Induction and mode of action of suppressor cells generated against human gamma globulin. I. An immunologic unresponsive state devoid of demonstrable suppressor cells.

A model of unresponsiveness to human gamma-globulin (HGG) which is maintained in the absence of demonstrable suppressor cells has been described. A/J mice were tolerized with deaggregated HGG purified from a variety of sources. The spleen cells from these tolerized mice were assessed for their ability to suppress the response of normal spleen cells to HGG when transferred into lethally irradiated mice. All of the HGG preparations obtained from commercial sources as Cohn fraction II of pooled, outdated plasma induced suppressor cells to HGG, although not of equal magnitude. However, suppressor cells could not be demonstrated in the spleens of mice tolerized with deaggregated HGG purified from the plasma of a healthy individual. This inability to detect suppression was independent of the method of purification of the HGG and of the time of assessment of the putative suppressor cells after tolerization. Similarly, deaggregated HGG isolated from an IgG1 lambda-myeloma protein induced unresponsiveness to HGG but did not stimulate demonstrable suppressor cells. These data suggest that suppressor T cells are not involved in the maintenance of tolerance to this antigen, although they may play a regulatory role in the immune response to HGG. Support for this concept was obtained by assessing the duration of unresponsiveness in the T and B lymphocytes of mice tolerized with the various HGG preparations. Mice tolerized with the HGG preparations that stimulated little or no suppression were among the last to recover responsiveness. Indeed, there was no consistent correlation between the level of suppressor cell activity and the degree of unresponsiveness in either the splenic T or B lymphocytes. Thus, although certain HGG preparations may provide a tool for the generation of antigen-specific suppressor T cells, the utilization of these suppressive preparations may be inappropriate for the investigation of the mechanisms of the induction and maintenance of the unresponsive state.

Regulation of integrin function by the urokinase receptor.

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.

Semirational design of a potent, artificial agonist of fibroblast growth factor receptors.

Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs. When fused with the c-jun leucine zipper domain, which binds heparin and forms homodimers, the polypeptide specifically reproduced the mitogenic and morphogenic activities of basic FGF with similar potency (EC50 = 240 pM). The polypeptide required interaction with heparin for activity, demonstrating the importance of heparin for FGFR activation even with designed ligands structurally unrelated to FGF. Our results demonstrate the feasibility of engineering potent artificial agonists for the receptor tyrosine kinases, and have important implications for the design of nonpeptidic ligands for FGF receptors. Furthermore, artificial FGFR agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.

An improved assay for the detection of interleukin 1.

A 24 h, highly sensitive assay for detection of interleukin 1 (IL-1) is described. A thioguanine-resistant mutant of the murine lymphoma cell line LBRM 33 was selected (LBRM TG6). When this cell line was incubated with low concentrations of PHA and IL-1 it produced interleukin 2 (IL-2). IL-2-dependent HT2 cells were co-cultured with the LBRM cells to measure the released IL-2. Prior to addition of tritiated thymidine to the co-culture, hypoxanthine and azaserine were added to metabolically block DNA synthesis by LBRM TG6 cells. This resulted in a sensitive, short term assay requiring minimal technical manipulations and characterized by a high signal to noise ratio.

Urokinase receptor antagonists: discovery and application to in vivo models of tumor growth.

Urokinase receptor antagonists based on the growth factor domains of both human and murine urokinase which show sub-nanomolar affinities for their homologous receptors have been expressed as recombinant proteins. Further modification of these molecules by preparing fusions with the constant region of human IgG has led to molecules with high affinities and long in vivo half-lives. Smaller peptidic inhibitors have been obtained by a combination of bacteriophage display and peptide analog synthesis. All of these molecules inhibit the binding of the growth factor domain of uPA to the uPA receptor and enhance binding of the uPA receptor to vitronectin. Protein uPA receptor antagonists were tested in an in vivo tumor model using the human breast carcinoma MDAmb231 in immunodeficient mice. Both human and murine receptor antagonists showed significant inhibition of primary tumor growth, demonstrating that in vivo, both tumor and stromal cell uPA receptor dependent plasminogen activation can modulate tumor growth.

Response of bovine and porcine peripheral blood mononuclear cells to human recombinant interleukin 2(125).

Bovine and porcine peripheral blood mononuclear cells (PBMC) were tested for their response to human recombinant interleukin 2(125) (rIL 2(125)). The rIL 2(125) used in these experiments was purified to homogeneity from Escherichia coli, contained a site-specific modification at amino acid #125 replacing a cysteine with a serine residue and had a specific activity of 4 X 10(6) units/mg. Human rIL 2(125) was shown to be directly mitogenic for bovine and porcine PBMC and was able to maintain the long-term growth of mitogen-activated PBMC of both species. Long-term cultures were highly sensitive to low levels of rIL 2(125) and showed dose-dependent responses when used in short-term IL 2 assays. Bovine and porcine PBMC preincubated with human rIL 2(125) for 1 and 5 days demonstrated enhanced levels of cell-mediated cytotoxicity against both allogeneic and xenogeneic cell lines.

Specific suppression of the immune response by HGG tolerant spleen cells. I. Parameters affecting the level of suppression.

After adoptive transfer, the spleen cells from mice made tolerant to human gamma-globulin (HGG) specifically suppress the immune response of normal spleen cells. However, this suppressive activity in the spleen cells of tolerant mice is only present for a brief period after treatment with tolerogen. Spleen cells from animals injected 10 days earlier with tolerogen reduce the immune response of an equal number of normal spleen cells by 75%. Spleen cells from mice made tolerant 40 days previously are, however, no longer suppressive, even though they remain completely unresponsive. These data suggest that active suppression of antigen-reactive cells is not the mechanism responsible for maintaining tolerance to HGG, but rather is only transiently associated with the tolerant state. Further evidence in favor of the separation of the tolerant state from suppressive activity is that complete suppression of the normal spleen cell response requires either a high ratio of tolerant to immune competent cells or a delay in the antigenic challenge of the reconstituted recipients. By contrast, such manipulations are not required to demonstrate the complete unresponsiveness of the tolerant cells after adoptive transfer.

Susceptibility of newborn mice with H-2k backgrounds to lymphocytic choriomeningitis virus infection.

Many strains of mice, when injected at birth with an ordinarily lethal dose of lymphocytic choriomeningitis virus (LCMV), grow to adulthood despite maintaining a persistent virus infection and chronic virus-induced immune complex disease. Because the susceptibility to LCMV infection changed over several years of observation, a number of murine strains with different histocompatibiity gene loci and genetic backgrounds were compared. Neonatal mice with H-2b, H-2d, and H-2q backgrounds were relatively insensitive to the effects of LCMV infection compared to mice with H-2k backgrounds, which had a high mortality rate in this situation. Expression of the H-2k gene locus itself did not affect the rate of mortality. Use of recombinant mice indicated that susceptibility was linked to H-2k backgrounds and not H-2k gene loci. The low survival rate of newborn mice with H-2k backgrounds infected with LCMV was not caused by cytotoxic natural killer cells, cytotoxic T lymhocytes, excessive amounts of virus in the organs, a unique distribution of virus or expression of viral antigens in vivo or unusual pathology in tissues.

Comparison of the biological activities of human recombinant interleukin-2(125) and native interleukin-2.

Human interleukin-2 proteins (IL-2), purified to homogeneity from both the Jurkat cell line and from genetically engineered Escherichia coli, were compared in a variety of biological systems. The gene coding for the recombinant IL-2 protein used in these studies contained a site-specific modification resulting in the replacement of a cysteine residue with a serine residue at position 125 in the encoded polypeptide. The specific activity was 2-4 X 10(6) units/mg for both the recombinant IL-2(125) and the native IL-2 molecules when measured by DNA synthesis in the murine HT2 cell line. The abilities of these two molecules to support the short-term proliferation and the long-term growth of mitogen- and alloantigen-activated peripheral blood mononuclear cells (PBMC) from humans and of mitogen-activated PBMC from cats, cows, sheep, and horses were equivalent. In addition, both molecules were directly mitogenic for human PBMC and induced the production of interferon-gamma. Human PBMC treated with IL-2 generated enhanced levels of cytotoxic cells against both natural killer (NK)-sensitive and NK-resistant targets. In all of these systems and assays, recombinant IL-2(125) had the same range of biological activity and potency as homogeneous native IL-2.

High level human interleukin 1 production by a hepatoma cell line.

The human hepatic adenocarcinoma cell line, SK-hep-1, was found to constitutively produce Interleukin 1. Addition of the ionophore A23187 and lipopolysaccharide resulted in a 30-fold enhancement in the release of biological activity. Serum supplementation did not affect the level of production. Interleukin 1 from these cells had a molecular weight of 10-20,000 daltons on gel exclusion chromatography. Polyadenylated RNA, when fractionated on sucrose density gradients and injected into Xenopus laevis oocytes, produced high levels of biological activity in the 14-16s region. An oligonucleotide probe, complementary to the coding sequence of the Interleukin 1 cDNA isolated from human monocytes, hybridized specifically to this part of the gradient. These results demonstrate that SK-hep-1 cells are a valuable source of material for studying the polypeptide and messenger RNA of Interleukin 1.

Quantitation of mRNA by the polymerase chain reaction.

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.

Specific, transient suppression of the immune response by HGG tolerant spleen cells. II. Effector cells and target cells.

In a previous report, it was shown that spleen cells from mice made tolerant to human gamma-globulin (HGG)5 could specifically inhibit the immune response of normal spleen cells after adoptive transfer to lethally irradiated recipients. However, that report also showed that the suppressive activity was only transiently associated with tolerant spleen cell populations. It was concluded from those experiments that while suppressive activity could be demonstrated in tolerant spleen cells under certain conditions, such activity was not obligatory for the maintainance of the tolerant state. The experiments presented here were performed to determine the nature of the effector cell(s) and the target cell(s) involved in this system of suppression of the immune response. Treatment of cells from tolerant animals with anti-thymocyte serum and complement to remove thymus-derived (T) cells completely abrogated suppresive activity. Removal of adherent cells from tolerant spleen cells by passage over glass wool columns resulted in partial loss of the suppression. The inhibitory activity of the suppressor cells was resistant to 900 R irradiation regardless of whether the tolerant spleen cells were irradiated before or after adoptive transfer. The cellular target(s) for the supprssor cells was examined by using lipopolysaccharide (LPS) as an alternative source of helper activity for the response to HGG. LPS, injected at the time of the initial antigenic challenge of mice that had been reconstituted with tolerant and normal spleen cells, prevented the expression of suppression against bone marrow-derived (B) cells. However, when LPS was presented only at the time of secondary antigenic challenge, it was unable to overcome suppression of the immune response of reconstituted recipients. Thus, LPS could produce a state where the B cells were resistant to suppression, but LPS could not rescue the responsiveness of B cells once the cells in the reconstituted recipient had been suppressed. In addition, the immune response to both the hapten dinitrophenol (DNP) and the carrier (HGG) were suppressed when recipients of tolerant and normal spleen cells were challenged with DNP6HGG. This indicates that T helper cells are also a target for suppression. The results presented in this paper are discussed in relation to a possible mechanism of suppression which proposes that suppressive activity represents the induction of tolerance in immunologically competent cells by HCG which is closely associated with the tolerant spleen cells.

Virus-induced immune complex disease: specific anti-viral antibody and C1q binding material in the circulation during persistent lymphocytic choriomeningitis virus infection.

Mice of several strains persistently infected with lymphocytic choriomeningitis virus (LCMV) mount continuous anti-LCMV immune responses leading to the formation and tissue deposition of immune complexes. Such mice carry infectious virus-immunoglobulin (presumably anti-LCMV antibody) complexes in the circulation. We have now determined that anti-LCMV antibody both complexed and free is found in the circulation of mice persistently infected with LCMV. This antibody reacts specifically against the three main LCMV structural polypeptides: nucleoprotein, 63,000 m.w. and two glycopeptides, GP-1 and GP-2 with m.w. of 45,000 and 35,000, respectively. A C1q binding assay was developed and found to be effective in measuring C1Q binding substances (presumably virus-anti-viral Ig complexes) in the circulations of several strains of mice persistently infected with LCMV. With different strains of mice, the levels, time of formation, and fate of C1q binding materials varied markedly. Formation of antibodies to LCMV was correlated with the detection of C1q binding materials. Mice (SWR/J) persistently infected with lactic dehydrogenase virus also form infectious virus-Ig in their sera but deposit minimal amounts of complexes in their tissues. In such mice, C1q binding substances did not form in the circulation.

High-affinity urokinase receptor antagonists identified with bacteriophage peptide display.

Affinity selection of a 15-mer random peptide library displayed on bacteriophage M13 has been used to identify potent ligands for the human urokinase receptor, a key mediator of tumor cell invasion. A family of receptor binding bacteriophage ligands was obtained by sequentially and alternately selecting the peptide library on COS-7 monkey kidney cells and baculovirus-infected Sf9 insect cells overexpressing the human urokinase receptor. Nineteen peptides encoded by the random DNA regions of the selected bacteriophage were synthesized and tested in a urokinase receptor binding assay, where they competed with the labeled N-terminal fragment of urokinase with IC50 values ranging from 10 nM to 10 microM. All of the isolated peptides were linear and showed two relatively short conserved subsequences: LWXXAr (Ar = Y, W, F, or H) and XFXXYLW, neither of which is found in urokinase or its receptor. Competition experiments demonstrated that the most potent peptide, clone 20, prevented binding of bacteriophage displaying the urokinase receptor binding sequence (urokinase residues 13-32). In addition, this peptide blocked other apparently unrelated receptor binding bacteriophage, suggesting overlapping receptor interaction sites for all of these sequences. These results provide a demonstration of bacteriophage display identifying peptide ligands for a receptor expressed on cells and yield leads for the development of urokinase receptor antagonists.

Increased apolipoprotein E and c-fms gene expression without elevated interleukin 1 or 6 mRNA levels indicates selective activation of macrophage functions in advanced human atheroma.

Cells found within atherosclerotic lesions can produce in culture protein mediators that may participate in atherogenesis. To test whether human atheromata actually contain transcripts for certain of these genes, we compared levels of mRNAs in carotid or coronary atheromata and in nonatherosclerotic human vessels by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from RNA. We measured PCR products (generated during exponential amplification) by incorporation of 32P-labeled primers. Levels of interleukin 1 alpha, 1 beta, or 6 mRNAs in plaques and controls did not differ. Compared to uninvolved vessels, plaques did contain higher levels of mRNA encoding platelet-derived growth factor A chain (42 +/- 24 vs. 12 +/- 10 fmol of product; mean +/- SD; n = 8 and 8, respectively; P = 0.007) and B chain (41 +/- 36 vs. 4 +/- 3 fmol of product, n = 14 and 6, respectively; P = 0.024). Atherosclerotic lesions consistently had much higher levels of apolipoprotein E (apoE) mRNA than did control vessels (131 +/- 71 vs. 5 +/- 3 fmol of product; n = 12 and 10, respectively; P less than 0.001). Direct RNA blot analyses confirmed elevated levels of apoE mRNA in plaque extracts. To test whether mononuclear phagocytes might be a source of the apoE mRNA, we studied a selective marker for cells of the monocytic lineage, the c-fms protooncogene, which encodes the receptor for macrophage colony-stimulating factor. Plaques also contained elevated levels of c-fms mRNA (30 +/- 17 vs. 5 +/- 3 fmol of product; n = 10 and 7, respectively; P = 0.002). Immunohistochemical colocalization demonstrated apoE protein in association with macrophages in plaques, whereas nonatherosclerotic vessels showed no immunoreactive apoE. ApoE produced locally in atheroma might modulate the functions of lesional T cells or promote "reverse cholesterol transport" by associating with high density lipoprotein particles, thus targeting them for peripheral uptake. Macrophages within the advanced human atheroma appear to exhibit a selective program of activation as they express high levels of apoE, whereas overall levels of interleukin 1 or 6 mRNAs in plaques are not elevated.

Interleukin 2 acts as an adjuvant to increase the potency of inactivated rabies virus vaccine.

Interleukin 2 (IL-2) occupies a central position in the cascade of events involved in the immune response. We were interested in determining whether IL-2 could function as an adjuvant to vaccination, to increase the immune response to vaccine immunogens. Using the National Institutes of Health test for rabies vaccine potency, we found that daily systemic administration of IL-2 in conjunction with inactivated rabies virus can increase the potency of vaccination in outbred mice at least 25-fold, as measured by survival following challenge with virulent rabies virus. Enhanced protection is not correlated with an increase in virus-neutralizing antibody titers, and we suggest that IL-2 acts to increase the cellular immune response to vaccination.

Comparison of two algorithms and their associated charges when evaluating adrenal masses in patients with malignancies.

OBJECTIVE: This study was performed to compare two proposed algorithms used when evaluating an adrenal mass discovered during staging evaluation of a patient with a known malignancy. Such evaluation was meant to lead to determination of the relative charges associated with each algorithm. SUBJECTS AND METHODS: Fifty-four patients with known malignancies who required evaluation of an adrenal mass underwent both chemical shift imaging (CSI) and CT-guided for CSI. The hospital charges incurred for each procedure and any associated complications were normalized using national relative-value scale charges and conversion factors. A decision analysis was performed to compare the relative charges that would have been incurred if adrenal MR imaging had been performed in all patients, followed by CT-guided biopsy only in those patients with MR findings not diagnostic of adrenocortical adenoma, and the relative charges incurred if only CT-guided adrenal biopsy had been performed in every patient. RESULTS: Twenty-three (43%) of 54 adrenal masses were shown to be metastases by CT-guided biopsy. The sensitivity and specificity of CSI for the diagnosis of adrenocortical adenoma were 94% and 100%, respectively. The charges incurred by performing MR imaging as the initial examination with subsequent CT-guided biopsy only in those patients with CSI findings not diagnostic of adenoma would have been similar to those incurred by first performing CT-guided adrenal biopsy in every patient. CONCLUSION: CSI is an accurate, noninvasive technique for evaluating adrenal masses in patients with cancer. If CT-guided biopsy is used only when CSI is not diagnostic of adrenocortical adenoma, the associated charges would be virtually the same as when CT-guided biopsy is performed as the first test in every patient. Moreover, biopsies could have been avoided in 54% of these patients.