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SDZ 280.636, a nontoxic diacyl glycerol derivative of muramyl dipeptide (MDP), a component of the inner bacterial cell wall, which is suitable for use in man, suppressed hapten specific IgE antibody forming cell (AFC) responses in spleen, serum levels of hapten specific IgE and hapten specific immediate hypersensitivity (i.h.) responses in skin, when fed to mice at the peak of a hapten specific IgE AFC response. In addition, serum levels of IL-6 appeared increased while IFN gamma was decreased. To induce these IgE responses, BALB/c mice were injected i.p. with BPO-KLH (benzylpenicilloyl-keyhole limpet hemocyanin) (10 micrograms) in aluminum hydroxide gel (alum) on days 0, 21 and 42. Mice were fed (gavage) with either MDP or SDZ 280.636 (1.0 or 10 mg/kg) on day 44, or on days 44, 46 and 48, and killed on days 46 or 50. Numbers of BPO specific AFC in spleen, and serum levels of BPO specific immunoglobulins (IgG1, IgE and IgA) were determined (ELISPOT assay, ELISA). In addition, BPO specific IH responses were measured in these animals. Mice were injected in the right pinna with BPO-BSA (0.1 microgram) and in the left pinna with an equal volume of saline (0.05 ml). At 2 hr, pinnae were measured using a micrometer caliper. We found that 1 feeding with either MDP or SDZ 280.636 abrogated IgE AFC responses and dramatically suppressed serum levels of IgE, both in isotype specific fashion, and suppressed IH responses (> 50%). 3 feedings with SDZ 280.636 also abrogated IgE AFC responses and further decreased serum levels of IgE. In contrast to SDZ 280.636, 3 treatments with MDP had opposite effects in that IgE AFC responses and serum levels of IgE dramatically increased. A single treatment with SDZ 280.636 appeared to increase serum levels of IL-6 up to three fold, while IFN gamma levels decreased. Our data suggest that SDZ 280.636 may be useful in the therapeutic and prophylactic management of human atopic disease such as allergic rhinitis, asthma, and other atopic diseases.

The ability of IL-6 or IFN alpha or antibodies to these cytokines to regulate serum levels of hapten specific immunoglobulins (IgM, IgG1, IgE, IgA) was studied in BPO-KLH (benzylpenicilloyl-keyhole limpet hemocyanin) sensitized BALB/c mice at the peak of a hapten specific IgE antibody forming cell (AFC) response. To induce peak IgE responses, mice were injected intraperitonealy (i.p.) with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on days 0, 21, and 42. On day 44, mice were injected s.c. with IL-6 (100-1000 U), IFN alpha (1000-10,000 U), anti-IL-6 (100-1000 neutralizing units [NU]), or anti-IFN alpha (1000-10,000 NU). On day 46, levels of BPO specific IgM, IgG1, IgE and IgA in serum were determined (ELISA). Data are expressed as micrograms/ml. IL-6 suppressed BPO specific IgE in serum in isotype specific fashion (to > 90%), increasing IgA (approximately 3 fold), and leaving IgM and IgG1 unchanged. Since removal of endogenous IL-6 with anti-IL-6 increased serum IgE, and suppressed IgG1 (approximately 50%), with IgM and IgA unchanged, this suggests that IL-6 is an isotype specific suppressor of peak IgE responses and as such may be useful in the therapeutic management of atopic disease. IFN alpha treatment increased serum IgE levels (60%), and potentiated IgA responses (> 30 fold), with IgM and IgG1 unchanged. Since removal of endogenous IFN alpha with anti-IFN alpha decreased IgE levels (approximately 50%), increasing IgA, with IgM and IgG1 unchanged, this suggests a role for IFN alpha as an isotype specific helper of peak IgE responses and in maintenance of IgA responses.

The ability of subcutaneously (s.c.) injected cytokines (IL-4, IL-5, IL-6, IFN alpha, IFN gamma, GMCSF) to regulate the induction of hapten-specific immediate hypersensitivity (IH) responses was studied in BPO-KLH (benzylpenicilloyl-keyhole limpet hemocyanin) sensitized BALB/c mice at the peak of a hapten-specific IgE antibody forming cell (AFC) response. To induce IH responses, mice were injected in the right pinna with either BPO-BSA (benzylpenicilloyl-bovine serum albumin), BPO-KLH (0.01-1.0 micrograms/ml) or mcAb anti-IgE (0.001 - 1.0 micrograms/ml); and in the left pinna with an equal volume of saline (0.05 ml). Pinnae were measured 5 min to 4 hr later using a micrometer caliper. Treatment of mice with IL-4 or IFN gamma dramatically suppressed the induction of IH responses in dose dependent fashion. In contrast, treatment of mice with IL-6 and IFN alpha increased these responses in dose dependent fashion, while GMCSF and IL-5 had no effect. The suppression obtained with IL-4 and IFN gamma, and the increases seen with IL-6 and IFN alpha, were transient since these cytokines, as well as GMCSF and IL-5, had no effect on IH responses elicited 21 days after the peak of BPO-specific IgE AFC responses. The data suggest that cytokine mediated effects on IH responses occur via changes in serum levels of BPO-specific IgG1 or IgE, through direct or indirect effects of cytokines on mast cells or other cell types, or by affecting the ability of BPO-specific homocytotropic antibodies to bind to mast cell surfaces.

Plasmin activity induced by different concentrations of added urokinase (UK) in serum from 20 patients with AIDS (P) and 10 healthy control (HC) subjects was measured using the fibrin plate assay. 125I-fibrin coated 24 well plates were exposed to UK (0.34-6.8 ng/ml) or to trypsin (T) (2.5 micrograms/ml) in the presence of serum (10%) from P or HC. Control wells were exposed to either T or tris buffer (pH 8.1) alone. Volumes were adjusted to 1 ml with buffer and after 1 hr at 37 degrees C, radioactivity (cpm) released into the medium was determined using a gamma counter. Data are expressed as % plasmin activity or as % inhibition of plasmin activity. All sera from HC totally abrogated (greater than 98%) the plasmin activating ability of UK (1.7 ng/ml). In contrast, sera from approximately 50% of P were less able to inhibit plasmin activation (to 26%). The inability of P sera to inhibit plasmin activation was specific since P and HC sera were equally capable of inhibiting T. Mixing experiments using P and HC sera demonstrated that P sera did not block the ability of HC sera to inhibit plasmin activation. The inhibitory activity of HC and active P sera eluted in the void volume of a spehadex G100 column (MW greater than 100,000 daltons) and is acid sensitive, however HC sera also contains an acid stable inhibitor(s) of plasmin activation not detected in P sera. These data suggest dysregulation of UK dependent proteolysis may be associated with AIDS.