibited blunted central venous pressure (CVP) and mean arterial pressure (MAP) responses to bolus i.v. doses of GTN in GTN-tolerant animals. The cGMP PDE inhibitor zaprinast (1 mg/kg) selectively reversed the blunted CVP response to GTN in tolerant animals but had no effect on the CVP response to GTN in nontolerant animals or on the MAP response in either group. These results suggest that increased PDE5 activity in the venous circulation contributes to the altered hemodynamic response to GTN following chronic GTN exposure.

Nitroglycerin-patch induced tolerance is associated with reduced ability of nitroglycerin to increase exhaled nitric oxide

Nitroglycerin (GTN), used in the treatment of ischemic heart disease, acts through the liberation of nitric oxide (NO). However, its clinical use is limited due to tolerance development. Expired NO was used as an indicator of GTN-bioactivation and was measured together with plasma nitrite and mean arterial pressure (MAP) during GTN indicator infusions. The model was applied in rabbits subjected to various time periods of low-dose GTN pretreatment by patch application for 1, 24 and 72 h. Pretreatment with GTN-patch resulted in significant attenuation of expired NO from the GTN indicator infusion in the 24 h and 72 h pretreatment groups compared to placebo (72 h). Dose–response curves with increasing GTN infusions after 24 h GTN-patch pretreatment revealed a significant attenuation of the MAP decrease compared to placebo. GTN-induced changes in plasma nitrite correlated to increases in expired NO and decreases in MAP. This indicates that expired NO could serve as an indicator of NO generation from GTN in the vascular system. We conclude that GTN tolerance is associated with reduced capacity to generate NO from GTN. Care should be taken in using MAP-reduction to evaluate tolerance since high indicator doses could liberate sufficient amounts of NO to elicit maximal MAP decrease even in tolerant animals.

Tolerance to Rat Heart Grafts Induced by Intrathymic Immunomodulation Is Mediated by Indirect Recognition Primed CD4+CD25+ Treg Cells

In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.

Lymph Node Occupancy Is Required for the Peripheral Development of Alloantigen-Specific Foxp3+ Regulatory T Cells

We previously demonstrated that L-selectin (CD62L)-dependent T cell homing to lymph nodes (LN) is required for tolerance induction to alloantigen. To explore the mechanisms of this observation, we analyzed the development and distribution of regulatory T cells (Treg), which play an important protective role against allograft rejection in transplantation tolerance. Alloantigen-specific tolerance was induced using either anti-CD2 plus anti-CD3 mAbs, or anti-CD40L mAbs plus donor-specific transfusion, in fully mismatched (BALB/c donor, C57BL/6 recipient) vascularized cardiac allografts. An expansion of CD4(+)CD25(+)CD62L(high) T cells was observed specifically within the LN of tolerant animals, but not in other anatomic sites or under nontolerizing conditions. These cells exhibited a substantial up-regulation of Foxp3 expression as measured by real-time PCR and by fluorescent immunohistochemistry, and possessed alloantigen-specific suppressor activity. Neither LN nor other lymphoid cells expressed the regulatory phenotype if recipients were treated with anti-CD62L mAbs, which both prevented LN homing and caused early allograft rejection. However, administration of FTY720, a sphingosine 1-phosphate receptor modulator that induces CD62L-independent T cell accumulation in the LNs, restored CD4(+)CD25(+) Treg in the LNs along with graft survival. These data suggest that alloantigen-specific Foxp3(+)CD4(+)CD25(+) Treg develop and are required within the LNs during tolerization, and provide compelling evidence that distinct lymphoid compartments play critical roles in transplantation tolerance.

Anti‐CD28 Antibody‐Induced Kidney Allograft Tolerance Related to Tryptophan Degradation and TCR− Class II− B7+ Regulatory Cells

B7/CTLA-4 interactions negatively regulate T-cell responses and are necessary for transplant tolerance induction. Tolerance induction may therefore be facilitated by selectively inhibiting the B7/CD28 pathway without blocking that of B7/CTLA-4. In this study, we selectively inhibited CD28/B7 interactions using a monoclonal antibody modulating CD28 in a rat model of acute kidney graft rejection. A short-term treatment abrogated both acute and chronic rejection. Tolerant recipients presented few alloantibodies against donor MHC class II molecules, whereas untreated rejecting controls developed anti-MHC class I and II alloantibodies. PBMC from tolerant animals were unable to proliferate against donor cells but could proliferate against third-party cells. The depletion of B7+, non-T cells fully restored this reactivity whereas purified T cells were fully reactive. Also, NK cells depletion restored PBMC reactivity in 60% of tolerant recipients. Conversely, NK cells from tolerant recipients dose-dependently inhibited alloreactivity. PBMC anti-donor reactivity could be partially restored in vitro by blocking indoleamine-2,3-dioxygenase (IDO) and iNOS. In vivo, pharmacologic inhibition of these enzymes led to the rejection of the otherwise tolerated transplants. This study demonstrates that an initial selective blockade of CD28 generates B7+ non-T regulatory cells and a kidney transplant tolerance sustained by the activity of IDO and iNOS.