Effect of cocaine and murine aids on lamina propria T and B cells in normal mice

We developed an experimental model to study the effect of daily cocaine administration on the mucosal immune system during murine acquired immune deficiency syndrome (MAIDS). Mice were infected with LP-BM5 murine leukemia virus, a retrovirus which causes immunosuppression with development of functional murine AIDS. Mice were given cocaine by daily intraperitoneal injection for 11 weeks. Our objective was to investigate if cocaine treatment could alter the mucosal immune system at the level of the intestinal lamina propria (ILP) and if it could further modify the already altered mucosal immunity when it was administered to MAIDS-mice. Daily cocaine administration induced a significant decrease in the number of IgA⁺ cells with a concomitant increase in the number of CD8⁺ cells per villi in the ILP. Murine retrovirus infection alone decreased the number of IgA⁺ and CD4⁺ cells in the ILP, and this decreased was even more marked when MAIDS mice also received cocaine. These data indicate that cocaine administration could potentiate the dramatic effect that MAIDS infection has in the mucosal-associated lymphoid tissues.     

Effect of a single administration of testosterone on the immune response and lymphoid tissues in mice.

Female mice were given a single intraperitoneal injection of testosterone immediately after irradiation and marrow reconstitution. Thirty days later testosterone had no suppressive effect on the recovery of thymus and spleen weights. Testosterone had no effect on the graft-versus-host reaction. Testosterone had no influence on the survival of the skin homografts. However, the plaque-forming cell response to sheep erythrocytes in the spleen was dramatically suppressed by testosterone. Histological observations revealed marked inhibition of lymphoid regeneration selectively in the thymus-independent areas of the peripheral lymphoid tissues. These results suggest that testosterone would act mainly on the differentiation of stem cells toward the population of bone marrow-derived B lymphocytes. The immune response to sheep erythrocytes was restored completely 90 days after testosterone administration. Testosterone given to normal adult mice can also have suppressive activity on the immune system 30 days after a single intraperitoneal injection.     

Retention of T cell reactivity to mitogens and alloantigens by Peyer's patch cells of aged mice

Immune function generally declines with advancing age. However, recent evidence suggests that this decline may differentially affect the lymphoid compartments comprising the immune system. The effect of age on the T cell-mediated competency of cells from two different lymphoid compartments, Peyer's patch (PP) and spleen, was studied. PP cells of aged C57BL/6 mice retained a greater capacity to proliferate in response to concanavalin A, phytohemagglutinin, and alloantigen and to generate alloimmune cytotoxic cells than did splenocytes of aged mice when compared with their young counterparts. These results could not be explained by significant shifts in the response kinetics or in the proportions of T cells in individual tissues. These results support the concept that lymphoid compartments are not equally sensitive to the deleterious effects of the aging process.     

Effect of carminomycin on the immunological reactivity of the body]

The morpho-functional state of the muscle lymphoid tissue of mice treated with karmionmycin (LD50, 1.1 mg/kg) was studied experimentally. Development of a number of changes evident of the cell disorganization in the lymphoid tissue was shown. The cytological shifts in the thymus and spleen were reversible, while the destructive period in the strumous gland was more prolonged. The morpho-structural normalization of the spleen did not coincide with reduction of its immunological function. The studies on the karminomycin effect on the kinetics of the primary immune response showed dependence of the antibiotic effect on the temporal ratio between the antigenic stimulus and the drug administration. The antibiotic had no effect on the inductive phase of the primary immune response but significantly suppressed its productive phase. On reproduction of the secondary immune response, a decrease in the content of the antibody-forming cells in the spleen was observed, production of the circulating antibodies remaining unchanged.     

Inhibition of the immune response by membrane-bound antibody.

Cells from the spleens of "normal" swine, which were pretreated with pronase to remove surface membrane-bound immunoglobulin, gave an enhanced hemolytic plaque-forming cell response to sheep red blood cells in vitro in comparison with untreated controls. The enhancement could be abrogated by preincubating pronase-treated spleeen cells in preparations containing antibody to sheep red blood cells. This effect was demonstrated by autologous sera, immune sera, and all three known classes of porcine serum immunoglobulins, including IgM, IgA, and IgG and could be removed by absorption with sheep red blood cells. Surface membrane-bound antibody exerted its effect by binding to the nonadherent cell population. The response of normal spleen cells was unaffected by antibody treatment. Pronase-treatment was not mitogenic, did not function as a polyclonal B cell activator, and did not selectively eliminate T or B cells. The results indicate that removal of antibody from the surface of lymphoid cells enhanced the humoral immune response invitro and confirm that membrane-bound antibody can inhibit response to antigen.     

Direct antigen presentation by a xenograft induces immunity independently of secondary lymphoid organs

The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.     

Antigenic markers on mouse lymphoid cells: origin of cells mediating immunologic memory

Specific antisera were used for the purification of thymus dependent and thymus independent or bursa equivalent lymphoid cells in the mouse. Spleen cells from mice immune to sheep erythrocytes, a thymus dependent antigen, or to E. coli 055:B5 lipopolysaccharide, a thymus independent antigen, were treated with anti-θ (C3H) serum or anti-MBLA serum and complement prior to their adoptive transfer into lethally irradiated syngeneic recipients. Syngeneic thymocytes, bone marrow cells, or spleen cells from nonimmune donors were appropriately added to antiserum treated cells prior to transfer. The secondary response to these antigens was assayed in recipient spleens six days after cell transfer. The kinetics of the primary response to SRBC was investigated as to its effect on origin of specific hyper-reactive T or B lymphoid cells.The adoptive response to CPS originated in the B lymphoid cell population. Immunologic memory to CPS was demonstrated in recipients of immune cells, compared to recipients of normal cells, by a five fold increase in antibody forming cells.The IgM and IgG adoptive immune response to high doses of SRBC depended upon an increased number of specifically hyper-reactive T-lymphoid cells to facilitate cooperation between T and B lymphocytes. High doses of SRBC initially stimulated T cell memory but at 42 days after priming an increased number of specifically hyper-reactive B lymphoid cells were present.     

Melatonin activates Th1 lymphocytes by increasing IL-12 production

Melatonin could act on immune system by regulating cytokine production of immunocompetent cells. The hormone enhances IL-2, IFN-gamma and IL-6 production by cultured human mononuclear cells. As enhancement of IL-6 production is related to monocyte activation by melatonin, the hormone acts on human lymphoid cells causing a Th1-type response. This paper shows that melatonin seems to promote a Th1-response by increasing IL-12 production. The hormone enhances IL-12 production by cultured monocytes under suboptimal stimulation in a dose-dependent way. The effect of the hormone increases when PBMCs are incubated with melatonin before monocyte isolation. Enhanced IL-12 production by melatonin can also be shown in cultured human mononuclear cells.     

Site of action of a soluble immune response suppressor (SIRS) produced by concanavalin A-activated spleen cells.

The cellular site of action of SIRS, a soluble immune response suppressor released by Con A-activated spleen cells which suppresses antibody responses to heterologous erythrocytes by murine spleen cells in vitro, was investigated. Exposure of spleen cells to SIRS for 2 hr at 37 degrees C or 1 hr at 4 degrees C was sufficient to suppress 5-day antibody responses in vitro. Similar exposure of splenic or peritoneal exudate macrophages to SIRS also suppressed antibody responses by untreated splenic lymphoid cells; exposure of splenic lymphoid cells to SIRS was without effect. SIRS did not act via T cells which might have contaminated the macrophage preparations. SIRS-mediated suppression could be partially overcome by an excess of normal peritoneal exudate macrophages, but not by an excess of T or B cells. These data indicate that the target cell of SIRS activity is the macrophage. The results are discussed in the context of macrophage functions that could be affected by SIRS.     

Potentiation of the primary humoral immune response in vitro by C5a anaphylatoxin

The effect of the anaphylatoxin C5a on the primary humoral immune response to SRBC was studied in culture of spleen cells from C3H mice. The addition of human C5a to antigen-stimulated cultures resulted in a significant, dose-dependent augmentation of the primary PFC response to antigen. The specificity of this effect was affirmed by the ability of C5ades Arg, but not of the structurally analogous C3a anaphylatoxin, to act in a parallel fashion. Enhancement could be observed over a range of doses of antigen. Brief preincubation of macrophages, but not of lymphoid cells, with C5a was sufficient to cause subsequent enhancement of the primary humoral immune responses. The duration of preincubation required for this effect closely paralleled that for binding of C5a to peritoneal cells. Immunopotentiation by C5a appears to involve the function of C5a receptor-bearing, Ia- accessory cells as well as Ia+ antigen-presenting cells. Immunopotentiation could be observed when the addition of C5a was delayed for up to 24 hr after initiation of culture. Additionally, augmentation of tritiated thymidine uptake in mixed lymphocyte reactions was enhanced by the addition of C5a in a fashion parallel to that for the primary response to SRBC. These observations support a role for C5a as a modulator of cellular immunity in addition to its role in acute inflammation. Possible cellular mechanisms and implications for immunomodulation of immune responses are discussed.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

Modulation of the adherence of human lymphocytes to measles-infected cells by corticosteroids

Corticosteroids have been shown to have an inhibitory effect on many aspects of the human inflammatory and immunologic response. It is largely unknown by what mechanism corticosteroids alter the biologic function of lymphoid cells. One possible mechansim by which steroids could alter lymphocyte function is by modulation of surface receptors. The present study was designed to examine this possibility by the study of the effect of hydrocortisone (HC) and other steroids on the lymphocyte surface binding site for measles virus antigens. These receptors were detected by an assay involving the rosetting of lymphocytes to measles-infected Hela cells (Hela-K11). We showed that HC and several other steroids inhibit the adherence of monocyte-depleted peripheral blood lymphocytes to Hela-K11 cells (HC inhibition, 34 ± 12.1%, mean ± 1 SD). Inhibition could only be observed after the removal of glass-adherent cells. Modulation of lymphocyte functional surface determinants may be important in explaining some of the pharmacologic effects of corticosteroids on the immune system.     

Lymphoid bone marrow cultures can reconstitute heterogeneous B and T cell-dependent responses in severe combined immunodeficient mice

Long-term lymphoid bone marrow cultures (LBMC) produce B lymphocytes and their precursors for several months in vitro. To assess their differentiative potential and determine their capacity to function as immune effectors, cells from the cultures were transplanted into mice with severe combined immune deficiency disease (SCID). SCID mice are deficient in T and B lymphocytes and are serum immunoglobulin (Ig) negative, but grafts of normal lymphoid precursors can expand and differentiate in them, thereby restoring immunocompetence. The results of these studies indicate that cells from LBMC are able to reconstitute splenic B lymphocytes in the SCID mice. Upon in vivo transfer, LBMC cells secreted Ig that displayed isotype distribution and a pattern of heterogeneity comparable with normal BALB/c mice, as determined by two-dimensional gel electrophoresis. The transplanted LBMC cells were functional, because reconstituted mice could respond to immunization with the T-independent antigen TNP-Ficoll. The results also indicate that cultured cells could reconstitute T cell activity in SCID mice. Splenocytes from approximately one-third of the recipients could generate a cytotoxic response to alloantigens after 5 days of sensitization in a mixed lymphocyte culture, and all reconstituted SCID mice could respond to immunization with the T cell-dependent antigen TNP-BSA. These results demonstrate that B cells, as well as T cell activity, are present in LBMC-reconstituted SCID mice, and show that LBMC cells have the capacity to mediate an immune response.     

Histology of the bone marrow antibody response

The histology of the specific and non-specific antibody response in mouse and rat bone marrow was studied after subcutaneous priming and intravenous boosting with horseradish peroxidase (HRP). Cells producing specific antibody against HRP were found only occasionally in the bone marrow after subcutaneous priming. After the intravenous boost injection their number gradually increased. These anti-HRP forming cells were found as single cells, randomly dispersed throughout the bone marrow. Such a random distribution was also found for cytoplasmic (non-specific) immunoglobulin containing cells. At no time point after immunization could lymphoid aggregates or trapping of immune complexes be observed in the bone marrow of either species. On the basis of these observations it is concluded that the bone marrow forms a suitable microenvironment for immigrating antibody-forming cells but does not contribute actively to the induction of the immune response.     

Rabbit tonsil-associated M-cells express cytokeratin 20 and take up particulate antigen.

M-cells are believed to play a pivotal role in initiation of the immune response. These cells, located in the epithelia that overlie mucosal lymphoid follicles, are responsible for the active uptake of particulate antigens and for their translocation to the underlying lymphoid tissue. The identification of reliable markers for M-cells is therefore extremely important for the study of the initial steps that lead to the immune response. For this purpose, we studied cytokeratin 20 (CK20) expression in the epithelium of rabbit palatine tonsils by immunofluorescence, confocal microscopy, and Western blotting. CK20+ cells were observed in all rabbit palatine tonsils examined. By Western blotting, one CK20-immunoreactive band was identified at 46 kD on samples of proteins from the intermediate filament-enriched cytoskeletal fraction of tonsil epithelium. Double labeling of CK20+ cells with cell-specific markers confirmed that such cells were actually M-cells. Moreover, CK20+ M-cells displayed a mature phenotype (they formed pockets harboring lymphoid cells) and were functionally competent because they could take up particulate antigens from the pharyngeal lumen. We conclude that CK20 is an M-cell marker for rabbit palatine tonsils. Moreover, we can hypothesize the use of M-cells as a possible site for antigen delivery of particle-based vaccines.     

Immunologic 'ignorance' of vascularized organ transplants in the absence of secondary lymphoid tissue

Secondary lymphoid organs (the spleen, lymph nodes and mucosal lymphoid tissues) provide the proper environment for antigen-presenting cells to interact with and activate naive T and B lymphocytes. Although it is generally accepted that secondary lymphoid organs are essential for initiating immune responses to microbial antigens and to skin allografts, the prevailing view has been that the immune response to primarily vascularized organ transplants such as hearts and kidneys does not require the presence of secondary lymphoid tissue. The assumption has been that the immune response to such organs is initiated in the graft itself when recipient lymphocytes encounter foreign histocompatibility antigens presented by the graft's endothelial cells. In contrast to this view, we show here that cardiac allografts are accepted indefinitely in recipient mice that lack secondary lymphoid tissue, indicating that the alloimmune response to a vascularized organ transplant cannot be initiated in the graft itself. Moreover, we demonstrate that the permanent acceptance of these grafts is not due to tolerance but is because of immunologic 'ignorance'.     

CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.

The proper function of immune surveillance requires well-coordinated mechanisms in order to guide the patrolling immune cells through peripheral tissues and into secondary lymphoid organs. Analyzing gene-targeted mice, we identified the chemokine receptor CCR7 as an important organizer of the primary immune response. CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and delayed type hypersensitivity reactions. Due to the impaired migration of lymphocytes, these animals reveal profound morphological alterations in all secondary lymphoid organs. Upon activation, mature skin dendritic cells fail to migrate into the draining lymph nodes. Thus, in order to bring together lymphocytes and dendritic cells to form the characteristic microarchitecture of secondary lymphoid organs, CCR7 is required to rapidly initiate an adoptive immune response.     

Immediate, long-lasting suppression of autoimmune encephalomyelitis by cell-bound neuroantigen

When lymphoid cells from rats recovered from experimental autoimmune encephalomyelitis (EAE) were incubated in vitro for 1 hr with myelin basic protein (BP), then washed and transferred along with anti-BP immune serum to naive recipients, those recipients immediately developed a solid, long-lasting resistance to active induction of EAE. To obtain this high level of suppression, both steps of BP-incubation of cells and transfer of immune serum were found to be essential, i.e., direct transfer of nonincubated cells plus immune serum had no comparable suppressive effect, nor had transfer of BP-incubated cells with nonimmune serum. Specificity of the suppressive effect was indicated by the finding that cells from BP-sensitized donors, incubated with BP, protected against BP-CFA-induced disease but not against disease induced with whole spinal cord homogenate (SCH-CFA). As expected, cells from SCH-CFA-sensitized donors incubated with SCH protected recipients against disease induced with either SCH-CFA or BP-CFA. The suppression appears to act early in the afferent stage of the immune response, since inoculation with incubated cells as little as 24 hr after active challenge was ineffective. There was no suppression of passively induced disease.     

Kinetics of transfer of immunity by immune leucocytes and PFC response to HRBC in isogeneic ginbuna crucian carp

Transfer of immunity to horse erythrocytes (HRBC) by immune lymphoid cells was performed to analyze the kinetics of adoptive immunity in the clonal ginbuna crucian carp, Carassius auratus langsdorfti . Immune lymphoid cells were intravascularly transferred to the unprimed recipients and then recipients were evaluated by measuring the antibody titre of the plasma. Antibody productivity was most successfully conferred by splenic cells, followed by pronephric and mesonephric cells, taken from immune donors 7 days post-immunization, while transferability by thymic cells was lacking or very low, even if possible. Peak response of plaque-forming cells (PFCs) was observed at 5–7 days after the first injection, and the maximum number of PFCs at peak response was almost the same in all organs examined, such as the pronephros, mesonephros, spleen and the thymus. Direct correlation between transferability and number of PFCs was not observed on individuals, although the peak of transferability corresponded to that of the PFC response. Preliminary experiments of cell transfer by separated pronephric cells showed that the lymphocyte-rich fraction was more effective than the bottom fraction containing fewer lymphocytes in transferring immune reactivity. These results suggest that cells involved in transferring immune reactivity are B lymphocytes, composed of different developmental stages and distributed differently in the different lymphoid organs.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.