Phenotypic distribution of T cells in human nasal mucosa differs from that in the gut

Phenotypic and functional studies are required to understand the immunoregulatory role of mucosal T cells. Information about T cells in the human upper respiratory tract is limited and conflicting. Therefore, we phenotyped T cells in nasal mucosa by means of multicolor in situ immunofluorescence. In normal mucosa, most CD3+ intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) (> 90%) expressed T-cell receptor (TCR)alpha/beta, and only approximately 5% expressed TCRgamma/delta. Although most IELs in the surface epithelium were CD8+ (64%), many expressed CD4 (30%) and the CD4 phenotype dominated (55%) only slightly in the lamina propria. This result was strikingly different from that obtained for comparable compartments in histologically normal jejunal mucosa, where IELs consisted of 83% CD8+ and LPLs of 73% CD4(+) T cells. Nasal CD3+ IELs and LPLs were mainly CD45RO+CD45RA- and usually expressed CD7. The integrin alphaEbeta7 was, as expected, more common on IELs than on LPLs (78 versus 20%). In conclusion, nasal T cells show several similarities to those of the normal jejunum but some notable differences exist, especially a relative increase in CD4+ T cells in the epithelium and a decrease in the lamina propria. It should be explored whether this disparity, together with an increased expression of epithelial adhesion molecules, might contribute to local immunological overstimulation and partly explain the relatively high frequency of airway allergy.     

Soluble and cell surface ICAM-1 as markers for disease activity in multiple sclerosis

Infiltration of a transplanted organ by host lymphoid cells is the hallmark of acute rejection. However, after intestinal transplantation, physiological lymphocyte migration may lead to host cell infiltration of the graft even in the absence of rejection. It is unclear whether this lymphocyte migration also involves the intraepithelial compartment of the graft or whether infiltration there is indicative of acute rejection. We demonstrate here that host cell infiltration of the intestinal mucosa occurs both during acute rejection of a small bowel allograft and, to a lesser extent, when rejection is prevented by immunosuppression with FK506. The infiltrating host cells consisted of CD3+ T cells with a predominant CD4-CD8+ phenotype resembling intraepithelial lymphocytes (IELs). Functional studies showed that the nonspecific cytolytic activity of IELs was not affected by acute rejection or by immunosuppression with FK506. These findings indicate that host cell infiltration of the intestinal mucosa does not connote an ongoing acute rejection. Furthermore, the decreased mucosal barrier function during acute rejection of intestinal allgrafts is probably not due to impaired cytolytic activity of IELs.     

Expression of IL-16 in allergen-induced late-phase nasal responses and relation to topical glucocorticosteroid treatment

Allergen-induced late nasal responses (LNRs) are associated with a cellular infiltrate in which CD4+ cells are prominent. These cells have been shown to be the major cellular source of Th2-type cytokines. Mechanisms responsible for the local accumulation of CD4+ cells in the nasal mucosa after allergen exposure are unclear. IL-16 is a potent chemoattractant for CD4+ cells in vitro and may play a significant role in recruiting CD4+ cells in LNRs. We investigated the expression of IL-16 messenger RNA and immunoreactivity in nasal biopsy specimens from 17 subjects with allergic rhinitis. A biopsy specimen of the nasal inferior turbinate was obtained before and 24 hours after local nasal provocation with grass pollen extract after 6 weeks of treatment with either topical fluticasone propionate (n = 9) or placebo (n = 8) nasal spray twice daily. IL-16 mRNA-positive cells and IL-16-immunoreactive cells were identified in both the epithelium and the subepithelial tissue at baseline. Within the placebo-treated group, the numbers of epithelial and subepithelial IL-16 mRNA-positive cells and IL-16-immunoreactive cells were significantly increased 24 hours after challenge compared with baseline (p < 0.001). Topical glucocorticoid therapy resulted in a decrease in allergen-induced epithelial immunoreactive cells and subepithelial IL-16 mRNA-positive cells. The numbers of CD4+ cells increased after antigen challenge compared with baseline (p < 0.05), and this increase was inhibited by glucocorticoid treatment. There were significant correlations between epithelial and subepithelial IL-16 immunoreactivity and CD4+ cell infiltration after antigen challenge. The upregulation of IL-16 expression in allergic nasal mucosa after antigen challenge may have critical implications in the accumulation of CD4+ cells in response to antigen exposure. Steroid-mediated inhibition of IL-16 may be partly responsible for the decrease in local CD4+ cells after topical glucocorticoid therapy.     

Activation and peripheral expansion of murine T-cell receptor gamma delta intraepithelial lymphocytes

BACKGROUND & AIMS: The intestinal epithelial compartment is populated by CD8(+) alpha beta and gamma delta intraepithelial lymphocytes (IELs), which monitor the integrity of the epithelial barrier. alpha beta IELs are activated by peptide antigens presented by class I major histocompatibility complex (MHC) molecules, but it is unclear how gamma delta IELs are activated. METHODS: G8 T-cell receptor (TCR) gamma delta transgenic (Tg) mice (specific for the class I MHC alloantigen, T22/10(b)) were crossed to class I MHC-deficient beta2-microglobulin-knockout (beta2m degrees) mice, and Tg+ IELs were examined for relative yields and surface and functional phenotype. RESULTS: Evidence for class I MHC-induced activation of Tg+ IELs was supported by the detection of 4-fold greater numbers of Tg+ IELs in G8 x beta2m+ mice that proliferated at 15-fold higher levels than IELs from G8 x beta2m degrees mice. However, expression of CD69, production of cytokine (interleukin 2 and interferon gamma), and detection of cytolytic function for IELs in G8 x beta2m degrees mice suggested that class I MHC was not required for gamma delta IEL development or maturation. CONCLUSIONS: These results suggest that CD8(+) TCR gamma delta IELs do not require class I MHC for development but support the notion that antigens presented by class I MHC molecules are involved in the peripheral expansion and differentiation of this subset.     

Duodenal intraepithelial and lamina propria T lymphocytes in human immunodeficiency virus-infected patients with and without diarrhoea

BACKGROUND: Diarrhoea is an important problem in human immunodeficiency virus (HIV)-infected patients. Intestinal pathologic conditions may arise from changes in local immunocyte populations. The aims of our study were to establish the histologic features of the duodenal mucosa of HIV-infected patients and to determine a) the phenotype of small-intestinal-intraepithelial (IELs) and lamina propria (LPLs)-lymphocytes; b) their degree of activation and differentiation within the lamina propria; and c) their relation to the presence of diarrhoea. METHODS: Distal duodenal biopsy specimens were obtained prospectively from 29 HIV-infected patients-11 patients with diarrhoea (group 1) and 18 patients without diarrhoea (group 2)- and from 42 patients who had neither any risk factor for HIV nor diarrhoea (group 3). Histopathologic and immunohistochemical studies were combined with flow cytometric analysis, after separation of the mucosal intraepithelial compartment from the lamina propria. RESULTS: The median number of IELs and the percentage of gamma delta IELs were both unchanged in HIV-infected patients as compared with controls. In HIV-infected patients LP CD4 cells were decreased, and LP CD8 cells increased. No significant difference was found in the expression of CD25 or CD27 within the LP CD8 populations of HIV-infected patients in groups 1 and 2. CONCLUSIONS: These findings suggest that the occurrence of diarrhoea in HIV-infected patients is unrelated to IEL and LPL phenotype.     

CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.     

Smoking status and relative weight by educational level in Finland, 1978-1995

Nasal administration of soluble antigens is an exciting means of specifically down-regulating pathogenic T-cell reactivities in autoimmune diseases. The mechanisms by which nasal administration of soluble antigens suppresses autoimmunity are poorly understood. To define further the principles of nasal tolerance induction, we studied the effects of nasal administration of myelin basic protein (MBP) on experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. EAE is a CD4+ T-cell-mediated animal model for human multiple sclerosis. Nasal administration of guinea-pig (gp)-MBP at a dose as low as 30 micrograms/rat can completely prevent gp-MBP-induced EAE, whereas nasal administration of bovine (b)-MBP is not effective even at a much higher dosage. Cellular immune responses, as reflected by T-cell proliferation and interferon-gamma (IFN-gamma)-ELISPOT, were suppressed in rats receiving the two different doses (30 and 600 micrograms/rat) of gp-MBP, but not after administration of b-MBP. Rats tolerized with both doses of gp-MBP had also abrogated MBP-induced IFN-gamma mRNA expression in popliteal and inguinal lymph node mononuclear cells compared with rats receiving phosphate-buffered saline nasally. However, adoptive transfer revealed that only spleen mononuclear cells from rats pretreated with a low dose, but not from those pretreated with a high dose, of gp-MBP transferred protection to actively induced EAE. Low-dose (30 micrograms/rat) gp-MBP-tolerized rats also had high numbers of interleukin-4 (IL-4) mRNA-expressing lymph node cells, while high-dose (600 micrograms/rat) gp-MBP-tolerized rats had low numbers of IL-4 mRNA-expressing lymph node cells. Our data suggest an exquisite specificity of nasal tolerance. Dose-dependent mechanisms also relate to nasal tolerance induction and protection against EAE in the Lewis rat.     

Bovine T cell responses to Cryptosporidium parvum infection

To better understand the immune mechanisms important for clearing of the primary infection and the subsequent development of resistance to Cryptosporidium parvum infection, several groups have recently characterised changes within the lymphoid cell population of the intestinal mucosa and associated lymphoid tissue in calves with cryptosporidiosis. In naive animals, infection results in a significant increase in the number of CD4+ and CD8+ T cells present within the intraepithelial lymphocyte population, lamina propria and Peyer's patch of the ileum. This is accompanied by a rapid and transient increase in the number of gamma/delta T cells present within the intestinal villi. In response to a challenge infection in immune calves, there is a substantial increase in the number of CD4+ T cells present in the Peyer's patch of the ileum and a specific localization of CD8+ T cells to the epithelium of the intestinal villi. Together, these data demonstrate that C, parvum elicits a strong cell-mediated response following both primary and secondary infections in calves, and that CD8+ T cells may play an important role in the bovine immune response to C. parvum infection.     

Migration resistant, blood-compatible plasticized polyvinyl chloride for medical and related applications

AIMS: To determine the morphology, immunophenotype and bcl-2 protein status of intraepithelial lymphocytes in HIV-positive lymphoepithelial lesions. METHODS AND RESULTS: Seventeen cases (from adults and children) of HIV-associated parotid and lung lymphoid lesions were examined. In addition, three lymphoepithelial cysts from HIV-negative patients were studied in parallel. Immunohistochemistry was performed on paraffin embedded tissue with the following antibodies: CD20, CD79a, CD3, CD4, CD8, bcl-2, CAM5.2, AE1/3, MIB1, kappa/lambda light chains and EBV-LMP-1. Heavy chain rearrangement was sought by polymerase chain reaction (PCR) in four of the cases. The lymphocytes participating in lymphoepithelial lesions of HIV-positive patients had the morphology of centrocyte-like cells with occasional cells resembling centroblasts. The majority of these cells were of B-cell lineage, but occasional intraepithelial T-cells (CD8 positive, CD4 negative) were also present. T-cells also formed a significant component of the infiltrative lymphoid cells outside the lymphoepithelial lesions. These were mainly CD8 positive, but very occasional CD4-positive T-cells were also noted. None of the cases showed light chain restriction and the four cases did not demonstrate heavy chain rearrangement by molecular biology. The interesting finding was the absence of bcl-2 expression by the intraepithelial lymphocytes. In contrast, the intraepithelial lymphocytes seen in the non-HIV setting were strongly bcl-2 positive. The majority of these were B-cells, and very occasional CD8 and CD4 positive T-cells formed the intraepithelial population. CONCLUSION: It is postulated that this finding is due to the HIV causing down-regulation of bcl-2 protein.     

Expression of perforin in nasal lymphoma. Additional evidence of its natural killer cell derivation

Eight patients with nasal lymphoma in whom fresh-frozen tissues were available were studied to elucidate the nature of the lymphoma cells. Two cases were diagnosed as diffuse, large cell lymphoma, and the remaining six cases as diffuse, mixed cell types. Immunohistochemical studies revealed that all of the cases were positive for perforin, which is a specific marker for cytotoxic T or natural killer (NK) cells. As all of the cases were CD8 negative, the perforin-positive finding further confirmed the concept that nasal lymphoma is a distinct neoplastic entity derived from NK or NK-related cells. Light microscopic immunohistochemical studies revealed that these nasal lymphoma cases could be classified into Leu19(CD56)+Leu4(CD3)+ (two cases) and Leu19(CD56)+Leu4(CD3)- (six cases) types according to the phenotypes of the proliferating cells. However, simultaneous staining for perforin and Leu4 (CD3) using immunoelectron microscopy on the Leu19+Leu4+ cases showed that the perforin-positive cells were different from the Leu4-positive cells. This finding suggests that the Leu4-positive cells are not neoplastic NK cells but reactive T cells. Six cases were positive for EBER-1 by in situ hybridization analysis. This finding reconfirms the previous studies that Epstein-Barr virus plays a significant role in the pathogenesis of nasal lymphoma.     

Effects of some phenylethylamines in rhesus monkeys trained to discriminate (+)-amphetamine from saline

The distribution of intraepithelial nerve fibres and neuroendocrine cells within the surface and glandular epithelium of human nasal mucosa and larynx was examined using immunohistochemical techniques. Neuronal structures were immunostained for the general neuroendocrine marker protein gene-product (PGP) 9.5, and the two neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) using immunofluorescence and streptavidin-biotin-peroxidase complex (S-ABC) methods. Intraepithelial nerve fibres with free nerve endings contained PGP 9.5 and were found within the respiratory surface epithelium of the nasal mucosa and the squamous epithelium of the larynx. A subpopulation of these nerve fibres showed positive immunoreactivties with antibodies against SP and CGRP. Nerve fibres within the ductal epithelium of subepithelial excretory ducts passing the basal membrane and reaching the luminal part were detected. These nerve fibres showed CGRP-like immunoreactivity but not for SP. A dense network of nerve fibres within the squamous surface epithelium was detected in the subglottic and epiglottic region containing CGRP and SP in a small subpopulation of nerve fibres. Single intraepithelial taste buds in the epiglottic region and neuroendocrine cells within the subglottic epithelium expressed PGP 9.5.     

Intraepithelial lymphocytes from villus tip and crypt portions of the murine small intestine show distinct characteristics

BACKGROUND & AIMS: Intraepithelial lymphocytes (IELs) are located between epithelial cells that are thought to display unique features and functions at the small intestinal villus tip and crypt levels. We have addressed whether the spatial differences in the intestinal epithelium extend to IELs and subsequent cross-talk between IELs and epithelial cells. METHODS: IELs were isolated from villus tip and crypt portions of mouse small intestine and then compared for spontaneous cytokine production and responsiveness to interleukin (IL)-2 and/or IL-7. RESULTS: No difference was observed between number of beta IELs in villus tips and crypts, whereas a trend toward increased frequencies of IELs bearing the gamma delta form of T-cell receptor was noted in villus tips. Interestingly, the number of beta IELs producing interferon gamma and IL-5 was significantly reduced in the cells from crypts compared with villus tips. Furthermore, villus tip beta IELs exhibited higher responses to stimulation signals provided by IL-2 and/or IL-7 than their crypt counterpart. Such functional differences were not observed with gamma delta IELs from the two intestinal sites. CONCLUSIONS: Distinct molecular cross-talk between IELs and epithelial cells occurs in intestinal villus tips and crypts.     

Analysis of myeloid and lymphoid markers on the surface and in the cytoplasm of mononuclear bone marrow cells in patients with myelodysplastic syndrome

Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.     

Dispensability of p53 degradation for tumorigenicity and decreased serum requirement of human papillomavirus type 16 E6

Tonsils and adenoids are secondary lymphoid organs exposed to the environment. The most important classifications of AIDS include the lymphocyte subsets of peripheral blood. We have studied the lymphocyte subsets in peripheral blood and secondary lymphoid organs in a control group of children suffering adenotonsillar pathology and in five children with AIDS and the same adenotonsillar pathology. The antigen surface markers were determined by flow cytometry in lymphocytes isolated from peripheral blood, and from tonsils and adenoids after tonsillectomy and adenoidectomy, in the control group and in children diagnosed with AIDS. The most important findings in tonsils and adenoids were a decrease of the total T lymphocytes, helper T lymphocytes and CD4/CD8 ratio; an increase of cytotoxic T lymphocytes and B lymphocytes, as well as a 200% increase in monocytes of AIDS-affected children. These observations show the value of analyzing the lymphocyte subsets of the tonsils and adenoids of AIDS-affected children, and establishing an earlier relation to clinical symptoms.     

Increased threshold for TCR-mediated signaling controls self reactivity of intraepithelial lymphocytes

To examine the effect of self Ag on activation requirements of TCR-alphabeta intestinal intraepithelial lymphocytes (IELs), we utilized the 2C transgenic (Tg) mouse model specific for a peptide self Ag presented by class I MHC, H-2Ld. CD8alpha alpha and CD4-CD8- IELs from syngeneic (H-2b, self Ag-) and self Ag-bearing (H-2b/d, self Ag+) strains were examined for their ability to respond in vitro to P815 (H-2d) cell lines expressing the endogenous antigenic peptide, p2Ca. Proliferation, cytokine production, and CTL activity were elicited in IEL T cells isolated from self Ag- H-2b mice when stimulated with P815 cells expressing basal levels of self Ag. These responses were enhanced following the addition of exogenous p2Ca peptide and ectopic expression of the costimulatory molecule, B7-1. By comparison, IEL from self Ag-bearing mice failed to respond to basal levels of self Ag presented by P815 cells even in the presence of B7-1-mediated costimulation. However, the addition of increasing amounts of exogenous p2Ca peptide induced a response from the in vivo "tolerized" T cells. These results suggest that exposure to self Ag in vivo increased the threshold of TCR activation of Ag-exposed self-reactive IELs. The dependence of increased signal 1 to activate self-reactive IELs suggests a defect in TCR signaling that may maintain self tolerance in vivo. These data suggest that conditions that overcome signal 1 IEL defects may initiate autoreactive responses in the intestine.     

Disclosing the cancer diagnosis: the patients'' experiences

To elucidate whether leukocytes in the surface secretion on the adenoid have a potential immunological function, imprint samples of surface secretion were obtained from the adenoid of 11 children and the corresponding mucosal area of 10 adults. May-Grünewald-Giemsa staining was used for evaluation of cell morphology and spatial relations, and immunohistochemical staining for identification of granulocytes, B cells, T cells and macrophages. The children's imprints showed large numbers of leukocytes, with mononuclear cells in the majority. The adults' imprints were characterized by moderate numbers of epithelial cells and few leukocytes. In six out of seven adenoid secretions successfully analysed with all four CD antigens, there was the simultaneous presence of granulocytes, B cells, 1 cells and macrophages. This was the case in one of nine analysable adult secretions. The CD-positive cells were often seen in juxtaposition, in clusters of two to 10 cells, as well as in contact with leukocytes of unknown CD antigenicity and epithelial cells. The simultaneous presence in the secretion of morphologically intact and CD antigen-presenting leukocytes in juxtaposition could indicate a potential immunological function of these cells.     

Intraepithelial lymphocytes and their recognition of non-classical MHC molecules

Recent studies of the TCR alpha and beta chains expressed by normal human IELs suggest that these intestinal lymphocytes are directed at a limited set of antigens, presumably on intestinal epithelial cells in view of their anatomic location. The direct sequence analysis of these cells has indicated that they are oligoclonal and cannot, therefore, be responding to the complex mixture of antigens which are present in the lumen. The abundant expression of the CD8 accessory molecule by the IELs, in addition, indicates that these putative intestinal epithelial cell antigens are presented by MHC class I or I-like molecules. The expression of CD8 also suggests that these cells function biologically in part as cytolytic T lymphocytes which is consistent with a variety of functional studies. Taken together with their expression of the CD45RO isoform, these phenotypic and functional observations suggest that iIELs are cytolytic, memory cells which are responsive to an extremely limited number of antigens bound to major histocompatibility complex (MHC) class I or class I-like molecules. Several non-polymorphic MHC class I-like molecules such as Qa, the thymus leukemia antigen (TL) and CD1 in the mouse and CD1 in human represent important candidate ligands for these oligoclonal iIELs. TL and CD1 are expressed specifically by murine intestinal epithelial cells. In humans, CD1d is constitutively expressed by intestinal epithelial cells. In addition, we have isolated iIEL T cell clones which specifically recognize members of the CD1 gene family when expressed on a transfected B cell line that lacks HLA-A and B and have shown that the proliferation of peripheral blood T cells to intestinal epithelial cells is CD1d dependent. Thus, the evidence to date strongly implicate the nonpolymorphic, class Ib molecules as novel restriction elements for unique populations of lymphocytes within the intestinal epithelium.