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Recent accumulating evidence suggests that innate immunity is associated with obesity-induced chronic inflammation and metabolic disorders. Here, we show that a Toll-like receptor (TLR) protein, radioprotective 105 (RP105)/myeloid differentiation protein (MD)-1 complex, contributes to high-fat diet (HFD)-induced obesity, adipose tissue inflammation, and insulin resistance. An HFD dramatically increased RP105 mRNA and protein expression in stromal vascular fraction of epididymal white adipose tissue (eWAT) in wild-type (WT) mice. RP105 mRNA expression also was significantly increased in the visceral adipose tissue of obese human subjects relative to nonobese subjects. The RP105/MD-1 complex was expressed by most adipose tissue macrophages (ATMs). An HFD increased RP105/MD-1 expression on the M1 subset of ATMs that accumulate in eWAT. Macrophages also acquired this characteristic in coculture with 3T3-L1 adipocytes. RP105 knockout (KO) and MD-1 KO mice had less HFD-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance compared with wild-type (WT) and TLR4 KO mice. Finally, the saturated fatty acids, palmitic and stearic acids, are endogenous ligands for TLR4, but they did not activate RP105/MD-1. Thus, the RP105/MD-1 complex is a major mediator of adipose tissue inflammation independent of TLR4 signaling and may represent a novel therapeutic target for obesity-associated metabolic disorders. [ABSTRACT FROM AUTHOR]

We studied the effect of passively administered anti-carrier arid anti- hapten antibodies on the primary anti-hapten antibody response to hapten-carrier conjugates in mice. Bacterial α-amylase (BαA), Taka-amylase A (TAA) and key-hole limpet haemocyanin (KLH) were used as carrier molecules, and 2,4-dinitro-phenyl (DNP) group was used as a haptenic determinant. Three groups of mice were injected intravenously with anti-carrier antiserum, anti-hapten antiserum and normal serum as the control, respectively, immediately after the immunization with the hapten-carrier conjugate. The primary anti-carrier antibody response was markedly suppressed by the passively administered anti-carrier antibody but not anti-hapten antibody. However, the primary anti-hapten antibody response was suppressed not only by passively administered anti-hapten antibody but also by the injection of anti- carrier antibody in an early period after the immunization. When anti-carrier antibody was given twice 0 and 7 days after immunization, the primary anti-hapten antibody response was markedly suppressed and the suppressive effect was observed even at a later period. In contrast, anti-hapten antibody given by the same schedule as above suppressed only the primary anti- hapten antibody response, but not the anti-carrier antibody response. Passively administered anti-carrier antibody did not suppress carrier-specific helper cell development, but still suppressed the development of B memory cells and antibody formation against carrier determinants. The antigen dose required for the development of B-cell memory was much higher than that necessary for the stimulation of' T cells. Passively administered anti-carrier antibody clearly inhibited the cellular cooperation between carrier-committed helper cells and hapten-specific B cells and the augmented primary anti-hapten antibody response induced by carrier-primed T cells was clearly abolished. Furthermore, mice preimmunized with carrier showed significantly lower anti-hapten antibody response following the hapten- carrier challenge. Moreover, development of hapten-specific memory cells was also suppressed. Thus, even in the non-specific antibody-induced suppression by anti-carrier antibody, negative feedback effect of a B-cell product with anti-carrier specificity exclusively regulates the B-cell line development and differentiation. [ABSTRACT FROM AUTHOR]

Human memory (CD45RO+) CD4+ T cells can be distinguished into two subpopulations on the basis of expression of the lymph node homing receptor, L-selectin (CD62L). In a prior study we showed that human L-selectin-positive memory T-helper (Th)cells promote the maturation of IgG- and IgA-producing cells by naive B cells. To further elucidate the contribution of memory CD4+ T cells to B-cell differentiation, human memory CD4+ T cells with or without L-Selectin expression were evaluated for production of cytokines that participate in regulation of immunoglobulin production. It was found that L-selectin-positive human memory CD4+ T cells produce mainly interleukin (IL)-4 and IL-5, whereas L-selectin-negative CD4+ T cells produce mainly interferon-γ (IFN-γ). This profile of cytokine expression coincides with the profile that distinguishes Th1 and Th2 subsets, in contrast to the routine system, IL-10 production was similarly contributed by human L-selectin-positive and -negative memory CD4+ T-cell subpopulations. These results suggest that the human L-selectin-negative and -positive subpopulations of human memory CD4+ T cells contain Th1-like and Th2-like cytokine- producing cells, respectively. [ABSTRACT FROM AUTHOR]

We studied cells in the lungs of BALB/c and BALB/c-nu/nu (nude) mice infected with Toxocara can/s, which produced interleukin-5 (IL-5) in in vitro culture with larval excretory-secretory antigen (ESAg). The proportion of CD4+/CD8+/CD4-CD8- cells in lungs of both BALB/c and nude mice was unchanged before and after infection with T. canis. Panning and complement mediated lysis using monoclonal antibody (mAb) to CD4 showed that CD4+ cells in the lung from both mice produced IL-5. Anti-CD4 mAb suppressed ESAg-stimulated IL-5 production in vitro. In vitro depletion or inhibition of CD8+ cells reduced IL-5 production significantly in some cases, suggesting involvement with IL-5 production. Anti-CD3 mAb enhanced IL-5 production when incubated with or without ESAg. Production of IL-5 was reduced by in vivo depletion of CD4+ cells only and both CD4+ and CD8+ T cells, by intraperitoneal injection with appropriate mAb; IL-5 production was stimulated by anti-CD3 mAb. In contrast, IL-5 production by lung cells of BALB/c mice decreased by more than 90% after simultaneous injection with anti-CD4, anti-CD8 and anti-CD3 mAb, and was not enhanced by anti-CD3 mAb. Similar results were obtained in nude mice. These results suggest that CD4- CD8- T cells, as well as CD4+ T cells, produce IL-5. [ABSTRACT FROM AUTHOR]

A novel monoclonal antibody (mAb) was established in an attempt to look for a cell-surface molecule that delivered a signal regulating apoptotic cell death of B cells. Because spleen cells in resting culture die from apoptosis, mAb were looked for that were able to prolong spleen cell survival in vitro. This screening selected mAb CS/2. CS/2 not only prolonged spleen cell survival in vitro, but also protected spleen cells from apoptotic cell death brought about by irradiation or dexamethasone. Moreover, stimulation of spleen cells with CS/2 mAb induced changes of cells to blastoid morphology, and a significant uptake of [³H]thymidine ([³H]TdR). The antigen recognized by CS/2 mAb (CS/2 Ag) was expressed on preB cells, B cells, and Mac-1+ cells. The cells surviving in vitro culture or irradiation in response to ligation with CS/2 mAb were mostly B cells expressing the CS/2 Ag. In addition, B cells from X-linked immunodeficient (XID) mice did not respond to CS/2 mAb. These results indicate that CS/2 mAb is agonistic to B cells, and that XID mice are deficient in this CS/2 mAb-mediated activation pathway. Determination of the amino-terminal 24 amino acid residues revealed that the CS/2 Ag appears to be identical to the murine CD38 homologue. These results are consistent with the conclusion that CS/2 mAb is directed against a murine CD38 homologue, and suggest a possible role of the murine CD38 homologue in controlling apoptotic cell death of B cells. [ABSTRACT FROM AUTHOR]

We depleted or neutralized interleukin-5 (IL-5) and IL-5 receptor of C57BL/6 mice, using rat antimurine IL-5 monoclonal antibody (NC17) and anti-murine IL-5 receptor monoclonal antibody (H7). Mice treated with these monoclonal antibodies were infected with Strongyloides venezuelensis larvae. The time-course of faecal egg output and peripheral eosinophilia were monitored. In a primary infection, anti-IL-5 treatment did not affect faecal egg output, although the eosinophil count in peripheral blood was markedly reduced. There was no difference in intestinal worm burden or faecal egg output between anti-IL-5 treated and non-treated mice. In a secondary infection, worms were expelled from the small intestine of anti-IL-5-treated mice as well as from non-treated mice. Worm recovery from the lungs of mice treated with either anti-IL-5 or anti-IL-5 receptor monoclonal antibody was the same as that of normal controls. However, a marked reduction in worm recovery was observed in re-infected mice that had not been treated with monoclonal antibodies. Treatment with anti-IL-5 or anti-IL-5 receptor monoclonal antibody suppressed blood and tissue eosinophilia. Thus the results suggested that the host's protective immunity against tissue-migrating larvae was IL5-dependent but intestinal immunity was not. [ABSTRACT FROM AUTHOR]

There is a body of evidence suggesting that IgA production is regulated by helper T cells or their products. To elucidate molecular mechanisms of IgA production, the role of lymphokines in the in vitro antigen-specific and polyclonal lgA responses was examined. Supernatants from antigen-stimulated T cells or mitogen-stimulated T-cell clones can enhance 2,4-dinitro phenyl (DNP)-specific IgM, IgGL and IgA production in cultures of DNP-ovalbumin (OVA)-stimulated T-cell-depleted spleen cells from DNP-keyhole limpet haemocyanin-primed mice. The IgA enhancement was inhibited by anti-IL-5 monoclonal antibody. Purified recombinant IL-5 could also enhance anti-DNP IgA production in a dose-dependent manner. This enhancing effect was not substituted by IL-1, IL-2, IL-3 or IL-4. Polyclonal IgA secretion of lipopolysaccharide-stimulated normal B cells was augmented preferentially by IL-5, but not by IL-4. Surface IgA-positive (sIgA+) B cells, but not surface IgA-negative B cells, responded to IL-5 for the development of IgA-secreting cells. Limiting-dilution analysis revealed that IL-5 increases the frequency of IgA-secreting cells in sIgA+ B-cell populations. These results indicate that IL-5 plays an essential role in the antigen-specific and polyclonal IgA formation as a maturation-inducing factor rather than class-switching factor. [ABSTRACT FROM AUTHOR]

Two distinct murine B-cell differentiation factors, designated B 151 -TRY 1 and B 151-TRF2, were described originally as B151 K12 T-cell hybridoma-derived lymphokines that induce immunoglobulin (Ig) secretion by antigen-activated B cells and unstimulated B cells, respectively. In the present study, we found that a highly purified B1 51-TRF1 fraction prepared by reversed-phase high-performance liquid chromatography (RP-H PLC) also has the ability to cause a polyclonal differentiation of unstimulated B cells into IgM-secreting cells in the apparent absence of co- stimulant. The activity of the B151-TRF1 fraction but not the B151-TRF2 fraction on unstimulated B cells was markedly inhibited by addition of a monoclonal antibody (mAb) specific for the B151- TRF 1/IL-S to the culture. To determine whether 8151 -TRF 1/IL-S and 8151 -TRF2 act on distinct populations among unstimulated B cells, the responsiveness of neonatal B cells and adult B cells that had been fractionated by Percoll density gradient centrifugation was assessed. B15 l-TRFI/IL-5 predominantly acted on lower density B cells, which appeared around 3 weeks after birth in the spleen. In contrast, B151-TRF2 could activate both lower and higher density B cells almost equally and B 151 -TRF2-responsive B cells were already present by 1 week of age. Thus, these results suggest that B151-TRFI/IL-5 and B151-TRF2 act on distinct subpopulations among antigen-unprimed normal B cells to induce IgM-secreting cells. [ABSTRACT FROM AUTHOR]

The B-cell differentiation-inducing activity of interleukin-1 (IL-1) was compared with that of T-cell replacing factor (TRF)/interleukin-5 (IL-5), which was originally described as a late-acting B-cell differentiation-inducing factor. Human recombinant IL-1 and murine recombinant TRF/IL-5 were used in this study. Purified B cells from non-primed or antigen-primed mice, LPS-stimulated B-cell blasts, and chronic B-cell leukaemia (BCL1) cells were used as the responding B-cell population. Addition of IL-1 to the culture of normal B-cells and sheep red blood cells (SRBC) induced a dose-dependent anti-SRBC IgM response, with maximal response at 100 U/ml, whereas the response induced by TRF/IL-5 was less than that induced by IL-1 and did not reach the maximum even at 100 U/ml. Addition of anti-IL-1 antibody, but not anti-TRF/IL-5 antibody or anti-IL-2 receptor antibody, inhibited IL-1-induced anti-SRBC responses. Depletion of cells adherent to Sephadex beads from splenic B cells showed no significant effect on the magnitude of the total responses. IL-1 could induce little, if any, differentiation in antigen-primed B cells, LPS-stimulated B-cell blasts, or BCL1 cells into antibody-secreting cells, whereas differentiation could be induced by low doses of TRF/IL-5 (1-2 U/ml). Of great interest is that suboptimal doses of IL-1 (10 U/ml) could synergize with TRF in the primary anti-SRBC PFC responses. Kinetic studies revealed that IL-1 acts on B cells for the first 2 days and TRF/IL-5 for the last 3 days in 5-day cultures of B cells. These results suggest that IL-1 acts primarily on resting B cells as a differentiation-inducing factor in the presence of antigen, and also acts as a 'priming' factor for TRF/IL-5. [ABSTRACT FROM AUTHOR]

We established three functionally distinct purified protein derivative (PPD)-reactive 1-cell clones (811.15. 812.F and D-2). Clone 811.15 could co-operate with DNP-primed B cells to induce anti- DNP lgG plaque-forming cell (PFC) responses only when high amounts of PPD were added to the culture, whereas stimulation of a low amount of DNP-PPD was ineffective (factor-mediated interaction). On the other hand, clone D-2 activated those B cells in a MHC-restricted manner only when DNP-PPI) was added to the culture (cognate interaction). 812.F could stimulate B cells with either PPD or DNP-PPD. Antigen non-specific helper factors (lymphokines) responsible for B-cell activation produced by cloned T cells upon stimulation with PPD and antigen-presenting cells were then investigated. Lymphokine activities determined in the present study were IL-2, BCGF I, BCGF II and TRF. BCGF I activity was determined by proliferation-inducing activity on purified B cells in the presence of anti-IgM antibody. BCGF II activity was measured by proliferation-inducing activity on purified B cells in the presence of dextran sulphate. TRF activity was determined on DNP-primed B cells for inducing further differentiation into anti-DNP IgG PFC. BCGF I active molecules were eluted in the fraction at apparent MW of 50,009-70,000 and 8,000- 10,000 in gel-permeation column chromatography. BCGF 11 active molecules were recovered in the fractions at an apparent MW of 30,000- 45,000. In addition, it was demonstrated that BCGF I acted on both activated and resting B cells. In contrast, BCGF II acted on blasted B cells but not resting B cells. TRF was hardly separable from the BCGF 11 active one on gel permeation column chromatography. In the comparative analysis of lymphokine activities in supernatants obtained from three functionally distinct T-cell clones stimulated by antigen, clone B12.F was the highest producers of all kinds of lymphokines tested. Clone B1 1.15 produced IL2, SCOF II and TRF. However, BCGF I was hardly detectable in cell-free supernatants of S 11.15. Clone D-2 produced relatively far less amounts of lymphokines except for IL-2. IL-2 was produced by all three 1-cell clones. The results suggest that multiple lymphokines are involved in S-cell activation through factor-mediated T-S cell interaction. Moreover, our data suggest that lymphokines such as BCGF I, BCGF II or TRF do not play important roles in B-cell activation through cognate T--B cell interaction. [ABSTRACT FROM AUTHOR]

Cooperative induction of secondary anti-hapten antibody responses was studied by using non-cross-reactive carrier proteins, bacterial α-amylase (BαA), Taka-amylase A (TAA) and keyhole-limpet haemocyanin (KLH), and the 2,4-dinitrophenyl (DNP) and benzylpenicilloyl (BPO) groups as haptenic determinants. Lymphoid cells obtained from mice primed with a hapten-carrier conjugate could be effectively stimulated with a hapten-heterologous carrier conjugate, provided that lymphoid cells primed to the heterologous carrier were also present. In the carrier-primed lymphoid cell population, helper activity of thymus-derived cells developed earlier following carrier immunization than did the capacity of antibody-forming cell precursors (AFCP) to produce an effective anti-carrier antibody response upon secondary stimulation. Attempts to generate hapten-specific helper cell activity were unsuccessful. Thus, cells primed to one haptenic determinant failed to exert a helper function with cells primed to a second hapten upon subsequent administration to the two haptens together on the same heterologous carrier molecule. In order to distinguish among carrier determinant specificities which react with thymus-derived helper cells from those which react with bone marrow-derived AFCP, the capacity of various anti-carrier antibodies or antibody fragments to suppress either anti-carrier antibody production alone or together with helper cell function in adoptive secondary anti-hapten antibody responses was tested. In this system, it was found that 7S anti-carrier antibody suppressed the reaction of helper cells and carrier-specific AFCP such that both the anti-hapten and anti-carrier antibody responses were abrogated. By contrast, passively administered 3.5S fragments of anti-carrier antibodies selectively prevented the stimulation of carrier-specific AFCP to produce anti-carrier antibodies, but had no effect on the capacity of carrier-specific helper cells to facilitate the secondary anti-hapten antibody response. As expected, passively administered 7S anti-hapten antibodies selectively abrogated the production of anti-hapten, but not anti-carrier antibodies. These data are discussed in the context of suggesting that distinct determinant sites on carrier molecules are recognized independently by thymus-derived helper cells and by bone marrow-derived AFCP. [ABSTRACT FROM AUTHOR]

Non-cross reactive antigens, bacterial α-amylase (BαA), Taka-amylase A (TAA) and keyhole-limpet haemocyanin (KLH), were used as the carrier proteins conjugated with 2,4-dinitrophenyl (DNP) and benzylpenicilloyl (BPO) groups as haptenic determinants. Lymphoid cells obtained from mice primed with these hapten-protein conjugates were stimulated in vitro with various DNP- and BPO-proteins as the second antigen and transferred into X-irradiated recipients. A good secondary antihapten antibody response was obtained when the cells primed with a hapten-carrier conjugate were stimulated with the same conjugate as that used for priming, but no response could be obtained if carrier for the secondary stimulus differed from that used for priming. However, stimulation with hapten-heterologous carrier gave a good secondary response to the hapten provided that cells committed to the heterologous carrier were also present. The activity of carrier-primed cells could not be replaced by circulating antibody. Co-operation between the carrier-primed cells and the hapten-primed cells was also observed when the antigenic stimulus was hapten-carrier conjugate incorporated into macrophages. The immunogenic molecule from the macrophage was considered to consist of the haptenic group and at least a portion of the carrier. When the primed cells were secondarily stimulated with hapten-carrier conjugates incorporated into macrophages and the mixtures transferred to recipient mice, the passive administration of antihapten antibody to these recipients specifically suppressed the corresponding antihapten antibody response only but passively administered anticarrier antibody completely suppressed both the anti-carrier and antihapten antibody responses. The suppression of antihapten antibody response by anticarrier antibody seems to be the result of failure of co-operation between carrier-primed cells and hapten-primed cells. [ABSTRACT FROM AUTHOR]

The suppressive effect of antibody on the secondary immune response was analysed using a technique of memory cell transfer into X-irradiated recipients. The response of memory cells stimulated by antigen-bearing cells was markedly suppressed when antibody was passively administered to recipients and this had its maximal effect when administered simultaneously with the cell transfer. The capacity of memory cells to respond to antigenic stimulation and the capacity of antigen-bearing cells to stimulate memory cells were not directly impaired by the suppressive antibody. The marked suppression was observed only when both memory cells and antigen-bearing cells were present together with antibody. These results suggest that antibody-induced suppression appears to be caused by blocking the effective antigenic stimulus from antigen-bearing cells to memory cells. Evidence is presented indicating that pre-existing antibody plays a regulatory role in the initiation of the secondary response and also controls the expression of memory. [ABSTRACT FROM AUTHOR]