ABSTRACT
BACKGROUND: Interleukin-12 (IL-12) is a cytokine that promotes type-1 helper T-cell responses and may have therapeutic utility in the treatment of cancer, asthma, and a variety of infectious diseases. METHODS: In a phase I trial, recombinant human IL-12 (rHuIL-12) was administered subcutaneously once a week at a fixed dose of 0.1 to 1.0 microg/kg to 24 patients with renal cell carcinoma. A similar study was later performed in mice to evaluate the mechanism of down-regulation of pharmacokinetic-pharmacodynamic response observed in patients with cancer. RESULTS: Adverse events, serum IL-12 levels, and serum levels of interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) produced in response to IL- 12 were all maximum in the week after the first dose of rHuIL-12 and decreased after long-term administration. Similar to these results, repetitive subcutaneous administration of recombinant mouse IL-12 (rMoIL-12) to normal mice led to down-regulation of serum levels of IL-12 and IFN-gamma measured 5 hours after rMoIL-12 injection. Down-regulation of IL-12 serum levels was inversely correlated with the up-regulation of IL-12 receptor expression and may be the result of increased clearance of rMoIL-12 from serum by binding to lymphoid cells expressing increased amounts of IL-12 receptor. The down-regulation of serum IFN-gamma levels correlated with decreased IFN-gamma messenger ribonucleic acid expression and may result from feedback inhibition of IL-12 signaling or from a more specific inhibition of IFN-gamma synthesis. CONCLUSION: Administration of rHuIL-12 in fixed weekly doses resulted in decreased serum levels of IL-12 and of IFN-gamma, a secondary cytokine believed to be critical to response of IL-12. A better understanding of the complex regulation of the pharmacokinetic-pharmacodynamic response to IL-12 should facilitate the development of more effective dosing regimens for its use in the clinic.
Subject(s)
Adjuvants, Immunologic/pharmacology , Carcinoma, Renal Cell/drug therapy , Gene Expression Regulation, Neoplastic , Interleukin-12/pharmacology , Kidney Neoplasms/drug therapy , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/adverse effects , Adjuvants, Immunologic/pharmacokinetics , Adult , Aged , Animals , Carcinoma, Renal Cell/blood , Down-Regulation , Drug Administration Schedule , Female , Humans , Interferon-gamma/blood , Interferon-gamma/genetics , Interleukin-12/administration & dosage , Interleukin-12/adverse effects , Interleukin-12/blood , Interleukin-12/pharmacokinetics , Kidney Neoplasms/blood , Male , Mice , Mice, Inbred C57BL , Middle Aged , RNA, Messenger/analysis , Recombinant Proteins/pharmacology , beta 2-Microglobulin/metabolismABSTRACT
Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.
Subject(s)
Interleukin-12/antagonists & inhibitors , Animals , Antibodies, Monoclonal/immunology , CHO Cells , Cricetinae , Cytotoxicity, Immunologic , Hypersensitivity, Delayed/immunology , Interferon-gamma/biosynthesis , Interleukin-12/chemistry , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Weight , Receptors, Interleukin/metabolism , Receptors, Interleukin-12 , Recombinant Proteins , Structure-Activity Relationship , Th1 Cells/immunologyABSTRACT
IL-12 is a cytokine that can exert regulatory effects on T and NK cells and promote Th1 responses. To delineate further the physiologic role of IL-12 in immunity, mice deficient for this cytokine were generated. IL-12-deficient mice were impaired but not completely lacking in the ability to produce IFN gamma following endotoxin administration and to mount a Th1 response in vivo, as measured by antigen-induced IFN gamma secretion by immune lymph node cells in vitro. In contrast, secretion of IL-4 was enhanced, while proliferation and secretion of IL-2 and IL-10 were normal following antigen stimulation. DTH responses were significantly reduced in IL-12-deficient mice, but no defect in allogeneic CTL responses was observed. These results indicate that IL-12 plays an essential role in regulating IFN gamma production and in facilitating normal DTH responses. However, other phenomena associated with Th1 responses and cell-mediated immunity, i.e., IL-2 secretion and CTL generation, were not compromised in the absence of IL-12.
Subject(s)
Interferon-gamma/biosynthesis , Interleukin-12/deficiency , Interleukin-12/genetics , Lymphokines/biosynthesis , Mice, Mutant Strains/immunology , Animals , Base Sequence , Genetic Vectors/immunology , Killer Cells, Natural/immunology , Lymphocyte Activation/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Mutant Strains/growth & development , Mice, Mutant Strains/metabolism , Molecular Sequence Data , Th1 Cells/immunology , Th1 Cells/metabolismABSTRACT
We previously described the cloning of a cDNA encoding an interleukin-12 receptor (IL-12R) subunit, designated beta, that bound IL-12 with low affinity when expressed in COS cells. We now report that a pair of monoclonal antibodies (mAb), 2B10 and 2.4E6, directed against different epitopes on the IL-12R beta chain, when used in combination, strongly inhibited IL-12-induced proliferation of activated T cells, IL-12-induced secretion of interferon-gamma by resting peripheral blood mononuclear cells (PBMC), and IL-12-mediated lymphokine-activated killer cell activation. The mAb had no effect on lymphoblast proliferation induced by IL-2, -4, or -7. Thus, the IL-12R beta chain appears to be an essential component of the functional IL-12R on both T and natural killer (NK) cells. We previously observed that high affinity IL-12R were expressed on activated T and NK cells, but not B cells. Studies using flow cytometry and reverse transcription-polymerase chain reaction analysis showed that IL-12R beta chain was expressed on several human T, NK, and (surprisingly) B cell lines, but not on non-lymphohematopoietic cell lines. The Kit225/K6 (T cell) and SKW6.4 (B cell) lines were found to express the greatest amounts of IL-12R beta chain (800-2500 sites/cell); however, Kit225/K6 but not SKW6.4 cells bound IL-12. Similar to SKW6.4 B cells, activated tonsillar B lymphocytes expressed IL-12R beta chain but, consistent with previous results, did not display detectable IL-12 binding. Likewise, up to 72% of resting PBMC from normal volunteer donors expressed IL-12R beta, but did not bind measurable amounts of IL-12. These results indicate that expression of IL-12R beta is essential, but not sufficient, for expression of functional IL-12R. We speculate that expression of functional, high-affinity IL-12R may require the presence of a second subunit that is more restricted in its expression than IL-12R beta.
Subject(s)
Interleukin-12/metabolism , Leukocytes, Mononuclear/chemistry , Receptors, Interleukin/metabolism , Receptors, Interleukin/physiology , Antibodies, Monoclonal/pharmacology , B-Lymphocytes/immunology , Base Sequence , Binding Sites/immunology , Binding, Competitive/immunology , Cell Line , Cell Separation , Humans , Interleukin-12/chemistry , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Molecular Sequence Data , Palatine Tonsil/cytology , Receptors, Interleukin/immunology , Receptors, Interleukin-12ABSTRACT
Although IL-12 has been reported to synergize with c-kit ligand (KL) in promoting hematopoietic stem cell proliferation in vitro, administration of recombinant mouse IL-12 (rIL-12) to normal mice caused a dose- and time-dependent anemia, leukopenia, and thrombocytopenia in vivo. Decreased numbers of bone marrow cells were recovered from the tibiae of IL-12-treated mice, and histologic examination of the marrow revealed a loss of mature neutrophils and red blood cell precursors. However, simultaneously with the suppression of hematopoiesis in the bone marrow, the IL-12-treated mice developed splenomegaly, which was largely caused by a marked enhancement of splenic extramedullary hematopoiesis of the erythroid, myeloid, and megakaryocytic lineages. These histologic observations were confirmed by colony-forming cell assays in which administration of IL-12 was shown to cause a time-dependent decrease in bone marrow CFU-GM, CFU-E, and BFU-E hematopoietic colony-forming cells while causing an increase in splenic CFU-GM and BFU-E colony-forming cells. All these effects were reversible upon cessation of IL-12 treatment. The observation that in IL-12-treated mice hematopoiesis was suppressed in the marrow but enhanced in the spleen suggests that myelosuppression was not caused by a direct effect of IL-12 on hematopoietic progenitors. It seems likely that myelosuppression was caused instead by an IL-12-induced alteration in the local environment of the marrow.
Subject(s)
Bone Marrow/drug effects , Hematopoiesis, Extramedullary/drug effects , Hematopoiesis/drug effects , Interleukin-12/pharmacology , Spleen/drug effects , Anemia/chemically induced , Animals , Dose-Response Relationship, Drug , Leukopenia/chemically induced , Mice , Mice, Inbred C57BL , Recombinant Proteins/pharmacology , Reference Values , Thrombocytopenia/chemically inducedABSTRACT
Mouse IL-12 acts on both mouse and human cells; human IL-12 acts only on human cells. This species specificity is determined by the p35 subunit of the IL-12 heterodimer. Since mouse and human p35 sequences are 60% identical, the determinants for the species specificity most likely residue in the nonhomologous sequences of mouse p35. To identify the regions on the p35 subunit interacting with the mouse IL-12 receptor, we constructed a series of chimeric mouse-human p35 molecules by replacing mouse sequences with the nonhomologous human counterparts. An IL-12 heterodimer containing a mouse-human p35 chimera with five residues changed in three discontinuous sites had drastically reduced (750-3000-fold) bioactivities on mouse cells. However, the competitive binding activity of the same mutant IL-12 heterodimer on mouse cells was only reduced 30-fold relative to wild-type IL-12. These findings therefore suggest that 1) the mouse p35 subunit participates in both receptor binding and signaling, 2) the mutations introduced into p35 affect signaling to a much greater extent than receptor binding, and 3) the five residues identified on p35 are required for interacting with the mouse, but not with the human IL-12 receptor and as such contribute extensively to the observed species specificity of IL-12.
Subject(s)
Interleukin-12/chemistry , Interleukin-12/pharmacology , Lymphocyte Activation/drug effects , Amino Acid Sequence , Animals , Binding, Competitive , Cell Line , Chlorocebus aethiops , Humans , Interleukin-12/biosynthesis , Kinetics , Macromolecular Substances , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Point Mutation , Protein Structure, Tertiary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Sequence Homology, Amino Acid , TransfectionABSTRACT
IL-12 is a heterodimeric cytokine that has been shown to enhance natural killer (NK) and cytotoxic T lymphocyte (CTL) responses, and to induce IFN-gamma production in vitro. In this study, we have examined the effects in vivo of administering purified murine rIL-12 to normal mice. Daily injections of rIL-12 i.p. (1 ng to 10 micrograms/day) caused dose-dependent enhancement of NK cell lytic activity in the spleens and livers of treated mice. Histologic examination of the livers of IL-12-treated mice revealed focal mononuclear cell infiltrates, and flow cytometry studies indicated that the livers of IL-12-treated mice contained increased numbers of NK cells, CD8+ T cells, and monocytes. Liver and splenic lymphoid cells from IL-12-treated mice, unlike liver and splenic lymphoid cells from control mice, spontaneously secreted IFN-gamma in vitro, suggesting that they had been induced by IL-12 to produce IFN-gamma in vivo. Consistent with this, IFN-gamma could be detected in the serum of IL-12-treated mice. In mice which had been immunized by footpad injection of allogeneic splenocytes, daily administration of rIL-12 i.p. was shown to enhance the specific CTL response in the draining lymph nodes. Thus, these studies demonstrate that IL-12 can enhance NK and CTL activity and induce IFN-gamma production in vivo, as well as in vitro, and suggest possible mechanisms by which IL-12 may exert therapeutic effects in the treatment of some tumors and infectious diseases.
Subject(s)
Interferon-gamma/biosynthesis , Interleukins/pharmacology , T-Lymphocytes, Cytotoxic/drug effects , Animals , Cytotoxicity Tests, Immunologic , Immunophenotyping , Interferon-gamma/drug effects , Interleukin-12 , Killer Cells, Natural/drug effects , Liver/drug effects , Lung/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Recombinant Proteins/pharmacology , Spleen/drug effects , Tumor Cells, CulturedABSTRACT
Se presenta un paciente evaluado en el PRN por presentar sexo ambiguo cuyo estudio hormonal fue normal pero la evaluación citogenética demostró la existencia de una translocación (Y;14) en mosaico: 45,X / 45,X, t (Y;14), posible de explicar por una translocación de media cromátide. Durante una hernioplastía se demostró la existencia de derivados mullerianos y dos gónadas de impresión equívoca que, histológicamente, correspondieron a testículo embrionario y a gónada veteada. Con el diagnóstico de disgenesia gonadal mixta del testículo fue ulteriormente extirpado. Se enfatiza la importancia de los estudios citogenéticos, hormonales e histopatológicos en la evaluación diagnóstica de pacientes con sexo ambiguo
Subject(s)
Infant, Newborn , Humans , Male , Gonadal Dysgenesis/diagnosis , Translocation, Genetic , Disorders of Sex Development , MosaicismABSTRACT
En los últimos años se han delineado tres entidades que involucran trisomía del cromosoma 22 y que comparten ciertas manifestaciones clínicas: el síndrome de ojo de gato, la trisomía parcial y la trisomía total 22, cuya existencia en recién nacidos aún está en controversia. Se describe un caso de trisomía 22 total, por translocación (15;22) "de novo", que falleció en el período de recién nacido con malformaciones pulmonares no descritas, y dos pacientes con una trisomía 22 parcial, (47, XX y XY, + der(22), t(11;22) (q25;q13) mat) debida a una segregación 3:1 en sus madres, quienes son portadoras de una translocación balanceada (11;22). Se discuten los hallazgos clínicos y citogenéticos de los probandos y sus familiares, así como los riesgos de recurrencia de estas trisomías