Insulin-like growth factor I messenger RNA and protein are expressed in the human lymph node and distinctly confined to subtypes of macrophages,antigen-presenting cells,lymphocytes and endothelial cells

Insulin-like growth factor I (IGF-I) is a potent hormone that stimulates growth and differentiation and inhibits apoptosis in numerous tissues. Preliminary evidence suggests that IGF-I exerts differentiating, mitogenic and restoring activities in the immune system but the sites of synthesis of local IGF-I are unknown. Identification of these sites would allow the functional role of local IGF-I to be clarified. The presence of IGF-I in non-immune cells suggests that it acts as a trophic factor, while its occurrence in subtypes of lymphocytes or antigen-presenting cells indicates paracrine/autocrine direct regulatory involvement of IGF-I in the human immune response. The present study investigated the location of IGF-I messenger RNA and protein on archival human lymph node samples by in situ hybridization, immunohistochemistry and double immunofluorescence staining using an IGF-I probe and antisera specific for human IGF-I and CD3 (T lymphocytes), CD20 (B lymphocytes), CD68 (macrophages), CD21 (follicular dendritic cells), S100 (interdigitating dendritic cells) and podoplanin (fibroblastic reticular cells). Numerous cells within the B- and T-cell compartments expressed the IGF-I gene, and the majority of these cells were identified as macrophages. Solitary follicular dendritic cells exhibited IGF-I. A few T lymphocytes, and no B lymphocytes, contained IGF-I immunoreactive material. Furthermore, IGF-I immunoreactive cells outside the follicles that did not react with CD3, CD20, S100 or podoplanin markers were identified as high-endothelial venule (HEV) cells. From this we conclude that the main task of IGF-I in human non-tumoral lymph node may be autocrine and paracrine regulation of the differentiation, stimulation and survival of lymphocytes, antigen-presenting cells and macrophages and the differentiation and maintenance of HEV cells.     

De novo expression of MECA-79 glycoprotein-determinant on developing B lymphocytes in gut-associated lymphoid tissues

Rabbit is one of several species that depend on development of B lymphocytes in gut-associated lymphoid tissues for primary immunoglobulin-repertoire diversification. The rabbit appendix is an important site of early B-lymphocyte development. We previously reported that peripheral lymph node addressin detected by monoclonal antibody (mAb) MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix. Here, we report expression of an approximately 127 000 MW O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulphation. The proportions of MECA-79(+) B lymphocytes gradually increased from < 0.5% at 3 days to > 70% at 6 weeks in appendix and Peyer's patches. The proportions of MECA-79(+) B lymphocytes in spleen and peripheral blood were very low (0.5-2%). However, the MECA-79 determinant was detected on B cells in splenic germinal centres after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix.     

Interleukin-4 regulates the interleukin-2 receptors on human peripheral blood B lymphocytes.

The high-affinity interleukin-2 (IL-2) receptor (IL-2R) consists of the non-covalent association of at least two subunits, p55 and p70-75, capable of binding IL-2 with low and intermediate affinity, respectively. We studied the effects of cytokines on the IL-2R expressed on human peripheral blood B lymphocytes using monoclonal antibodies specific for IL-2R p55 and IL-2R p70-75, by means of two-colour flow cytometric analysis. In freshly isolated peripheral blood B lymphocytes, the p55 subunit was expressed only in a small population (7.0% of CD20+ cells), whereas the p70-75 subunit was expressed in a large population (89.0% of CD20+ cells). Of the cytokines studied, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were involved in the regulation of IL-2R on B cells. After a 2-day incubation with IL-4, expression of IL-2R p55 was markedly induced, but expression of IL2-R p70-75 was profoundly suppressed in a dose-dependent manner. These abilities of IL-4 to promote IL-2R p55 expression and suppress IL-2R p70-75 expression were inhibited by the presence of IFN-gamma. Other cytokines, including IL-1, IL-2, IL-5, and IL-6, had little effect on the expression of these two subunits. These findings suggest that IL-4 is a cytokine modulating B cell response through the regulation of IL-2R.     

Accessory cell function of dendritic cells from lymph nodes containing Mycobacterium leprae induced granulomas

Dendritic cells were enriched from guinea pig auricular lymph nodes containing Mycobacterium leprae induced granulomas by immunomagnetic depletion of other cells. These cells were strongly positive for major histocompatibility complex class II antigens and labelled with an antidendritic cell monoclonal antibody, but not with an antimacrophage antibody. Interdigitating dendritic cells were identified in the granulomatous lymph node by staining with the antidendritic cell antibody and by transmission electron microscopy. When cultured in vitro with purified T lymphocytes, these cells acted as accessory cells for both purified protein derivative and concanavalin A induced proliferation. Although previous studies have shown that macrophages from these lymph nodes do not act as accessory cells, the present results indicate that dendritic cells from M. leprae granuloma containing lymph nodes may act as antigen-presenting cells.     

Comparison of antigen presentation by lymph node cells from protein and peptide-primed mice.

Lymph node cells from mice primed with peptides from the allergens Der p I and Der p II (the group I and II allergens of Dermatophagoides pteronyssinus) were unable to recall responses to the protein antigen when cultured in vitro despite being able to mount large responses to the peptides. The T cells could however recall responses to the protein when spleen-adherent cells were added into culture. Treating the spleen accessory cells with the monoclonal antibody (mAb) 33D1 and complement largely abrogated the protein response of peptide-primed T cells which indicates that dendritic cells were mainly responsible for the antigen-presenting function. If mice were primed with two injections of peptide the lymph node cells obtained could respond to both protein and peptides in vitro without the need for exogenous accessory cells. Using either negative depletion with the J11D mAb or positive purification, it was found that the presentation of protein antigen to lymph node T cells primed with either protein or peptide was limited to antigen-specific B cells. Peptide antigens could however be presented by both B and non-B populations. In one case the peptide 105-129 from Der p II which contains a T-cell epitope could not be shown to induce T-cell responses in the lymph node unless presentation was mediated by spleen-adherent or B-specific cells. These results are important for peptide-based immunomodulation and in interpreting results obtained from lymph node cultures.     

Characterization of a monoclonal antibody that recognizes a lymphocyte surface antigen for the cetacean homologue to CD45R.

As part of our current efforts to develop assays and reagents to study the immune system of marine mammals, and in view of the effort currently made to develop monoclonal antibodies to cell surface proteins of lymphocyte subsets in different species, the present paper reports on the characterization of a monoclonal antibody against the homologue of CD45R on cetacean lymphocytes. The specificity of this antibody has been characterized on the basis of immunoprecipitation of the antigen it recognized, immunoperoxidase staining on cetacean lymph node and thymus sections, as well as one and two-colour flow cytometric analysis of cetacean peripheral blood mononuclear cells and single-cell suspensions of thymus, lymph node and spleen. Anticetacean CD45R (F21.H) immunoprecipitated proteins of 180, 200 and 220 x 10(3) MW, with the 180 x 10(3) MW from being predominantly expressed on T cells and the 220 x 10(3) MW form expressed predominantly on B cells and thymocytes F21.H labelled all B cells and a proportion of T cells on single-cell suspensions of spleen cells. CD45R- killer whale peripheral blood lymphocytes expressed a higher density of CD2 than CD45R+, a characteristic of memory T cells. Killer whale T lymphocytes also lost the expression of CD45R upon activation with concanavalin A (Con A) and phytohaemagglutinin (PHA). This is the first report of a monoclonal antibody to CD45R in cetaceans, and this antibody is foreseen as a possible valuable diagnostic and research tool to assess immune functions of captive and wild cetaceans as part of the evaluation of their health status.     

Collaboration of bovine T lymphocytes and macrophages in T-lymphocyte response to Brucella abortus

Brucella abortus-induced bovine macrophage-T-lymphocyte collaboration was studied as a prerequisite for the eventual clearance of this infectious organism. Esterase-positive, peripheral blood monocytes functioned as an adherent antigen-presenting cell population. A dual requirement for expression of bacterial antigens in combination with self major histocompatibility complex class II products was required by adherent cells for the activation of T lymphocytes. Comparison of antigen-presenting cell populations that were either trypsinized or nontrypsinized following B. abortus ingestion substantiated the need for phagocytosis and antigen processing. A monoclonal antibody (H4) directed against major histocompatibility complex class II determinants was able to block or, with complement, to abrogate T-lymphocyte responses. Measurement of both proliferation and interleukin 2 production via [3H]thymidine incorporation confirmed specific activation of an enriched T-lymphocyte population. These results indicate that in vivo-primed T lymphocytes of peripheral blood origin recognize phagocytized bacterial components of the facultative intracellular bacterium B. abortus and may contribute to the removal of the bacteria. Furthermore, bovine peripheral blood-adherent cells function as classic antigen-presenting cells, which suggests that macrophages are capable of processing this bacteria. Therefore, any lymphocyte-mediated dysfunction attributable to B. abortus most likely occurs at some point in the cascade of immune events following initial macrophage-T-lymphocyte collaboration.     

Porcine DC-SIGN: molecular cloning, gene structure, tissue distribution and binding characteristics

DC-SIGN, a human C-type lectin, is involved in the transmission of many enveloped viruses. Here we report the cloning and characterization of the cDNA and gene encoding porcine DC-SIGN (pDC-SIGN). The full-length pDC-SIGN cDNA encodes a type II transmembrane protein of 240 amino acids. Phylogenetic analysis revealed that pDC-SIGN, together with bovine, canis and equine DC-SIGN, are more closely related to mouse SIGNR7 and SIGNR8 than to human DC-SIGN. pDC-SIGN has the same gene structure as bovine, canis DC-SIGN and mouse SIGNR8 with eight exons. pDC-SIGN mRNA expression was detected in pig spleen, thymus, lymph node, lung, bone marrow and muscles. pDC-SIGN protein was found to express on the surface of monocyte-derived macrophages and dendritic cells, alveolar macrophages, lymph node sinusoidal macrophage-like, dendritic-like and endothelial cells but not of monocytes, peripheral blood lymphocytes or lymph node lymphocytes. A BHK cell line stably expressing pDC-SIGN binds to human ICAM-3 and ICAM-2 immunoadhesins in a calcium-dependent manner, and enhances the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) to target cells in trans. The results will help better understand the biological role(s) of DC-SIGN family in innate immunity during the evolutionary process.     

CD1d structure and regulation on human thymocytes, peripheral blood T cells, B cells and monocytes

Human T cells expressing CD161 and an invariant T-cell receptor (TCR) alpha-chain (Valpha24invt T cells) specifically recognize CD1d and appear to have immunoregulatory functions. However, the physiological target cells for this T-cell population, and whether alterations in CD1d expression contribute to the regulation of Valpha24invt T-cell responses, remain to be determined. A series of antibodies were generated to assess CD1d expression, structure and regulation on human lymphoid and myeloid cells. CD1d was expressed at high levels by human cortical thymocytes and immunoprecipitation analyses showed it to be a 48 000-MW glycosylated protein. However, after solubilization, the majority of the thymocyte CD1d protein, but not CD1d expressed by transfected cells, lost reactivity with monoclonal antibodies (mAbs) against native CD1d, indicating that it was alternatively processed. Moreover, thymocytes were not recognized by CD1d-reactive Valpha24invt T-cell clones. Medullary thymocytes and resting peripheral blood T cells were CD1d-, but low-level CD1d expression was induced on activated T cells. CD1d was expressed by B cells in peripheral blood and lymph node mantle zones, but germinal centres were CD1d-. Resting monocytes were CD1d+ but, in contrast to CD1a, b and c, their surface expression of CD1d was not up-regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) activation. These results demonstrate constitutive CD1d expression by human professional antigen-presenting cells and that post-translational processing of CD1d may contribute to regulation of the activity of CD1d-specific T cells.     

In situ expression of B7/BB1 on antigenpresenting cells and activated B cells: an immunohistochemical study

B7/BB1 is a physiological ligand for CD28, a receptor expressedon a major subset of T lymphocytes. B7/BB1 has been shown tobe expressed on human blood dendritic cells and on in vitroactivated (but not resting) B cells and monocytes. Llgatlonof CD28 with B7/BB1 upregulates cytoklne production and preventsthe Induction of anergy in T cells activated through TCR/CD3.We examined the in situ expression of B7/BB1 by immunohlstochemlstrywith a novel mAb B7-24. Dendritic cells in skin (Langerhanscells), lymph node sinuses (veiled cells), and T cell zonesof spleen and lymph nodes (Interdlgltatlng dendritic cells)were strongly positive for B7/BB1. B7/BB1 was also present onfetal thymus dendritic cells located at the cortlco-medullarJunction and the medulla, but absent in normal adult thymuses.Resident macrophages and endothellal cells did not stain, butin granulomatous inflammations B7/BB1 was found on macrophagesand epltheloid cells. A subset of B immunoblasts and of germinalcenter B cells In lymph node and spleen was also found to expressB7/BB1. Our findings on the distribution of B7/BB1 expressionIn tissues, in particular its expression on professional antigen-presentingcells, further substantiate the putative co-stlmulatory roleof B7/BB1 in T cell activation in vivo. The presence of B7/BB1in fetal but not adult thymlc medulla suggests a role for B7/BB1in thymocyte maturation.     

Nitric oxide synthase: expression of the endothelial,Ca2+/calmodulin-dependent isoform in human B and T lymphocytes

Nitric oxide (NO) is a pleiotropic mediator of a variety of cellular processes such as vasorelaxation, neurotransmission, and cytotoxicity. We studied the expression of the human endothelial, Ca²⁺/calmodulin-dependent NO synthase (NOS) isoform (ecNOS) in highly purified human lymphocytes from peripheral blood and tonsillar tissue. ecNOS mRNA was detected in IgD⁺ or IgD^? B cells, peripheral blood and tonsillar T cells. Upon stimulation, ecNOS mRNA expression decreased in all these lymphocyte subpopulations. Germinal center T cells and follicular dendritic cells did not express ecNOS mRNA either in an unstimulated or in a stimulated state. ecNOS expression by human lymphocytes was further substantiated by its mRNA detection in lymphoid cell lines such as Raji, Daudi, and Jurkat. By the use of a specific monoclonal antibody, ecNOS was shown to be present in T cells from peripheral blood and in various germinal center cells of frozen tonsillar sections. These data are the first to demonstrate the expression of the endothelial ecNOS at mRNA and protein level in human lymphocytes.     

Development of a fusion partner cell line for efficient production of human monoclonal antibodies from peripheral blood lymphocytes

We developed a unique fusion partner cell line that is capable of fusing with both human peripheral blood and lymph node lymphocytes at a high efficiency. The cell line was generated by fusing a murine myeloma cell line with a human myeloma cell line, producing a heteromyeloma (B6B11), which was subsequently fused with a human lymph node lymphocyte to produce a trioma (MFP-2). B6B11 and MFP-2 fuse well with human lymphocytes from both spleen and lymph nodes. Interestingly, MFP-2 also fuses with a high efficiency to peripheral blood lymphocytes. The resulting hybrids are stable for extended periods of time and produce human monoclonal antibodies at significant levels. The utility of MFP-2 as a fusion partner was demonstrated by the isolation of several hybridoma cell lines using lymph node and peripheral blood lymphocytes from patients with breast cancer. These hybridomas produce monoclonal antibodies displaying specificity to breast cancer tissue and cell lines.     

Monoclonal antibody binding to congophilic elements in human Alzheimer brain.

Immunohistochemical studies, using a monoclonal antibody, SMP, raised in a mouse against aged human peripheral blood white cells, are described. This antibody reacts with dendritic reticulum cells in lymph node and tonsil, and, in Alzheimer brain, with argyrophilic plaques, neurofibrillary tangles, and cerebral vascular amyloid.     

Characterization of CMRF-44, a novel monoclonal antibody to an activation antigen expressed by the allostimulatory cells within peripheral blood, including dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) involved in the initiation of primary T-lymphocyte responses. However, despite their importance, no DC-specific surface marker has been identified in humans and many aspects of their ontogeny and the mechanisms underlying their potent functional activity remain unknown. In this report we describe a novel monoclonal antibody (mAb), CMRF-44, which recognizes an early activation antigen expressed by human DC and acts as a marker of allostimulatory activity within preparations of peripheral blood mononuclear cells (PBMC). The CMRF-44 antigen was expressed strongly on DC isolated from blood and tonsil by standard techniques, but was not detectable on Langerhans' cells within skin or on DC isolated directly from blood using a cell-sorting method which involves minimal DC manipulation/activation. Normal resting peripheral blood leucocytes did not label with CMRF-44, although weak staining of a small subpopulation (15%) of blood B lymphocytes was identified by double labelling. However, following overnight culture at 37 degrees, moderate staining of a subpopulation of PBMC was detected. Confirmation that CMRF-44 recognized an early marker of activated DC and hence allostimulatory activity was obtained by sorting cultured cell preparations on the basis of CMRF-44 reactivity. A marked enrichment of allostimulatory activity was observed in the CMRF-44-positive cellular population, whereas the CMRF-44-negative cells showed only minimal stimulatory activity. Activation studies established that the CMRF-44 antigen was an early activation marker, expressed constitutively on the majority of tonsil B lymphocytes, which can be induced on peripheral blood B lymphocytes and subpopulations of monocytes. Expression of the CMRF-44 antigen on cell lines was similarly restricted, CMRF-44 antigen being detected only on Hodgkin's disease-derived and B-lymphoid lines. The cellular distribution, expression kinetics and biochemical characteristics of the CMRF-44 antigen identify it as a new early marker of activated allostimulatory (DC) populations.     

Production of a monoclonal antibody reactive with human dendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue.

A murine monoclonal antibody (designated R4/23) which reacts strongly with human dendritic reticulum cells (DRC) is described. Immunoperoxidase staining of tissue cryostat sections revealed that this antibody reacts strongly with DRC in lymphoid follicles (both primary and secondary), and also weakly with marginal zone splenic B cells and with some peripheral follicular mantle B lymphocytes in lymph node cortical follicles. The value of antibody R4/23 is that it allows the distribution of DRC in reactive and neoplastic lymphoid tissue to be clearly delineated. Of particular interest is the fact that all cases of follicular lymphoma of germinal centre cell origin are consistently accompanied by a proliferation of DRC, even when the neoplasm is present in non-lymphoid tissue--for example, in the kidney. In contrast, DRC in B cell lymphomas of non-germinal centre origin are partially or totally obliterated.     

Histamine-receptor leucocytes (HRL). Organ and lymphoid subpopulation distribution in man.

The frequency of lymphoid cells with a membrane receptor for histamine was determined in various lymphoid organs in man using a histamine-rosette assay. Thymus had very low numbers of histamine-receptor cells while lymph node and peripheral blood had increasing percentages. Through a combination of cell separation techniques, we demonstrated that about one third (1/3) of peripheral blood B lymphocytes and macrophages carry histamine receptors. Immature B cells or null cells (E-rosette and membrane-immunoglobulin-negative) do not have this receptor. Only 10% of peripheral blood T lymphocytes formed histamine rosettes. That these histamine receptor T lymphocytes are a subpopulation representing the differentiated suppressor/cytotoxic T cells is suggested by evidence showing complete removal of histamine receptor T lymphocytes on nylon wool adherence columns. Thus, the histamine receptor is expressed on differentiated B and T lymphocytes and may serve as a marker for developed suppressor/cytotoxic T cells in man.     

Monoclonal antibody to a human leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat

The monoclonal antibody (F10–89–4) described in this study recognizes an antigen which by quantitative absorption analysis is absent from human brain, kidney, liver, heart, erythrocytes, platelets and normal serum, but is present on spleen, lymph node, chronic lymphatic leukemia cells, bone marrow, thymus and granulocytes at a ratio of 1 : 1 : 0.8 : 0.3 : 0.3 : 0.1, respectively. Analysis with the fluorescence-activated cell sorter showed that 100% of thymocytes, lymph node lymphocytes, blood mononuclear cells and granulocytes carry the antigen, while 83% of bone marrow cells are positive. There was marked heterogeneity in the amount of labeling of thymocytes, with 3 major peaks. There was also heterogeneity of labeling of blood mononuclear cells and lymph node lymphocytes, with a weakly staining hump containing approximately 20% of the cells in the case of lymph node lymphocytes. Double labeling experiments demonstrated that the weakly staining cells of blood and lymph node were B lymphocytes, while frozen sections of thymus showed that the antigen was expressed most weakly in subcapsular cortical thymocytes, and most strongly on medullary thymocytes. Biochemical studies established that the antigen bound to lentil lectin columns, and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies using NaB³ H₄-labeled blood mononuclear cells established that the antigen was a major glycoprotein of lymphocytes, and that its molecular weight was in the region of 190 000 to 215 000.     

A murine monoclonal antibody strictly reactive with human peripheral blood monocytes

We have produced and characterized a novel murine monoclonal antibody (LAM7) of IgG1 isotype which appears specific for peripheral blood monocytes (PBM) on the basis of histochemical and functional studies. By indirect immunofluorescence, including FACS analysis, the antibody reacts with 90 +/- 6% of PBM and with monocytic leukemias, while it is totally unreactive with B and T lymphocytes, platelets, granulocytes, peripheral macrophages, dendritic cells, large granular lymphocytes, and nonmonocytic leukemias. The antigen-presenting capacity of peripheral blood mononuclear cells is abolished by treatment with MoAb LAM7 in an antiglobulin-complement-mediated cytotoxicity test, and restored by addition of purified PBM. The progressive disappearance of the antigen recognized by LAM7 from PBM within approximately 3 days in culture, and its absence from both bone marrow precursors and tissue macrophages, define it as a line-specific and stage-specific differentiation.     

Human lymph node lymphocytes fail to effect lysis of antibody-coated target cells.

Human lymphocytes prepared from peripheral blood, lymph nodes, spleen and thymus were titrated for ability to mediate lysis of human target cells coated with rabbit anti target antibody. Lymphocytes from blood and spleen produced efficient lysis of targets in the presence of antibody. Lymph node cells and thymocytes were essentially non-reactive in this system. Lymph node preparations from non-cancer patients contained approximately 25% of non-T cells with receptors for Fc,C3 and/or Ig. Regional lymph nodes from patients with primary tumours contained 37-50% non-T cells by the same criteria. Failure of lymph node lymphocytes to effect lysis of antibody-coated targets did not therefore correlate with content of Fc or C3 bearing cells per se. The effector cell in antibody-dependent cytotoxicity in other systems has been shown to carry Fc and C3 receptors, but not surface Ig. This cell type appears to be absent or non-functional in human lymph nodes.