Glycyrrhizin inhibits the manifestations of anti-inflammatory responses that appear in association with systemic inflammatory response syndrome (SIRS)-like reactions

In association with the systemic inflammatory response syndrome (SIRS), anti-inflammatory response syndrome is commonly manifested in patients with trauma, burn injury, and after major surgery. These patients are increasingly susceptible to infection with various pathogens due to the excessive release of anti-inflammatory cytokines from anti-inflammatory effector cells. Recently, CC-chemokine ligand 2 (CCL2) found in the sera of mice with pancreatitis was identified as an active molecule for SIRS-associated anti-inflammatory response manifestation. Also, the inhibitory activity of glycyrrhizin (GL) on CCL2 production was reported. Therefore, the effect of GL on SIRS-associated anti-inflammatory response manifestation was investigated in a murine SIRS model. Without any stimulation, splenic T cells from mice 5 days after SIRS induction produced cytokines associated with anti-inflammatory response manifestation. However, these cytokines were not produced by splenic T cells from SIRS mice previously treated with GL. In dual-chamber transwells, IL-4-producing cells were generated from normal T cells cultured with peripheral blood polymorphonuclear neutrophils (PMN) from SIRS mice. However, IL-4-producing cells were not generated from normal T cells in transwell cultures performed with PMN from GL-treated SIRS mice. CCL2 was produced by PMN from SIRS mice, while this chemokine was not demonstrated in cultures of PMN from SIRS mice treated with GL. These results indicate that GL has the capacity to suppress SIRS-associated anti-inflammatory response manifestation through the inhibition of CCL2 production by PMN.     

Metabolism of nitric oxide by Neisseria meningitidis modifies release of NO-regulated cytokines and chemokines by human macrophages

Macrophages produce nitric oxide (NO) via the inducible nitric oxide synthase as part of a successful response to infection. The gene norB of Neisseria meningitidis encodes a NO reductase which enables utilization and consumption of NO during microaerobic respiration and confers resistance to nitrosative stress-related killing by human monocyte-derived macrophages (MDM). In this study we confirmed that NO regulates cytokine and chemokine release by resting MDM: accumulation of TNF-alpha, IL-12, IL-10, CCL5 (RANTES) and CXCL8 (IL-8) in MDM supernatants was significantly modified by the NO-donor S-nitroso-N-penicillamine (SNAP). Using a protein array, infection of MDM with N. meningitidis was shown to be associated with secretion of a wide range of cytokines and chemokines. To test whether NO metabolism by N. meningitidis modifies release of NO-regulated cytokines, we infected MDM with wild-type organisms and an isogenic norB strain. Resulting expression of the cytokines TNF-alpha and IL-12, and the chemokine CXCL8 was increased and production of the cytokine IL-10 and the chemokine CCL5 was decreased in norB-infected MDM, in comparison to wild-type. Addition of SNAP to cultures infected with wild-type mimicked the effect observed in cultures infected with the norB mutant. In conclusion, NorB-catalysed removal of NO modifies cellular release of NO-regulated cytokines and chemokines.     

Human immunodeficiency virus type 1 (HIV-1)-induced GRO-alpha production stimulates HIV-1 replication in macrophages and T lymphocytes

We examined the early effects of infection by CCR5-using (R5 human immunodeficiency virus [HIV]) and CXCR4-using (X4 HIV) strains of HIV type 1 (HIV-1) on chemokine production by primary human monocyte-derived macrophages (MDM). While R5 HIV, but not X4 HIV, replicated in MDM, we found that the production of the C-X-C chemokine growth-regulated oncogene alpha (GRO-alpha) was markedly stimulated by X4 HIV and, to a much lesser extent, by R5 HIV. HIV-1 gp120 engagement of CXCR4 initiated the stimulation of GRO-alpha production, an effect blocked by antibodies to CXCR4. GRO-alpha then fed back and stimulated HIV-1 replication in both MDM and lymphocytes, and antibodies that neutralize GRO-alpha or CXCR2 (the receptor for GRO-alpha) markedly reduced viral replication in MDM and peripheral blood mononuclear cells. Therefore, activation of MDM by HIV-1 gp120 engagement of CXCR4 initiates an autocrine-paracrine loop that may be important in disease progression after the emergence of X4 HIV.     

M2b monocytes predominated in peripheral blood of severely burned patients

Severely burned patients were shown to be carriers of M2 monocytes, and all of the monocytes isolated from peripheral blood of severely burned patients (19 of 19 patients) were demonstrated as M2b monocytes (IL-12(-)IL-10(+)CCL1(+) monocytes). Low levels of M2a (IL-12(-)IL-10(+)CCL17(+) monocytes) and M2c monocytes (IL-12(-)IL-10(+)CXCL13(+) monocytes) were demonstrated in peripheral blood of severely burned patients (M2a, 2 of 19 patients; M2c, 5 of 19 patients). M2b, M2a, and M2c monocytes were not detected in peripheral blood of healthy donors. However, M2b monocytes appeared when healthy donor monocytes were cultured in media supplemented with burn patient serum (15%). CCL2 was detected in sera of all burn patients, and M2b monocytes were not generated from healthy donor monocytes cultured with media containing 15% burn patient sera that were previously treated with anti-CCL2 mAb. In addition, M2b monocytes were generated from healthy donor monocytes in cultures supplemented with rCCL2. These results indicate that M2b monocytes are predominant in peripheral blood of severely burned patients who are carriers of CCL2 that functions to stimulate monocyte conversion from resident monocytes to M2b monocytes.     

C5a and C5a(desArg) enhance the susceptibility of monocyte-derived macrophages to HIV infection

Mononuclear phagocytes, which include circulating blood monocytes and differentiated tissue macrophages, are believed to play a central role in the sexual transmission of HIV infection. The ability of HIV to productively infect these cells may be influenced by action of exogenous or host-derived substances at the site of viral entry. Given the potent capacities of inflammatory mediators to stimulate anaphylatoxic and immunomodulatory functions in mucosa, the effects of complement-derived anaphylatoxins on the susceptibility of monocytes and monocyte-derived macrophages (MDM) to HIV-1 infection were examined. In our in vitro system, the susceptibility to infection was up to 40 times increased in MDM that had been exposed to C5a or C5a(desArg), but not to C3a or C3a(desArg), for 2 days before adding of virus. By contrast, the treatment with complement anaphylatoxins did not affect HIV replication in fresh monocytes. Stimulatory effect of C5a and its desArg derivative on HIV infection correlated with the increase of TNF-alpha and IL-6 secretion from MDM. All these functional effects of C5a and C5a(desArg) were reversible by treatment of cells with the mAb that functionally blocks C5aR. Taken together, these results indicate that C5a and C5a(desArg) may increase the susceptibility of MDM to HIV infection through stimulation of TNF-alpha and IL-6 secretion from these cells.     

Glycyrrhizin inhibits neutrophil-associated generation of alternatively activated macrophages

Patients with severe burn injuries are extremely susceptible to infection, and the host's antibacterial responses are frequently suppressed by alternatively activated macrophages (M2Mphi), commonly demonstrated in these patients. An immunosuppressive subset of neutrophils (PMN-II), demonstrated in the peripheral blood of thermally injured patients, has been described as an inducer of M2Mphi. In the present studies, the inhibitory effect of glycyrrhizin (GL) on M2Mphi generation stimulated by PMN-II was examined. M2Mphi were generated from resident Mphi (R-Mphi, lower chamber) after cultivation with PMN-II (upper chamber) in a dual-chamber transwell. However, M2Mphi were not generated from R-Mphi when the same transwell cultures were performed in the presence of GL. M2Mphi were not generated from R-Mphi after cultivation with PMN-II previously treated with GL, while R-Mphi previously treated with GL converted to M2Mphi after they were cultured with PMN-II in transwells. Interleukin-10 and CCL2 released from PMN-II were shown to be effector molecules responsible for the generation of M2Mphi. However, these soluble factors were not produced by PMN-II treated with GL. These results indicate that GL inhibits PMN-II-stimulated M2Mphi generation through the inhibition of CCL2/interleukin-10 production by PMN-II.     

Interferon gamma induces actin polymerization, Rac1 activation and down regulates phagocytosis in human monocytic cells

IFNγ is a potent activator and IL-10 a powerful inhibitor of macrophage functions. However, neither all cellular functions are enhanced by IFNγ nor IL-10 inhibits all cellular responses. Thus, FcγRs-mediated phagocytosis in monocyte-derived macrophages (MDM) increases after IL-10 treatment, and decreases after treatment with IFNγ, although both IL-10 and IFNγ up regulate FcγRI expression. In this work we investigated the effect of IFNγ and IL-10 on phagocytic signaling by FcγRs in MDM. Treatment with IFNγ diminished phagocytosis of IgG-opsonized SRBC (IgG-SRBC) while treatment with IL-10 increased it. These opposite effects cannot be attributed to changes in FcγR expression induced by each cytokine. Early biochemical responses mediated by FcγRs were distinctly affected by cytokine treatment. Syk phosphorylation and the rise in [Ca²⁺]_i were higher after IL-10 treatment, whereas IFNγ treatment also increased Syk phosphorylation but had no effect on the rise in [Ca²⁺]_i. IFNγ treatment led to increased basal levels of F-actin and this effect correlated with the decrease in phagocytosis of both IgG-SRBC and non-opsonized Escherichia coli. IL-10 did not alter F-actin basal levels, and enhanced the phagocytosis of E. coli and IgG-SRBC. The level of F-actin reached after IFNγ treatment was not further increased after stimulation with IgG-SRBC or CCL5, whereas MDM treated with IL-10 showed a slightly higher response than control cells to CCL5. IFNγ increased Rac1-GTP levels. Inhibition of PI3K with LY294002 prevented IFNγ-mediated actin polymerization. Our data suggest that IFNγ induces a higher basal level of F-actin and activation of Rac1, affecting the response to stimuli that induce cytoskeleton rearrangement such as phagocytic or chemotactic stimuli.     

Impact of cytokines on replication in the thymus of primary human immunodeficiency virus type 1 isolates from infants

Early infection of the thymus with the human immunodeficiency virus (HIV) may explain the more rapid disease progression among children infected in utero than in children infected intrapartum. Therefore, we analyzed infection of thymocytes in vitro by HIV type 1 primary isolates, obtained at or near birth, from 10 children with different disease outcomes. HIV isolates able to replicate in the thymus and impact thymopoiesis were present in all infants, regardless of the timing of viral transmission and the rate of disease progression. Isolates from newborns utilized CCR5, CXCR4, or both chemokine receptors to enter thymocytes. Viral expression was observed in discrete thymocyte subsets postinfection with HIV isolates using CXCR4 (X4) and isolates using CCR5 (R5), despite the wider distribution of CXCR4 in the thymus. In contrast to previous findings, the X4 primary isolates were not more cytopathic for thymocytes than were the R5 isolates. The cytokines interleukin-2 (IL-2), IL-4, and IL-7 increased HIV replication in the thymus by inducing differentiation and expansion of mature CD27(+) thymocytes expressing CXCR4 or CCR5. IL-2 and IL-4 together increased expression of CXCR4 and CCR5 in this population, whereas IL-4 and IL-7 increased CXCR4 but not CCR5 expression. IL-2 plus IL-4 increased the viral production of all pediatric isolates, but IL-4 and IL-7 had a significantly higher impact on the replication of X4 isolates compared to R5 isolates. Our studies suggest that coreceptor use by HIV primary isolates is important but is not the sole determinant of HIV pathogenesis in the thymus.     

Role for IL-4 nonproducing NKT cells in CC-chemokine ligand 2-induced Th2 cell generation

NKT cells from C57Bl/6 mice are known to be the initial cellular source of IL-4 that acts as a trigger for Th2 cell differentiation. CC-chemokine ligand 2 (CCL2) has been described as an initial stimulator of IL-4 production by these cells; however, IL-4 was not produced by NKT cells from BALB/c mice even when Th2 cell responses were established in these mice. In this study, we found a new pathway for CCL2-associated Th2 cell generation in BALB/c mice. Splenic T cells from BALB/c mice produced IL-4 in response to CCL2 stimulation. However, IL-4 production was not seen in cultures of splenic T cells from CD1-/- mice (BALB/c origin), whereas, in the presence of CCL2, splenic T cells from CD1-/- mice produced IL-4 when NKT cells from wild-type mice were added. CCL2 induced IL-4 in cultures of NKT cells cocultured with naive T cells, but IL-4 was not produced by these cells cultured separately with CCL2. Interestingly, IL-4 was produced by naive T cells cocultured with NKT cells that were previously treated with CCL2 (CCL2-NKT cells). In addition, IL-4 was produced by naive T cells supplemented with a culture supernatant of CCL2-NKT cells. These results indicate that, through the production of a soluble factor(s) other than IL-4, NKT cells play a role in the CCL2-associated generation of Th2 cells.     

CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages

Classically activated macrophages (CAMphi) have been described as a major effector cell on the host's innate immunities. However, CAMphi are not generated in immunocompromised hosts whose alternatively activated macrophages (AAMphi) predominate. In this study, the mechanism by which AAMphi suppress the ability of resident macrophages (RMphi) to generate CAMphi was investigated. AAMphi were isolated from peritoneal exudates of mice 2 days after third-degree thermal injuries affecting 15% total body surface area. CAMphi were generated from RMphi (peritoneal Mphi from normal mice) through stimulation with CpG DNA, a typical CAMphi inducer. RMphi did not polarize to CAMphi when they were cultured with AAMphi in a dual-chamber Transwell even when supplemented with CpG DNA. In addition, RMphi stimulated with CpG DNA did not convert to CAMphi when they were cultured with the culture fluids of AAMphi (AAMphi Culture-Sup). AAMphi Culture-Sup contained IL-6, IL-10, CCL17, PGE(2), and TGF-beta. Among these, CCL17 and IL-10 inhibited CAMphi generation. The ability of AAMphi Culture-Sup to inhibit CAMphi generation was eliminated when the Culture-Sup was treated with a mixture of mAbs directed against CCL17 and IL-10. These results indicate that CCL17 and IL-10 released from AAMphi inhibit CAMphi generation from RMphi stimulated with CpG DNA.     

HIV infection of placental macrophages: effect on the secretion of HIV stimulatory cytokines.

Vertical transmission of HIV-1 can occur at three different stages: during gestation, delivery and breast feeding. To determine the role of cytokines in vertical transmission of HIV during gestation, we studied the secretion of IL-1beta, TNF-alpha and IL-6 from in vitro infected and Mock-infected placental macrophages (Hofbauer cells) in comparison to blood monocyte derived macrophages (MDM). Hofbauer cells stimulated with lipopolysaccharide (LPS) secreted lower levels of HIV stimulatory cytokines (6-8 ng/ml) in the supernatants than MDM (26 ng/ml, p<0.005). Cytokine levels in MDM decreased upon HIV infection to 7 ng/ml. IL-6 was the major cytokine produced after LPS stimulation by the two cell populations (p<0.005), being MDM the major cytokine producer. In vitro infection studies with a M-tropic virus (HIV-BaL) indicated that MDM were 10x more susceptible to HIV than placental macrophages (p=0.001). Our results indicate that although macrophages from term placenta secrete lower amount of HIV stimulatory cytokines than MDM, there was no correlation between the levels of cytokines and HIV production by both cells.     

Persistent CCR5 utilization and enhanced macrophage tropism by primary blood human immunodeficiency virus type 1 isolates from advanced stages of disease and comparison to tissue-derived isolates

Viral phenotype, tropism, coreceptor usage, and envelope gene diversity were examined in blood isolates collected from 27 individuals at different stages of human immunodeficiency virus type 1 (HIV-1) disease and tissue derived isolates from 10 individuals with AIDS. The majority (89%) of blood and all tissue HIV-1 isolates from all stages of infection were non-syncytium inducing and macrophage (M) tropic. Tropism and productive infection by HIV isolates in both monocytes and monocyte-derived macrophages (MDM) increased in advanced disease (HIV tropism for monocytes, 1 of 6 from categories I and II versus 11 of 21 [P = 0.05] from category IV and II [CD4 < 250]; and high-level replication in MDM, 1 of 6 from categories I and II versus 16 of 21 from categories IV and II [P = 0. 015]). There was a high level of replication of blood and tissue isolates in T lymphocytes without restriction at any stage. Overall, the level of replication in MDM was 5- to 10-fold greater than in monocytes, with restriction in the latter occurring mainly at entry and later stages of replication. Only three blood isolates were identified as syncytium inducing, and all had a dualtropic phenotype. There was a significant increase of HIV envelope gene diversity, as shown by a heteroduplex mobility assay, in advanced disease; this may partly underlie the increase of HIV replication in MDM. Unlike blood isolates (even those from patients with advanced disease), tissue isolates displayed greater similarities (90%) in productive infection between MDM and monocytes. The majority (87%) of all isolates, including those from patients with advanced disease, used CCR5, and only 5 of 37 isolates showed expanded coreceptor usage. These results indicate that in the late stage of disease with increasing viral load and diversity, CCR5 utilization and M-tropism persist in blood and tissue and the replicative ability in macrophages increases. This suggests that these characteristics are advantageous to HIV and are important to disease progression.     

Uncoupled anti-HIV and immune-enhancing effects when combining IFN-alpha and IL-7

Cytokine-based therapies have been examined for purging viral reservoirs and immunomodulation in HIV infection. However, single cytokines did not result in either HIV eradication or an efficient HIV-specific immune response. We hypothesize that cytokines with distinct biologic effects need to be combined for immunotherapy of HIV infection. In this study, we investigated the anti-HIV activity and immune-enhancing effects of the combination of IFN-alpha and IL-7. In human lymphocyte aggregate cultures infected ex vivo with the X4 HIV strain NL4-3, IFN-alpha/IL-7 potently inhibited HIV replication and preserved CD4(+) T cells, probably by up-regulating Bcl-2. IFN-alpha/IL-7 also strongly inhibited R5 HIV replication. Furthermore, in allogeneic MLRs, IFN-alpha/IL-7 increased T cell proliferation and IFN-gamma production. IFN-alpha alone also had strong anti-HIV activity, but neither preserved CD4(+) T cells nor increased T cell responses in MLRs. IL-7 alone maintained T cells and enhanced T cell activation in MLRs, but only moderately inhibited or increased HIV replication. Thus, coadministration of IFN-alpha/IL-7 combines the potent anti-HIV activity of IFN-alpha with the beneficial effects of IL-7 on T cell survival and function. We speculate that IFN-alpha will block viral replication, activate APCs, and up-regulate MHC molecules, thus allowing IL-7 to display its effects for generating an efficient immune response. In this scenario, the known reactivation of latent HIV by IL-7 may be advantageous.     

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.     

Cross-linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immunosuppressive program

Immature monocyte-derived dendritic cells (DC) strongly express the endocytic mannose receptor (MR). Addition of a specific anti-MR mAb (clone PAM-1) for 24 h to cultures of immature DC induced phenotypical and functional maturation of the cells, assessed as up-regulation of costimulatory molecules and CD83, and chemotactic response to CCL19. A different isotype-matched anti-MR mAb (clone 19.2) had no significant effect. Engagement of MR with mAb PAM-1 induced the production of the anti-inflammatory cytokines IL-10, IL-1R antagonist, and of the nonsignaling IL-1R type II. In contrast IL-1beta, TNF, and IL-12 were not produced. PAM-1-treated DC were unable to polarize Th1 effector cells and did not secrete the chemokines CXCL10 and CCL19; in turn, they produced large amounts of CCL22 and CCL17, thus favoring the amplification of Th2 circuits. T cells cocultured with PAM-1-matured DC initially proliferated but later became anergic and behaved as suppressor/regulatory cells. Natural ligands binding to MR had differential effects. MUC III (a partially purified mucin), biglycan (a purified complex proteoglycan), and mannosylated lipoarabinomannan from Mycobacterium tuberculosis affected cytokine production with high IL-10, IL-1R antagonist, IL-1R type II, and inhibition of IL-12. In contrast, mannan, dextran, and thyroglobulin had no significant effect. In conclusion, the appropriate engagement of the MR by mAb PAM-1 and selected natural ligands elicit a secretory program in mono-derived DC characterized by a distinct profile of cytokines/chemokines with the ability to dampen inflammation and to inhibit the generation of Th1-polarized immune responses.     

Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.