Inability of memory T cells to induce graft-versus-host disease is a result of an abortive alloresponse

Several groups, including our own, have independently demonstrated that effector memory T cells from non-alloantigen-primed donors do not cause graft-versus-host disease (GVHD). In the current study, we further investigated whether this approach could be extended to all memory T cells, and we studied the underlying mechanisms. Neither total memory T cells nor purified central memory T cells were able to induce GVHD. Memory T cells were at least 3-log less potent than bulk T cells in mediating GVHD. As expected, memory T cells failed to elicit cytotoxicity and proliferated poorly against alloantigens in standard 5-day mixed-lymphocyte cultures. However, the proliferative responses of memory T cells were more comparable with those of bulk and naive T cells when the culture time was shortened. Moreover, the frequencies of IL-2-secreting cells measured by 42-hour enzyme-linked immunosorbent spot (ELISPOT) assay were similar among naive, memory, and bulk T cells. These data indicated that memory T cells are able to respond to alloantigens initially but fail to develop to full potential. The abortive immune response, which was mediated by non-alloantigen-specific memory T cells in response to alloantigens, may explain why memory T cells from unprimed and non-alloantigen-primed donors could not induce GVHD.     

Age-dependent changes in intraepithelial lymphocytes (IELs) of the small intestine, cecum, and colon from young adult to aged mice

We previously reported the regional differences in the IELs present in the proximal (P), middle (M), and distal (D) parts of the small intestine, cecum (Ce), and colon (Co) of mice. In this study, we investigated the age-dependent changes in the regional differences of IELs from young adult to aged mice. In this experiment, 3-, 6-, 12-, 18-, and 24-month-old mice were examined. IELs were separately isolated from 5 parts of the intestines and analyzed by flow cytometry. Regional differences in the number and phenotype of IELs showed the same trends in all age groups. The number of IELs was highest in 6-month-old mice and then gradually decreased with age. As to IEL subsets, age-related changes were not seen except for a few subsets among the age groups. We conclude that age-related decreases in IELs in mouse small intestine may be one of the aging phenomena of the intestinal immune system. Such age-related decreases in IELs may be concerned with the increased liability to intestinal infections in the elderly.     

Antigen-specific helper T-cell clone supernatant is sufficient to induce both polyclonal proliferation and differentiation of small resting B lymphocytes.

Gradient-purified resting B lymphocytes can be polyclonally stimulated by antigen-specific major histocompatibility complex (MHC)-restricted helper T lymphocytes as well as by antigen-activated helper T-cell supernatant. In contrast to what has been described so far, we show that helper T-cell supernatant (in the absence of any other added stimulus, such as that provided by anti-mu antibodies) is sufficient to induce both proliferation of resting B cells and their differentiation into IgM-secreting cells. The stimulation induced by the helper T-cell supernatant takes place in serum-free medium and is not MHC-restricted. Our findings strongly support the existence of a B-cell activating factor acting on the resting B cell and causing it to enter the G1 phase of the cell cycle in a MHC-unrestricted manner.     

Short DNA sequences from the cytoplasm of mouse tumor cells induce immortalization of human lymphocytes in vitro.

Cytoplasts of mouse L929 and Ehrlich ascites tumor cells harbor DNA sequences that induce unlimited proliferation ("immortalization") of human lymphocytes after transfection in vitro. By equilibrium centrifugation of cytoplasmic lysates in a neutral CsCl gradient, the immortalizing activity was recovered together with extramitochondrial fractions at high salt densities (1.85-1.87 g/cm3). Unexpectedly, these fractions contain linear DNA molecules of 50-500 bp in length. In contrast, cytoplasts of primary, senescent cells (mouse embryo fibroblasts, human lymphocytes) do not harbor DNA in the corresponding fractions. Cytoplasmic DNA isolated from high-density fractions of mouse tumor cells was cloned in subset libraries, and of 45 DNA sequences we identified 2 clones--one from L929 cytoplasts (203 bp) and another one from the cytoplasm of Ehrlich ascites cells (372 bp)--that induce unlimited proliferation of human lymphocytes in vitro. Immortalized lymphoid cells harbor 1-5 copies of transfected DNA integrated into chromosomal DNA, whereas about 100 copies were found as episomal DNA in the cytoplasmic fraction. No immortalization could be induced by transfection of nuclear DNA randomly fragmented to 200-500 bp. Although the cloned DNA sedimented at 1.70 g/cm3, after transient transfection into lymphocytes, these DNA sequences form salt-stable complexes that sediment in fractions at the same high density (1.82-1.88 g/cm3) from which they were originally cloned. The high-density banding of these cytoplasmic DNA sequences may be due to association with RNA and/or with (metallo-) proteins in vivo. Since both cloned DNA sequences with immortalizing activity have stop codons for protein translation in all possible reading frames, immortalization may be induced by insertional inactivation or functional suppression of genes that are needed to be expressed during cellular senescence or programmed cell death.     

Features of small intestinal pathology (epithelial cell kinetics, intraepithelial lymphocytes, disaccharidases) in a primary Giardia muris infection.

In an attempt to correlate host and parasite-related events occurring during the course of a primary Giardia infection in the mouse we have measured epithelial cell kinetics, enzymes, and intraepithelial lymphocytes at different stages of the infection. New methods were developed for the accurate measurement of parasite numbers and distribution within the gut. In jejunum a modest decrease in villus length and intraepithelial lymphocytes at week 1 preceded a pronounced disaccharidase deficiency at week 2, the time of maximum trophozoite numbers, whereas crypt lengthening and increased cell production became maximal at week 3. As trophozoite numbers fell the intraepithelial lymphocyte count and disaccharidase values rose. With the exception of the intraepithelial lymphocyte count, which followed the same pattern as in jejunum but two weeks later, the changes seen in the ileum were the opposite of those in jejunum, suggesting rapid ileal adaptation. The results indicate that the disaccharidase deficiency associated with giardiasis is likely to represent a direct effect of the parasite on the brush border rather than enterocyte immaturity, whereas the intraepithelial lymphocyte response reflects host immunity to the parasite. Profound adaptive changes occur throughout the small intestine in response to a relatively localised insult.     

Acute epithelial injury in the rat small intestine in vivo is associated with expanded expression of transforming growth factor alpha and beta.

BACKGROUND--Previous studies have shown the importance of transforming growth factors alpha and beta (TGF alpha and TGF beta) in modulating epithelial cell restitution after injury in vitro. AIM--To investigate the role of the growth factors TGF alpha and TGF beta after acute epithelial injury in vivo. METHODS--An in vivo model of phytohaemagglutinin (PHA) induced acute epithelial injury in rat small intestine was used. Epithelial cell turnover was assessed by autoradiography and liquid scintillation counting of thymidine uptake. Expression of TGF alpha and TGF beta was assessed by immunohistochemistry. RESULTS--An expansion of the proliferative compartment and increased turnover of intestinal epithelial cells was seen in rats with PHA induced intestinal epithelial injury. Expression of TGF alpha and TGF beta peptides was shown in both the epithelial cell and lamina propria compartment. Different patterns of TGF alpha and TGF beta expression were seen, however, within the epithelium of rats with acute intestinal injury compared with untreated controls, while the expression of these peptides within the lamina propria was not changed. CONCLUSIONS--These findings suggest that acute intestinal epithelial cell injury in vivo is associated with compensatory changes in expression of TGF alpha and TGF beta in the epithelial cell compartment, while the lamina propria does not seem to be significantly affected.     

Survival of mouse pancreatic islet allografts in recipients treated with allogeneic small lymphocytes and antibody to CD40 ligand.

Combined treatment with allogeneic small lymphocytes or T-depleted small lymphocytes plus a blocking antibody to CD40 ligand (CD40L) permitted indefinite pancreatic islet allograft survival in 37 of 40 recipients that differed from islet donors at major and minor histocompatibility loci. The effect of the allogeneic small lymphocytes was donor antigen-specific. Neither treatment alone was as effective as combined treatment, although anti-CD40L by itself allowed indefinite islet allograft survival in 40% of recipients. Our interpretation is that small lymphocytes expressing donor antigens in the absence of appropriate costimulatory signals are tolerogenic for alloreactive host cells. Anti-CD40L antibody may prevent host T cells from inducing costimulatory signals in donor lymphocytes or islet grafts.     

Intestinal permeability to polyethyleneglycol 600 in Crohn's disease. Peroperative determination in a defined segment of the small intestine.

Ileal permeability to different sized polyethyleneglycols (590-942 dalton PEG) was investigated peroperatively in 11 patients with Crohn's disease and seven with colonic carcinoma. A 15 cm ileal segment was converted into a tied loop, in which the PEG's were deposited. Absorption from the ileal segment was then measured as six-hour urinary recovery of the PEg dose. Polyethyleneglycol absorption in Crohn's disease was greater than in cancer patients and similar throughout the weight range, but in the cancer patients it was inversely proportional to molecular weight. Thus there was significantly greater absorption of the higher weights (greater than or equal to 678 dalton) in the Crohn's, than in the cancer patients. The apparently increased permeability of the small intestine in Crohn's disease may be an important factor in its pathogenesis.     

Anti-endothelial cell IgG fractions from systemic lupus erythematosus patients bind to human endothelial cells and induce a pro-adhesive and a pro-inflammatory phenotype in vitro.

Affinity purified immunoglobulin G (IgG) fractions from systemic lupus erythematosus (SLE) patients positive for anti-endothelial cell antibodies (AECA) bind human umbilical vein endothelial cell (HUVEC) monolayers. In vitro incubation of serial protein concentrations of SLE AECA IgG induces a dose-dependent endothelial activation: i) increase of functional adhesion of the monocytic cell line U937; ii) upregulation of E-Selectin, ICAM-1, VCAM-1 expression evaluated by a cell solid-phase enzyme linked immunoassay; and iii) increased secretion of interleukin (IL)-6 in the culture supernatants. Control experiments carried out with HUVEC monolayers incubated with IgG fractions from normal healthy controls or from AECA negative SLE sera do not affect at all endothelial adhesion molecule expression or pro-inflammatory cytokine secretion. The AECA IgG effects are not related to both anti-phospholipid or anti-DNA activities. Taken together the findings suggest that these autoantibodies might be important in recruiting and in activating mononuclear leukocytes responsible for vessel wall infiltration and raise the possibility that AECA might display a pathogenic role in SLE vessel damage.     

A proposal concept of a polygene network in systemic vasculitis: lessons from MRL mouse models.

In addition to the studies of cellular and molecular events in the pathogenesis of systemic vasculitis, a genome analysis of mouse models may shed some light on the complex clinicopathological manifestations of systemic vasculitis. In the study of susceptibility loci to vasculitis in MRL mouse models, we learned that systemic vasculitis developed in a cumulative effect of multiple gene loci, each of which by itself did not have a significant effect to induce the related phenotype, thus indicating a polygenic system. The mice developed vasculitis in an additive manner of multiple genes with a hierarchical effect. Some of the susceptibility loci seemed to be common to those in other collagen diseases as well. Moreover, the loci controlling tissue specificity of vasculitis were present. One of the positional candidate genes for vasculitis showed an allelic polymorphism in the coding region, thus possibly causing a qualitative difference in its function. As a result, a particular combination of polygenes with such an allelic polymorphism may thus play a critical role in leading the cascade reaction to develop vasculitis, and also a regular variation of systemic vasculitis. This is designated as the polygene network in systemic vasculitis.     

Motor responses of the small intestine to intraluminal distension in normal volunteers and a patient with visceral neuropathy.

The motor responses of the small intestine to intraluminal distension were studied proximal and distal to an inflatable balloon in 13 normal volunteers. During fasting, distension rapidly induced a persistent localised inhibition of distal contractile activity with a small proximal increase. Proximally, phase III activity was unaffected during distension but its propagation across and appearance below the balloon was inhibited. Upon deflating the balloon a normal motor pattern rapidly returned. Similar changes were observed during distension in the fed state. The changes in the motor pattern resemble those of the intrinsically mediated 'peristaltic reflex', studied in animals, and suggest that in man the response to balloon distension may also be mediated through an intrinsic mechanism. A patient with a visceral neuropathy, studied in a similar manner, had no inhibition of distal motor activity during distension, suggesting a functional defect of the enteric nerves. Further observations of the motor responses to distension in similar patients seem indicated to determine the usefulness of this technique for evaluating enteric nervous system function when an abnormality is suspected.     

Mast cell-dependent proteolytic modification of HDL particles during anaphylactic shock in the mouse reduces their ability to induce cholesterol efflux from macrophage foam cells ex vivo

ObjectiveWe have found previously that proteolytic modification of HDL by mast cell chymase in vitro reduces cholesterol efflux from cultured macrophage foam cells. Here, we evaluated whether mast cell-dependent proteolysis of HDL particles may occur in vivo, and whether such modification would impair their function in inducing cellular cholesterol efflux ex vivo.MethodsSystemic activation of mast cells in the mouse was achieved by intraperitoneal injection of a high dose of the mast cell-specific noncytotoxic degranulating agent, compound 48/80. Serum and intraperitoneal fluid were then evaluated for degradation of HDL apolipoproteins and for their potential to act as cholesterol acceptors from cultured mouse macrophage foam cells.ResultsLysates of isolated mouse peritoneal mast cells containing active chymase partially proteolyzed apoA-I in α- and preβ-HDL particles in mouse serum in vitro, and, when injected into the mouse peritoneal cavity, the lysates also degraded endogenous apoA-I in peritoneal fluid in vivo. Systemic activation of mast cells in mast cell-competent mice, but not in mast cell-deficient (W-sash c-kit mutant) mice, reduced the ability of serum and intraperitoneal fluid derived from these animals to promote efflux of cellular cholesterol. This inhibitory effect was related to mast cell-dependent proteolytic degradation of apoA-I, apoA-IV, and apoE, i.e., the HDL-associated apolipoproteins that are efficient inducers of cholesterol efflux.ConclusionThe present results document a role for extracellular mast cell-dependent proteolysis in the generation of dysfunctional HDL, and suggest an inhibitory role for mast cells in the initial step of reverse cholesterol transport in vivo.     

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-gamma production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and IFN-gamma mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-alpha mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-alpha/beta. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.     

Different capabilities of monocytes from patients with systemic lupus erythematosus and rheumatoid arthritis to induce glycosylation alterations of acute phase proteins in vitro.

The effect of conditioned medium on the biosynthesis and glycosylation profile of acute phase proteins secreted by the human hepatoma cell line Hep G2 was studied. Conditioned medium was prepared from nonactivated [CM-LPS(-)] and ex vivo lipopolysaccharide activated [CM-LPS(+)] monocytes from eight patients with active rheumatoid arthritis (RA), five patients with active systemic lupus erythematosus (SLE), and seven healthy subjects. The biosynthesis of albumin, alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor and the profile of glycosylation of proteinase inhibitor were analysed. CM-LPS(-) from patients with SLE had a similar effect to CM-LPS(-) from healthy subjects. In contrast, CM-LPS(-) from patients with RA had the same effect as CM-LPS(+) from healthy donors. A similar effect to that of CM-LPS(+) of healthy subjects was seen with CM-LPS(+) from patients with SLE and with CM-LPS(+) from patients with RA. The treatment of CM-LPS(+) with antibodies against interleukin 6 neutralised most of its ability to induce changes in the biosynthesis and glycosylation of acute phase proteins. Antibodies to interleukin 1 and tumour necrosis factor alpha had only a limited effect on the ability of CM-LPS(+) to induce changes of albumin and alpha 1-antichymotrypsin syntheses, whereas they had no effect on the biosynthesis and glycosylation of proteinase inhibitor. These results indicate that: (a) monocytes isolated from patients with active SLE and active RA have different capabilities of inducing alterations of acute phase proteins in vitro; (b) ex vivo activation of monocytes from patients with SLE leads to the full induction of its capabilities to change acute phase proteins, whereas the activation of monocytes from patients with RA has no additive effects; and (c) interleukin 6 seems to be a major cytokine involved in the regulation of the glycosylation pattern of acute phase proteins.     

Atrial natriuretic peptide is a physiological inhibitor of ACTH release: evidence from immunoneutralization in vivo.

The brain is thought to exert a predominantly stimulatory action on ACTH secretion mediated mainly by corticotrophin-releasing factor-41 (CRF-41) and arginine vasopressin (AVP). Several data, however, also point to the existence of an ACTH-inhibiting factor. Atrial natriuretic peptide (ANP), at concentrations found in hypophysial portal blood, inhibits ACTH release in vitro. The aim of the present studies was to use ANP immunoneutralization to determine whether ANP does in fact inhibit ACTH release in vivo. Intracerebroventricular infusion (1 microliters/min for 30 min) of sheep anti-ANP serum into male rats anaesthetized with sodium pentobarbitone had no significant effect on jugular venous plasma concentrations of ACTH or LH but did decrease significantly the plasma concentrations of prolactin. Intravenous infusion of 0.8 ml sheep anti-ANP serum but not control (non-immune) sheep serum, through an indwelling intra-atrial cannula in conscious male rats resulted in a marked and significant increase in plasma ACTH and corticosterone concentrations. The ACTH and corticosterone response to a 30-s ether stress was not significantly potentiated in the same conscious rats infused with anti-ANP serum. Intra-atrial infusion of anti-ANP did not significantly affect plasma prolactin, LH, glucose or sodium concentrations or plasma osmolality. These results show for the first time that ANP is a potent inhibitor of ACTH secretion in the conscious male rat and that, therefore, ANP is a hypothalamic neurohormone which is likely to play an important inhibitory role in the neural control of ACTH release.     

Failure of cholinergic stimulation to induce a secretory response from the rectal mucosa in cystic fibrosis.

The secretory response to cholinergic stimulation was investigated in rectal biopsy specimens from children with cystic fibrosis and a control group using a modified Ussing chamber technique. Acetylcholine (10(-3) mol/l) increased the short circuit current in 12 control specimens by mean (SEM) 83.0 (16.4) microA/cm2, but samples from five children with cystic fibrosis failed to exhibit such a response (-1.4 (3.2) microA/cm2). Amiloride (10(-4) mol/l), which will inhibit electrogenic sodium absorption in viable tissues, caused similar reductions in the short circuit current of both control and cystic fibrosis tissues (control = -37.7 (7.7) microA/cm2; cystic fibrosis = -44.0 (9.3) microA/cm2). Thus, the failure of chloride secretion observed in the small intestine also exists in the rectal mucosa. This observation could be used both to aid diagnosis and to study the basic defect.