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In the present study we demonstrate that both X4- and R5-tropic HIV-1 strains are able to infect the human epithelial cell line HT-29. Infection was enhanced 2-fold when HIV was added to semen before contact with the cell cultures. The enhancing effect of semen was complement dependent, as evidenced by blockage of generation of C3a/C3a(desArg) in semen by heat or EDTA treatment of semen and suppression of semen-dependent enhancement with mAbs directed to complement receptor type 3 (CD11b/CD18) and soluble CD16. Infection of HT-29 cells was assessed by the release of p24 Ag in cultures and semiquantitative PCR of the HIV-1 pol gene. Inhibition of infection of HT-29 by stromal cell-derived factor 1 was decreased in the case of semen-opsonized X4- and R5-tropic virus compared with unopsonized virus. In contrast, inhibition of infection by RANTES was increased for opsonized X4-tropic HIV-1 compared with unopsonized virus. Taken together these observations indicate that activation of complement in semen may play an enhancing role in mucosal transmission of HIV-1 by facilitating infection of epithelial cells and/or enhancing infection of complement receptor-expressing target cells in the mucosa.

In the present study, we demonstrate that normal human IgG for therapeutic use (i.v. Ig) contains natural Abs directed against the CCR5 coreceptor for HIV-1. Abs to CCR5 were isolated from i.v. Ig using an affinity matrix consisting of a synthetic peptide corresponding to the N-terminus of CCR5 coupled to Sepharose. Natural anti-CCR5 Abs inhibited the binding of RANTES to macrophages, demonstrating their interaction with the coreceptor of R5-tropic HIV-1. Affinity-purified anti-CCR5 Ig further inhibited infection of lymphocytes and monocytes/macrophages with primary and laboratory-adapted strains of HIV-1, but did not inhibit infection with X4-tropic HIV. Our results suggest that anti-CCR5 Abs from healthy immunocompetent donors may be suitable for development of novel passive immunotherapy regimens in specific clinical settings in HIV infection.

We investigated the effect of IL-10 on replication of primary CXCR4-dependent (X4) HIV-1 strains by monocyte-derived dendritic cells (DCs) and macrophages (M Phis). M Phis efficiently replicated CXCR4-dependent HIV-1 (X4 HIV-1) strains NDK and VN44, whereas low levels of p24 were detected in supernatants of infected DCs. IL-10 significantly increased X4 HIV-1 replication by DCs but blocked viral production by M Phis as determined by p24 levels and semiquantitative nested PCR. IL-10 up-regulated CXCR4 mRNA and protein expression on DCs and M Phis, suggesting that IL-10 enhances virus entry in DCs but blocks an entry and/or postentry step in M Phis. The effect of IL-10 on the ability of DCs and M Phis to transmit virus to autologous CD4(+) T lymphocytes was investigated in coculture experiments. DCs exhibited a greater ability than did M Phis to transmit a vigorous infection to CD4(+) T cells despite their very low replication capacity. IL-10 had no effect on HIV-1 replication in DC:T cell cocultures but markedly decreased viral production in M Phi:T cell cocultures. These results demonstrate that IL-10 has opposite effects on the replication of primary X4 HIV-1 strains by DCs and M Phis. IL-10 increases X4-HIV-1 replication in DCs but does not alter their capacity to transmit virus to CD4(+) T lymphocytes. These findings suggest that increased levels of IL-10 observed in HIV-1-infected patients with disease progression may favor the replication of X4 HIV-1 strains in vivo.

We demonstrate that soluble CD16 (sCD16; soluble Fc gamma RIII), a natural ligand of CR3, inhibits the infection of monocytes by primary R5 HIV-1 strain opsonized with serum of seronegative individuals. Inhibition of monocyte infection by sCD16 was similar to that observed with anti-CR3 mAbs, indicating that opsonized HIV may use a CR3-dependent pathway for entry in monocytic cells. Cultured human monocytes express both CR3 (CD11b/CD18) and CCR5 receptors. RANTES, the natural ligand of CCR5, inhibited infection of monocytes with unopsonized HIV particles and partially that of monocytes infected with HIV particles opsonized with complement-derived fragments. Although HIV-infected monocytes from homozygous CCR5 Delta 32/Delta 32 (CCR5(-/-)) individuals produce low levels of p24, cells infected with opsonized particles produced higher levels of p24 than cells infected with unopsonized particles. Our results thus suggest that CR3 may represent an alternative coreceptor to CCR5 of opsonized primary R5 virus entry into monocytes/macrophages. We also observed that the concentration of sCD16 is greatly decreased in sera of HIV-infected patients with low lymphocyte CD4(+) counts. Taken together, our findings suggest that sCD16, present in plasma, may play an important role in controlling HIV-1 spread.

To investigate the responses of dendritic cells (DC) during Gram-negative infections, we analyzed the effects of graded doses of LPS on the cytokine profile, phenotype, and allostimulatory potential of human DC generated by culturing plastic-adherent PBMC in presence of IL-4 and granulocyte-macrophage-CSF. First, we found that LPS stimulates the production of high levels of TNF-alpha, IL-6, IL-8, IL-12 by DC and up-regulates their expression of HLA-DR, B7-1, B7-2, and CD40. The effects of LPS were dose dependent, with a significant stimulatory effect already observed at a concentration of 0.1 ng/ml and a plateau being reached at 10 ng/ml. These phenotypic changes correlated with increased allostimulatory properties of LPS-activated DC because DC treated with LPS were significantly more efficient than untreated DC in eliciting IL-2 and IFN-gamma synthesis by alloreactive T cells and stimulating their proliferation. Experiments using neutralizing anti-IL-12 mAb indicated that LPS-induced IL-12 is responsible for the increased production of IFN-gamma but not for the increased proliferation during MLR. Finally, we observed that the DC responses to low levels of LPS (1 ng/ml) were dramatically inhibited by a blocking anti-CD14 mAb, although DC do not express CD14 molecules on their membrane. Experiments using serum depleted of soluble CD14 (sCD14) and sCD14 either purified from human serum or in recombinant form further established that DC respond to LPS via a soluble CD14-dependent pathway.

Several studies have pointed to a link between immune and endocrine systems, including a regulatory function of GH on monocyte activation. The present study demonstrates that human THP-1 promonocytic cells, engineered by gene transfer to constitutively produce human growth hormone (hGH), secreted depressed amounts of TNF-alpha in response to challenge by LPS. The effect of GH appears to occur in an autocrine fashion, since the inhibitory effect on TNF-alpha secretion by constitutive GH production could be abolished in the presence of anti-hGH mAb. The GH-induced inhibitory effect was also observed using normal human monocytes and monocyte-derived macrophages. Inhibition of TNF-alpha production by THP-1-hGH-transfected cells cultured in the presence of LPS is dependent on a selective pathway, since no inhibition of TNF-alpha production was observed when cells were cultured in the presence of PMA. Inhibition of TNF-alpha secretion by LPS-stimulated THP-1-hGH cells was associated with a decrease in nuclear translocation of nuclear factor-kappaB. The capacity of GH to inhibit LPS-induced TNF-alpha production by monocytes without altering other pathways leading to TNF-alpha production may be of potential relevance in septic shock, since GH is available for clinical use.

Monocyte/macrophages may harbor HIV in a nonproductive fashion for prolonged periods of time. Viral gene expression may be reactivated by stimulation of the cells with LPS or cytokines such as TNF-alpha in vitro. The effect of LPS and TNF-alpha is mediated by their ability to induce nuclear translocation of the DNA-binding heterodimer NF-kappa B (p50/p65), which binds to a specific sequence in the HIV-long terminal repeat. The present study demonstrates that triggering of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) enhances viral replication in HIV-infected human monocytic cells. Monocytic cell lines and normal peripheral blood monocytes were infected with HIV-1 in vitro and cultured in the presence or absence of F(ab')2 fragments of monoclonal anti-CR1 or anti-CR3 Abs or with C3 fragments. Stimulation of CR1 or CR3 induces a two- to fourfold increase in the amount of cell-associated and released p24 Ag in cell cultures that was equivalent to that observed in control cultures triggered with LPS. We further observed that stimulation of CR1 or CR3 induces the nuclear translocation of NF-kappa B p50/p65 in infected cells. Translocation of NF-kappa B p50/p65 was also observed following stimulation of CR1 or CR3 of uninfected peripheral blood monocytes from HIV-seronegative donors. The amount of protein translocated was similar to that observed when cells were stimulated with rhTNF-alpha. TNF-alpha did not mediate the translocation of NF-kappa B p50/p65 induced by triggering of complement receptors. Taken together, these observations suggest that HIV gene expression may be activated in infected monocytes through interaction of the cells with complement-opsonized particles and that enhanced viral replication is associated with C3 receptor-mediated nuclear translocation of the NF-kappa B complex.

The nature of the binding sites for LPS on human monocytes was investigated using [3H] labeled intact LPS from Neisseria meningitidis and from Salmonella minnesota R7, and the [3H] labeled purified inner core region (PS-OMe) of S.m. R7 LPS. In the presence of serum, intact LPS from enterobacterial and nonenterobacterial strains bound to monocytes in a dose-dependent, saturable, and displaceable fashion. N.m. LPS and LPS from the enterobacterial strain of Escherichia coli 0111-B4 bound to the same sites on monocytes as assessed in competitive binding experiments. Specific binding of intact LPS to monocytes occurred through the CD14 molecule as shown by the ability of mAb and of F(ab')2 fragments of mAb directed against specific epitopes of CD14 to inhibit the binding of [3H]-LPS to cells and by the lack of binding of intact LPS to CD14-deficient cells from patients with paroxysmal nocturnal hemoglobinuria. Specific binding of LPS to monocytes was not mediated by the CD11/CD18 complex because mAb to the alpha and beta chains of the Leu-CAM molecules did not alter the binding of LPS to cells and because LPS did not inhibit the binding of labeled mAb to monocytes. [3H]-PS-OMe also bound in a dose-dependent and displaceable fashion to monocytes involving an unidentified, non-CD14, binding site on the cells. Binding of LPS to monocytes also involved nonsaturable binding sites for hydrophobic structures of LPS as evidenced in binding experiments performed in the absence of serum. These observations indicate that intact LPS may interact with the monocyte membrane in at least three ways including serum-dependent binding to CD14 and to a lectin-like receptor, and serum-independent hydrophobic interactions.

Soluble polymerized human C3b and C3b bound to Sepharose induced the production of cell-associated IL-1 and the extracellular release of IL-1 activity from cultured human adherent monocytes. Cultures were performed in serum-free conditions in the presence of polymyxin B and of the PG synthesis inhibitor indomethacin. Induction of intracellular IL-1 activity was associated with that of IL-1 alpha and IL-1 beta Ag. Biologically active released IL-1 was IL-1 beta. Monomeric C3b induced cell-associated IL-1 but not the release of IL-1 from monocytes. IL-1 production was also induced by stimulation of CR1 on adherent monocytes with anti-C3b receptor (CR1) antibody. The ability of multivalent C3b to induce IL-1 production could be dissociated from that of possibly contaminating LPS by several criteria including: the lack of detectable LPS in purified C3b; functional inhibition of LPS in cultures with polymyxin B; the lack of induction of extracellular IL-1 by C3b monomer; a correlation between the expression of CR1 on monocytes and the monocytic response to C3b; a dissociation between the expression of CR1, and the expression of LPS binding sites and the IL-1 response of monocytes to endotoxin. Induction of IL-1 represents a novel pathway by which C3b-CR1 interactions may modulate the immune response.

Binding studies of human IgG proteins to murine P388D1 cells indicated that they bind to an apparently homogeneous Fc receptor population. The association constant was 0.89 x 10(6)M-1 at 22 degrees C and was comparable to the binding affinities of homologous murine IgG2a and IgG2b. The number of receptor sites was found to be approximately 6 x 10(5)/cell. Fc gamma 1 and Fc gamma 3 fragments bound with an affinity comparable to that of the parent proteins. The P388D1 receptors could discriminate between the human IgG subclasses; the relative cytophilic activity was IgG3 greater than IgG1 greater than IgG4 and IgG2 was devoid of binding activity. Fragments corresponding to the C gamma 2 and C gamma 3 domains of human IgG1 were both unable to bind to the P388D1 receptors either alone or in equimolar combination. This suggests that the cytophilic site may be formed cooperatively by interaction between the two domains. The integrity of the hinge region appeared to be essential for full expression of cytophilic activity since reduction of the hinge-region disulfides in both human IgG1 and its Fc fragment markedly decreased their binding affinity. In addition, a mutant IgG1 molecule lacking the hinge region was significantly less cytophilic than its normal counterpart.

The Bordetella pertussis endotoxin, labeled with tritium ((3H)-LPS), bound irreversibly and nonspecifically to rabbit lung macrophages, but bound reversibly and specifically to both resident and elicited rabbit peritoneal macrophages. The specific binding capacity of the macrophages was saturated with about 3 X 10(4) LPS molecules per cell. The binding was inhibited with the homologous unlabeled endotoxin, but not at all with endotoxin from Proteus mirabilis, thus assessing ligand specificity. Endotoxins from other bacteria gave intermediate inhibition value. Binding of tritium-labeled pertussis endotoxin was significantly inhibited by one of the two polysaccharides (PS-1) present in this endotoxin, but neither the other polysaccharide (PS-2) nor the Lipid A fragment exhibited such activity. These results strongly suggest the presence of a lectin-like receptor for LPS on the membrane of rabbit peritoneal macrophages.

Purified human C3a(C3adesArg) induced dose-dependent generation of intracellular IL 1 activity and release of IL 1 in cultures of human mononuclear adherent cells in serum-free conditions. Concentrations of C3a(C3adesArg) of 10(-8) M and 6 hr of culture were sufficient to induce production of cell-associated IL 1, as detected in monocyte lysates. Ten- to 100-fold higher concentrations of C3a(C3adesArg) and 24 hr of culture were required for induction of IL 1 release. Release of IL 1 induced by suboptimal amounts of C3a(C3adesArg) was greatly enhanced by the addition of indomethacin to the culture medium. Contamination with C5a of the C3a(C3adesArg) preparation did not account for C3a(C3adesArg)-induced IL 1 production. Induction of IL 1 activity by C3a(C3adesArg) was not due to contaminating LPS, as indicated by the following observations: the amount of contaminating LPS in C3a(C3adesArg) was below that which could induce IL 1 release from human monocytes in serum-free conditions; induction of IL 1 by C3a(C3adesArg) was not suppressed by polymyxin B; kinetics of IL 1 production and release in the presence of C3a(C3adesArg) differed from those observed in the presence of LPS; and sialated gangliosides, which inhibit IL 1 release induced by LPS, had no effect on the induction of IL 1 by C3a(C3adesArg). The C3a(C3adesArg) preparation used in this study mostly contained the desArg derivative, suggesting that, in contrast with the requirement for an intact C-terminal arginyl residue for the spasmogenic activity of C3a, both C3a and its C3adesArg derivative may interact with receptors on human monocytes. By inducing IL 1 production and release, C3a(C3adesArg) may contribute to the generation of the inflammatory process and the regulation of the immune response.

The response to anti-allotype (anti-Ab4), Nocardia Water Soluble Mitogen (NWSM), pneumococcal polysaccharide type III (SSS III), and human Fc fragments of various purified and unfractionated rabbit spleen cell populations was determined in terms of 3H-thymidine up-take. B cells were isolated either from untreated suspensions of spleen cells or from suspensions from which adherent and phagocytic cells were removed. The purification factor was greater than the enhancement of 3H-thymidine uptake by anti-Ab4, NWSM, and SSS III as compared with the response of unfractionated spleen cells. It thus appears that a helper cell was involved: the mitogen response of purified B cells was enhanced by the addition of T cells. B subpopulations were separated by sedimentation or by rosetting, which allowed us to separate Fcgamma receptor-bearing cells from cells that did not possess this receptor. There were differences between cells responding to B mitogens not only in sedimentation velocity but also in the absolute number of cells. B cells bearing the Fcgamma receptor were less responsive to anti-Ab4 and more responsive to SSS III, NWSM, and human Fc than were B cells lacking the Fcgamma receptor.