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.     

Polypeptide fragments of horse cytochrome c activate a small subset of secondary B lymphocytes primed against the native protein

Horse cytochrome c (cyt c) and two large, overlapping cyanogen bromide-cleaved fragments (1-80 and 66-104), together encompassing the entire length of the polypeptide chain, were examined for their abilities to stimulate into antibody production individual secondary B lymphocytes primed against the intact protein. T cell help was provided against the carrier protein, hemocyanin, to which cyt c and its peptides were conjugated by using glutaraldehyde. All the B cells activated by both of the fragments elicited antibodies that reacted with intact cyt c in enzyme-linked immunosorbent assay, whereas only a fraction of the antibodies elicited by the intact protein reacted with the peptides. However, in general, antibodies reactive with the polypeptide fragments, whether elicited by the intact protein or by the fragments, could not be effectively inhibited from binding plate-bound cyt c in enzyme-linked immunosorbent assay in the presence of soluble native cyt c. This indicates that these antibodies are specific for denatured forms of cyt c that apparently arise during the chemical coupling of cyt c to carrier molecules for immunization and/or during emulsification of the immunogen in adjuvant. Whereas, at most, 5% of the secondary B cells specific for native cyt c could be activated by the 1-80 fragment, even fewer were activated by the 66-104 fragment. Therefore, it is unlikely that smaller peptides which fail to assume native conformation would be effective. Antibodies elicited in vivo in a primary response to the 1-80 fragment also failed to bind native cyt c. These results suggest that linear peptides intended to mimic epitopes on globular proteins, and which have not been engineered to adopt native conformation, will not be very effective either as primary or as secondary vaccines for B cell activation.     

The molecular basis of the hyaluronic acid-mediated stimulation of granulocyte function

Previous investigations have demonstrated that the function of polymorphonuclear leukocytes (PMN) is stimulated by hyaluronic acid (HA). The aim of the present investigation was to study the molecular basis for the effect of HA. HA fragments of m.w. in the range from 792 (tetrasaccharide) to 3,000,000 all stimulated the chemotactic and phagocytic function of PMN. The active concentration ranged from 4 to 64 pmol/liter, irrespective of the molecular size. Further investigations demonstrated that N-acetyl-D-glucosamine (NAGA) was the smallest active fragment of HA. NAGA is one of the components from which HA is built up; the other component glucuronic acid was without effect, and so were the other glycosaminoglycans, N-acetyl-D-mannosamine, N-acetyl-D-galactosamine, and D-glucosamine. Finally, Con A, the glucopyranosyl and glucomannosyl binding lectin, inhibited the stimulatory effect of NAGA. As is the case with HA, fibronectin also acts as a necessary cofactor to NAGA when incubations are made in the absence of whole blood or serum. The present results strongly indicated that the combined action of NAGA and fibronectin worked directly on the PMN by an interaction at the cellular membrane level. We conclude that the stimulatory action of HA on granulocyte functions is mediated through one of its two structural components, i.e., NAGA.     

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.     

Improved method for the synthesis of 1- or 3-acyl-sn-glycerols

Optically active 1- or 3-acyl-sn-glycerols were synthesized from 2,3- or 1,2-isopropylidene-sn-glycerols, respectively. The 2,3- or 1,2-isopropylidene-sn-glycerols were condensed with appropriate long saturated or unsaturated fatty acids and the resulting acyl isopropylidene compounds were treated with dimethylboronbromide at - 50 degrees C to give the title compounds. The ketal cleavage of acyl isopropylidene-sn-glycerols by dimethylboronbromide to produce the long 1- or 3-acyl-sn-glycerols was effective and gave good yields (70-90%). The reaction conditions were mild and there was no acyl migration, as shown by optical rotation of the monoacyl-sn-glycerols. The synthesis of 2,3-isopropylidene-sn-glycerol was improved to give an overall yield of 40% from L-arabinose. L-Arabinose was first converted to its 1,1'-diethylmercapto derivative and then condensed with 2-methoxypropene to yield 1,1'-diethyl-mercapto-4,5-isopropylidene-L-arabinose. Oxidation of this compound with sodium periodate followed by reduction with sodium borohydride under alkaline conditions yielded 2,3-isopropylidene-sn-glycerol [alpha]22D = -14.90 degrees, neat (Lit. 8 [alpha]22D = -14.5 degrees, neat; 14 [alpha]25D = -10.8 degrees; methanol C, 16.9). The optical purity of isopropylidene-sn-glycerols was determined as benzoyl derivatives on a high performance liquid chromatographic column packed with a chiral stationary phase.     

Synthesis of a thiophosphate analog of dioctanoylphosphatidylcholine: a phospholipase C substrate

Dioctanoylthiophosphatidylcholine, a racemic thiophosphate analog of L-alpha-dioctanoylphosphatidylcholine, has been synthesized and isolated by flash chromatography. In contrast with the didecanoylthiophosphatidylcholine synthesized previously, the analog is easily dispersed on sonication in aqueous media and is rapidly hydrolyzed to produce a free thiol group in the presence of the extracellular phospholipase C from either Bacillus cereus or Clostridium perfringens. When 5,5'-dithiobis (2-nitro-benzoic acid) was included as a thiol reactive chromogenic agent, the resultant measurement of product release, as an increase in absorbance at 412 nm, showed a linear relationship with added enzyme.     

Effect of Quinolinic Acid in the Nucleus Basalis Magnocellularis on Cortical High-Affinity Choline Uptake

A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific [3H]hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.