Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.     

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.     

Cytotoxic T lymphocytes from mice with soluble class I Q10 molecules in their serum are not tolerant to membrane-bound Q10

Q10 is a class I Qa-2 region-encoded molecule that is secreted by the liver and present in serum at high concentrations (about 10 to 60 micrograms/ml) in most strains of mice. The amino terminal portion of this molecule can also be expressed as an integral membrane protein by splicing the 5' end of the Q10 gene to the 3' end of H-2Ld and transfecting the hybrid gene into murine L cells. Because CTL primarily recognize polymorphic determinants controlled by the alpha 1 and alpha 2 domains of class I molecules and because the Q10d/Ld product expressed by transfected L cells includes the alpha 1 and alpha 2 domains of Q10d, we could address whether mice bearing serum Q10 were tolerant to this molecule at the CTL level. The results of these experiments demonstrate that Q10+ mice are able to generate H-2-unrestricted CTL activity against Q10d expressed on transfected L cells, and this response was not inhibitable by the addition of Q10-containing normal mouse serum. It is unlikely that this CTL activity is due to possible polymorphic differences in Q10 alleles, since semisyngeneic BALB/c (H-2d) mice, from which the Q10d hybrid gene construct was derived, are able to generate anti-Q10d effector cells. The Q10d molecule was shown to cross-react with H-2Ld, lending support to the concept that Qa genes can serve as donors for polymorphic sequences found in H-2K, -D, and -L. That mice can generate anti-Q10 CTL activity suggests that this soluble class I protein does not act as a toleragen for these cells. The implications of these findings for an understanding of self-tolerance are discussed.     

Biologic-response-modifier-induced indoleamine 2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures

Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been augmented in human epithelial cell lines treated with human interferon-gamma (HuIFN-gamma). Several human biologic response modifiers, including HuIFN-gamma, HuIFN-beta, HuIFN-alpha, interleukin 2 (HuIL-2), and tumor necrosis factor alpha, have now been assessed for their ability to enhance tryptophan degradation in human peripheral blood mononuclear cell (PMC) cultures. PMC were isolated from normal donors, cultivated in RPMI 1640 medium containing [3H]tryptophan, and treated with individual biologic response modifiers. At various intervals, culture supernatants were removed, fractionated by reversed-phase high performance liquid chromatography, and radioactivity in resultant fractions was determined. Significantly increased amounts of tryptophan catabolites were observed after treatment with HuIFN-gamma, HuIFN-beta, HuIFN-alpha, and HuIL-2, but not human tumor necrosis factor alpha. Often, greater than 30% of available tryptophan was degraded by treated PMC cultures. Although antibodies to HuIFN-alpha, HuIFN-beta, and HuIFN-gamma specifically neutralized the induction of IDO activity in PMC by their respective HuIFN, only anti-HuIFN-gamma antibody also neutralized HuIL-2-induced IDO activity. Furthermore, T24 bladder carcinoma cells, in which IDO was induced by HuIFN-gamma but not by the other biologic response modifiers, were induced to degrade tryptophan by supernatants of HuIL-2-stimulated PMC cultures, but not by HuIFN-beta-stimulated PMC culture supernatants. Thus, whereas HuIL-2 indirectly induced IDO in PMC cultures by stimulating production of HuIFN-gamma, all cases of interferons appeared to induce IDO directly in PMC cultures.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

Qc-1, a novel Q-region-controlled CTL determinant expressed on B lymphocytes

Using cloned cytotoxic T lymphocytes (CTL), we have identified a Q region controlled determinant with a unique strain and tissue distribution. Several strains that express the classically defined Qa-2 determinant and other Q region controlled determinants do not express the CTL determinant. In addition, strain BALB/cByJ, which does not normally express any Q region controlled cell surface determinant, expresses this new determinant. Cross-reactivity between the Q region controlled CTL determinant and a Kk region controlled class I product (probably H-2Kk) was observed. Finally, among lymphocytes, the CTL determinant is expressed preferentially (if not exclusively) on B cells, thus distinguishing it from all previously described Q region controlled determinants, which are expressed predominantly on T cells. We provisionally designate this novel Q region controlled CTL determinant Qc-1. The possibility that Qc-1 is recognized together with a self antigen is discussed.     

Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors

Two experimental situations have been studied, and the results provide evidence for a negative regulatory role for the low affinity interleukin 2 receptor (LA-IL 2R). The IL 2-dependent T helper cell line L-14, deprived of IL 2, becomes quiescent and expresses comparable numbers of high affinity IL 2R (HA-IL 2R) and LA-IL 2R. After activation by recombinant IL 2, this cell line preferentially expresses LA-IL 2R. The IL 2 responsiveness of the L-14 cell line was found to vary according to the ratio of LA-IL 2R to HA-IL 2R: the relative predominance of the LA-IL 2R coincides with a hyporeactivity of cells to IL 2. In contrast, a predominance of HA-IL 2R is accompanied by an increase in cellular IL 2 reactivity. Treatment of three IL 2-dependent T cell lines (L-14, HT-2, and C30.1) with limited amounts of recombinant IL 2 and moderate concentrations of anti-IL 2R monoclonal antibodies stimulates T cell growth. This treatment was shown to selectively diminish the expression of membrane LA-IL 2R. The stimulation was attributed to the decrease of expression of LA-IL 2R.     

A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1

The synthetic peptide CKS-17 has homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, to envelope proteins of HTLV I, II, III, and to that encoded by an endogeneous C-type human retroviral DNA. CKS-17 inhibits the immune response of lymphocytes and the respiratory burst of human monocytes. Because P15E-related antigens are present in human malignant cell lines and cancerous effusions, we sought to determine the effect of CKS-17 on monocyte-mediated tumor cell lysis. Lysis of A375 tumor cells by lymphokine or lipopolysaccharide-activated human monocytes was inhibited by 10 microM CKS-17 (control, 79%; CKS-17-treated, 19%). Another synthesized peptide, CS-2, which has partial homology to CKS-17, failed to block monocyte-mediated killing. Thus, the inhibition by CKS-17 appeared to be specific. Because interleukin 1 (IL-1) is a cytocidal factor produced by activated monocytes, we also investigated the effect of CKS-17 on IL-1 production by monocytes and on direct IL-1-mediated cytotoxicity. CKS-17 did not block production or secretion of IL-1 by lipopolysaccharide- or interferon-gamma-activated monocytes. However, the direct cytocidal activity of both recombinant IL-1 alpha and IL-1 beta against A375 tumor cells was blocked by CKS-17. The cytotoxic activity of IL-1 was inhibited by CKS-17 if (a) IL-1 was preincubated with CKS-17 for 1 hr at 37 degrees C or (b) the A375 cells were incubated with CKS-17 for 1 hr prior to the addition of IL-1. CKS-17 also blocked IL-1-induced proliferation of murine thymocytes, the D10 T cell line, and an IL-1-responsive astrocytoma cell line. These data suggest that CKS-17 may be a potent inhibitor of IL-1.     

Influence of neighbouring base sequence on N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the lacI gene of Escherichia coli

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations within the first 540 base-pairs of the lacI gene of Escherichia coli were cloned and sequenced. In total, 167 MNNG-induced independent mutations were characterized, with G.C to A.T transitions accounting for all but three of the mutations. This mutagenic specificity is consistent with the mispairing predicted by the methylation of the O6 position of guanine. The characterization of such large numbers of mutations permitted an analysis of the influence of local DNA sequence on mutagenesis. This analysis revealed a strong influence by the 5' flanking base. On average, guanine residues preceded (5') by a guanine or an adenine residue were, respectively, nine times and five times more likely to mutate after treatment with MNNG than those preceded by a pyrimidine residue.