Ontogeny of culture-generated suppressor cells.

Culture of murine lymphoid cells without added antigen results in the generation of cells which suppress a variety of in vitro immune responses, such as the mixed lymphocyte response (MLR) and the generation of alloreactive cytotoxic T cells (CTL). The ontogeny of this phenomenon was studied. Cells which suppressed the MLR after preculture were isolated from spleens and hematopoietic livers of fetal and young (less than 1 wk old) mice. On the other hand, the generation of alloreactive CTL could be suppressed only by precultured spleen cells taken from 1-w-old or older mice. The parallel between the development of the suppressor functions and the maturation of the responses they regulate, suggests a possible biological significance of the effect.

Antibody response of immunodeficient (xid) CBA/N mice to Escherichia coli 0113 lipopolysaccharide, a thymus-independent antigen.

CBA/N mice, which possess an X-linked immunodeficiency (xid), produce a convincing antibody response to lipopolysaccharide derived from Escherichia coli 0113 (LPS 0113), a thymus-independent antigen. The antibody response produced was shown to be specific for the O-polysaccharide moiety of LPS 0113, rather than lipid A or lipid-A-associated protein. The relevance of this finding to the nature of the genetic defect of xid-mice is discussed.

Glomerular immune complex deposits associated with mouse mammary tumor.

Adult Paris R-III mice with localized mamary tumors were found to have subendothelial glomerular immune complex deposits by electron microscopy and immunofluorescence. In addition to IgG adn B-1-C, the glomeruli contained antigenic material that reacted with an antiserum to mouse mamary tumor virus. These findings support the hypothesis that many animals with congenitally or neonatally induced virus-related tumors are not immunologically tolerant to the oncogenic virus.

Electron microscopy and immunofluorescence of glomerular immune complex deposits in cancer patients.

Kidneys of 29 patients without clinical renal disease were studied by electron microscopy for the presence of glomerular basement membrane deposits: those from 11 of 20 patients with cancers of various sites, but only 1 of 9 patients without cancer contained electron-dense subendothelial deposits. The majority of these kidneys gave positive immunofluorescent reactions for immunoglobulin and complement. However, among a number of the cases studied, a lack of correlation between electron microscopic and immunofluorescence findings has yet to be investigated. Although the number of patients in this study is small because of the difficulty in obtaining tissue for electron microscopy, it is postulated that the deposition of immune complexes in the kidney occurs with a high frequency among cancer patients. The kidneys may thus be a valuable source for isolating tumor-associated antigens and corresponding antibodies.

Multiple responses to administration of liposome-encapsulated hemoglobin (LEH): Effects on hematopoiesis and serum IL-6 levels.

Liposome-encapsulated hemoglobin (LEH) has been tested in animals as an oxygen-carrying red cell substitute and has been shown to be beneficial in the treatment of hemorrhagic shock. The effects of LEH on immune responses have not been studied thoroughly in any well-controlled model. Using a murine model, we evaluated nephrotoxicity and hepatotoxicity as well as immune function parameters following LEH administration. Following intravenous administration of LEH, 1) a serum spike of interleukin-6 (IL-6) occurred in mice at 4-8 hours, with no elevation of IL-1, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma); 2) the serum liver function enzymes SGOT (AST, aspartate aminotransferase) and SGPT (ALT, alanine aminotransferase) were elevated at 48 hours; 3) only a slight increase in serum antibody to bovine hemoglobin was observed; and 4) increased hematopoietic activity was observed in the spleen and bone marrow. The finding that only IL-6 but not the associated TNF, IL-1, or IFN-gamma is secreted in vivo following LEH administration is novel and may have significance in defining the mechanisms underlying specific adverse responses observed with LEH administration in animals.

An improved model for the examination of biological effects of locally administered cytokines.

Cytokines incorporated into agarose blocks and implanted subcutaneously into mice establish an in vivo gradient which can be used to mimic a local inflammatory process. We have developed a model in which cellular influx into cytokine impregnated blocks parallels the normal cellular reaction to infections or wounds. Agarose blocks containing supernatants from ConA activated rat spleen cells attracted neutrophils within 4 h. These cells were followed by lymphocytes and macrophages in 24 h. Flow cytometry analysis of lymphoid cells on day 1 revealed that 38% were Ig+ (B cell marker), 60% MAC-2,3+ and 20% Thy 1.2+ of which only a small fraction were expressing CD4 on their surface. These numbers changed with time following implantation of the blocks. Initially, isolated adherent cells (macrophages) were resting, with low phagocytic activity. Cells isolated from blocks at later time points were activated, as evidenced by their increased ability to ingest fluoresceinated beads. The secretion patterns of cells trafficking to murine rIL-1 containing agarose blocks were examined. TNF, IL-6 and antibody secreting cells were found. No IL-2 was detected at any time. We believe that this model will be of value in studies of local actions of cytokines.

Gut damage during hemorrhagic shock: effects on survival of oral or enteral interleukin-6.

It has been reported that oral interleukin (IL)-6, without deleterious systemic side effects, prevents bacteremia and gut epithelial apoptosis after hemorrhagic shock (HS) in rodents. The goal of this study was to explore potential benefit of oral or enteral IL-6 on the gut and, consequently, on survival in a long-term outcome model of HS in rats. In Study A, 20 rats (control and IL-6, n = 10 per group) were anesthetized by spontaneous breathing of halothane and N2O. The left femoral vein and artery were cannulated. HS was initiated with withdrawal of 3 mL of blood per 100 g body weight over 15 min, and mean arterial pressure was maintained at 40 to 50 mmHg for another 75 min (total HS 90 min) by blood withdrawal or infusion of Ringer's solution. At HS 90 min, resuscitation included reinfusion of shed blood and additional Ringer's solution to restore normotension for 30 min. After awakening at resuscitation time 30 min, the rats received either 300 units IL-6 or the same volume of vehicle (controls) injected into the stomach via a feeding cannula. In Study B, 20 rats (control and IL-6, n = 10 per group), fasted overnight, were prepared and treated as in Study A, except that HS was initiated with withdrawal of 2 mL blood per 100 g over 10 min, and mean arterial pressure was maintained at 35-40 mmHg. IL-6 rats received 3,000 units IL-6 in 5 mL of normal saline injected directly into the ileum lumen 20 min after induction of shock and again at resuscitation time 60 min. Control rats received normal saline alone. In both studies, survival was observed to 72 h. In Study A, 7 of 10 rats in the control group and 5 of 10 in the IL-6 group survived to 72 h (NS). Macroscopic assessment of gut injury was not different between the two groups. In Study B, 6 of 10 rats survived to 72 h in each group. Frequency of bacteria growth in liver tissue of 72 h survivors was not different between the two groups. IL-6, administered into the stomach or directly injected into the small intestine lumen, did not protect the gut from ischemic injury, nor did it improve survival following severe HS in rats.

Recruitment of tissue resident cells to hydrogel composites: in vivo response to implant materials.

A model of local cellular recruitment was established using hydrogel matrices composed of alginate implanted subcutaneously into mice. Cells which trafficked to the matrix blocks were recovered and characterized for surface phenotype using fluorescently labelled antibodies and flow cytometry (fluorescence activated cell sorting). Temporal information of the differential recruitment of cells was determined. The basic pattern of recruitment in response to the hydrogels was established and mimicked that seen in a local inflammatory response. Neutrophils (PMN) were rapidly recruited (1 d) followed by macrophages and lymphocytes (1-3 d). Cell surface phenotype studies included the determination of CD3+, CD4+ and CD8+ cells, Mac-1+ cells, and immunoglobulin bearing cells. Microscopic analysis revealed numerous activated PMNs and monocyte derived foamy macrophages. Fluorescence immunocytochemistry of frozen sections of the block revealed that macrophages, CD3+ and natural killer cells were all recruited to the interior of the block. Ultrastructural analysis (transmission electron microscopy) showed highly activated macrophages, with abundant rough endoplasmic reticulum and secretory vesicles. Cells which remained on the surface of the matrix block were CD44 positive migratory cells. Electron microscopic evidence showed foamy macrophages with a varying degree of involvement with the hydrogel material. Surface scanning electron microscopy revealed numerous fibroblast-like cells coating the surface of the block. We suggest that these methods may be used to address the inflammatory response elicited with a a variety of implanted materials such as hydrogels, silicones, ceramics and metals. Furthermore, this model has been useful in determining cellular responses to cytokines and growth factors under similar conditions.

Biocompatibility of lipid microcylinders: effect on cell growth and antigen presentation in culture.

The authors are developing a lipid-based microcylinder for the controlled release of biological response modifiers and as templates for cellular migration and differentiation. These structures are comprised of a photopolymerizable phosphatidylcholine (1,2-ditricosa-10,12-diynoyl-sn-glycero-3-phosphocholine) and form spontaneously as a result of a thermotropic phase transition in aqueous solution or in a cosolvent solution of 70:30 ethanol:water. The hollow cylinders are helically wrapped lipid bilayers, variable in length (50-250 microns, depending on conditions of formation) and are 0.5-1.0 microns in diameter. The interaction has been examined of three types of lipid microcylinders: (1) monomeric, (2) photopolymerized by exposure to 254 nm light, and (3) surface-modified by incorporation of 6 mol% gangliosides, with different human cell lines and peripheral blood leucocytes to evaluate the biocompatibility of these structures. The proliferative status of U937 (a histiocytic monocyte), K562 (an erythroleukaemic cell), and Jurkat's derivative (a T-lymphoblast) as measured by pulsed tritiated thymidine was unaffected by the presence of up to 100 micrograms/ml of lipid microcylinders after 3 d in culture. Adherent human peripheral blood monocytes were shown to form adhesive contacts with the lipid microcylinders. An 'association' index from this interaction shows that after 3 d in culture, the association was much lower for those microcylinders that had incorporated ganglioside compared with monomeric or polymerized structures. The lipid microcylinders do not activate T-cells isolated from human peripheral blood, nor do they inhibit the activation of T-cells by phorbol esters or other mitogens.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic sepsis following hemorrhagic shock: alleviation with oral interleukin-6.

Gut-origin sepsis is a serious medical complication of military injuries following hemorrhage. Splanchnic ischemia induces intestinal necrosis leading to systemic bacteremia. Rat and mouse models of hemorrhagic shock were used to investigate bacterial translocation from the gut. Orally administered ameliorative treatments using the cytokine interleukin-6 (IL-6) were able to reduce or eliminate sepsis following hemorrhage. To mimic battlefield wounds and hemorrhage, anesthetized mice were bled from the femoral artery, held at a mean arterial blood pressure of 35 mm Hg for 1 hour, and then resuscitated with shed blood and 2-fold volume lactated Ringer's solution. Anesthetized rats were bled from the carotid artery at a rate of 15 ml/kg at 1 ml/minute. Bacteriological cultures of livers and mesenteric lymph nodes from hemorrhaged animals given recombinant IL-6 had significantly fewer colonies per gram of tissue than saline-fed controls. 125I-labeled IL-6 remained in the gut for up to 6 hours giving regional protection, whereas labeled interleukin-2 was disseminated throughout the body in the same time. In vivo and vitro studies of IL-6 showed that long incubations with high doses of trypsin, chymotrypsin, or intestinal contents were necessary to inactivate the bioactivity of this cytokine. Electron microscopy showed that epithelial cells from hemorrhaged mice fed saline had sparse or missing villi and vacuolated cytoplasm. Epithelial cells from control mice or mice hemorrhaged and fed cytokine appeared completely normal. Oral administration of IL-6 on the battlefield may be an important treatment for the prevention of sepsis following hemorrhage.

IL-6 protects enterocytes from hypoxia-induced apoptosis by induction of bcl-2 mRNA and reduction of fas mRNA.

Interleukin-6 (IL-6) has been shown to rescue enterocytes from hypoxia-induced apoptosis when given orally following hemorrhagic shock. In vitro models using an intestinal epithelial cell line (IEC-6) cultured with lipopolysaccharide (LPS) under low O2 conditions, to mimic intestinal conditions, show that these cells also undergo apoptosis, which can be reduced by subsequent culture with IL-6. To examine further the mechanisms of rescue, we cultured normal rat intestinal epithelial cells (IEC-6) under both normoxic and hypoxic conditions and analyzed their responses to LPS and IL-6. We showed that IEC-6 expressed IL-6 receptor on its surface. Further, IEC-6 cells could be rescued from hypoxia-induced apoptosis by co-culture with IL-6. RNase protection assay (RPA) examination revealed that under hypoxic conditions, IEC-6 cells that were resistant to apoptosis showed reduced fas expression and increased bcl-2 expression after co-culture with LPS+IL-6.

Modulation of mucosal immunity against Campylobacter jejuni by orally administered cytokines.

The effect of oral recombinant interleukin (rIL) treatment on the course of Campylobacter jejuni infection and the development of mucosal immunity in mice was investigated. rIL-2, rIL-5, and rIL-6 were administered to mice at 24 and 6 h before infection and at 0, 24, and 48 h after infection with C. jejuni HC, and the subsequent development of an immune response and intestinal colonization resistance were determined. In this model, orally administered cytokines retained their biological activities with no apparent side effects. Following infection, initial bacterial counts in fecal samples collected from cytokine-treated and untreated mice were similar. However, within 48 h of infection a greater than 3-log-unit reduction in the number of C. jejuni shed in the feces was found for rIL-6-treated animals. Colonization levels were similarly reduced in rIL-5-treated mice, although the rate of clearance was somewhat slower. In contrast, rIL-2 treatment had no significant effect on colonization levels compared with that in controls. Oral rIL-6 treatment was also associated with enhanced intestinal and systemic Campylobacter-specific immunoglobulin A responses compared with those observed in either rIL-5- or rIL-2-treated animals. Upon rechallenge, initial colonization in all cytokine-treated groups was approximately 2 log units lower than that in controls. However, local infection was controlled only in rIL-2-treated mice over time. rIL-5 and rIL-6 treatment had only a marginal effect on colonization resistance following rechallenge. On the basis of these results, it appears that rIL-5 or rIL-6 may function to modulate the induction and/or expression of anti-C. jejuni immunity through different mechanisms.

Rapid induction of protective tolerance to potential terrorist agents: a systematic review of low- and ultra-low dose research.

OBJECTIVE: To systematically review the literature on the ability of low-dose (LD) and ultra-low-dose (ULD) toxin exposure to prevent and treat biological and chemical threats. METHODS: Laboratory research articles on protection or treatment from LD or ULD exposure for the 13 high-risk chemical and biological warfare threats were collected and systematically evaluated for quantity and scientific quality using pre-defined methodological criteria. RESULTS: Over 2600 articles were screened. Only five studies met the inclusion criteria examining stimulation and protective effects of LD- or ULD-exposures to the 13 pre-identified biological and chemical agents. The quality evaluation (QE) of these studies was above average with a mean QE score of 70.6% of maximum. Two articles of fair to good quality reported both protective and treatment efficacy from exposure of animals or humans to LD- and ULD-exposures to toxins of risk in biochemical warfare. CONCLUSION: There is little research on agents of biological and chemical warfare investigating the possible use of LD- and ULD-toxins for protection and treatment. The existing literature is generally of good quality and indicates that rapid induction of protective tolerance is a feasible but under-investigated approach to bioterrorist or biowarfare defense. In our opinion, further research into the role of induced protection with LD- and ULD-toxic agents is needed.

Mechanisms of immunity to rickettsial infection: characterization of a cytotoxic effector cell.

Rickettsiae, as other intracellular bacteria, are relatively sequestered from the effects of antibody and local antibody-independent responses. Considering the obligate intracellular nature of rickettsia, the exact mechanisms by which lymphocytes and macrophages encounter rickettsial antigens and eliminate the infection depends upon the appropriate presentation of antigen to the immune system. We demonstrate here that cells taken from the spleens of Rickettsia typhi- or R. tsutsugamushi-infected mice are able to lyse specifically tissue culture targets infected with the homologous organism. This effect was eliminated upon treatment of the spleen cells with anti-Thy-1.2 + complement. Furthermore such T cells exhibit H-2-restricted killing when tested on infected targets of different genetic backgrounds. We propose that a T cell-mediated cytotoxic immune mechanism exists that may play an important role in the elimination of rickettsial organisms during infection.

Induction of T cell activity in athymic (nu/nu) mice infected with Trypanosoma rhodesiense.

Infection of C57BL/10 (B10)3 nu/nu mice with Trypanosoma rhodesiense results in the development of significant T-cell reactivity in spleen and lymph nodes. The proliferative responses to mitogens, such as concanavalin A (Con A) and phytohemagglutinin (PHA), and in mixed-lymphocyte reactions (MLR) to alloantigens are enhanced compared with control uninfected nu/nu mice. These results serve to emphasize the stimulatory nature of trypanosomes on the immune system.

Positive selection of T-cell subsets. I. Proliferative responses of Lyt 2 separated thymocytes and splenic T cells.

Flow microfluorometry analysis of peanut lectin non-agglutinable (PNA-) thymocytes (ThC) reveals the existence of 30%-50% Lyt 1,2,3+ and 50%-70% Lyt 1+,2,3- subpopulations. Using positive selection on anti-immunoglobulin-coated (Mage) plates, we selected PNA- Lyt 2+ and PNA- Lyt 2- ThC as well as their peripheral counterparts in the spleen. These populations were tested in parallel for their ability to respond to concanavalin A (Con A) and phytohaemagglutinin (PHA), to respond to allogeneic stimulation in the mixed lymphocyte reaction (MLR); ThC subpopulations were also tested for their ability to provide synergy with lymph node cells (LNC) in the MLR. It was found that (a) Lyt 2- cells of both thymic and splenic origin responded to all doses of Con A or PHA; (b) PNA- Lyt 2+ ThC were unresponsive to Con A or PHA, whereas splenic Lyt 2+ T cells responded to low doses of mitogens; and (c) PNA- ThC of both Lyt phenotypes responded in a MLR and provided synergy with LNC in the MLR. These data support the notion that Lyt 2+ cells of either PNA- or PNA+ subpopulations must undergo post-thymic maturation before becoming responsive to low doses of T-cell mitogens.

The abnormal function of T cells in chronically anti-mu-treated mice with no mature B lymphocytes.

T cells from anti-mu-treated mice, normal goat serum ( NGS )-treated mice or untreated control mice were compared with respect to their surface antigenic phenotypes, T cell mitogenic responses, helper function and precursor frequency of helper T cells. Anti-mu treatment arrested the development of B cells at an immature stage, as determined by flow microfluorometry; it resulted in no serum IgM, but detectable levels of IgG by solid-phase radioimmunoassay. Proliferative responses to phytohemagglutinin and concanavalin A were significantly decreased in T cells obtained from mu-suppressed C57BL/6 mice, but not from control mice. When T cells from anti-mu-treated mice were tested in vitro for their helper activity to collaborate with B cells from nu/nu C57BL/6 mice to give plaque-forming cells to sheep red blood cells, they provided far less help than did T cells from control mice. The frequency of T helper cells, as measured by limiting dilution analysis, was much lower in the anti-mu- than in the NGS -treated mice. Cell mixing experiments provided evidence for active suppression of T helper function among splenocytes taken from mu-suppressed mice.