Jack R. Battisto

Chemokine expression in trinitrochlorobenzene-mediated contact hypersensitivity

The expression of the murine IP‐10 and MCP‐1 genes has been examined in the skin of mice during contact hypersensitivity reactions to the hapten trinitrochlorobenezene (TNCB). In both naive and passively sensitized animals, challenge with TNCB resulted in elevated expression of both genes as early as 4 h as detected by Northern hybridization analysis. Twenty‐four hours after challenge, expression was markedly reduced in naive animals but remained elevated in sensitized animals. This prolonged expression of chemokine gene products correlates with the tissue swelling response generally used as a measure of delayed‐type hypersensitivity (DTH) in this model and suggests that the continued expression of these genes results from the stimulation of hapten‐specific T helper cells. Examination of cell type expression patterns by in situ hybridization using 3H‐radiolabeled riboprobes confirmed the results of Northern hybridization experiments. Both genes were expressed predominantly in cells exhibiting the morphology of connective tissue fibroblasts, although the distribution of cells positive for IP‐10 mRNA expression differed from that of cells expressing MCP‐1 mRNA. IP‐10 expression was localized almost exclusively to a population of connective tissue cells surrounding the fur follicle. MCP‐1 expression was rarely found associated with fur follicles but instead was distributed throughout the dermis in cells embedded in the collagenous extracellular matrix. Surprisingly, neither endothelial cells lining the small vessels located deep within the dermis nor epidermal keratinocytes were positive under any of the conditions utilized in the present study. Expression of both IP‐10 and MCP‐1 has been previously reported in a variety of distinct cell types in vitro. The present results indicate that only a subset of the cell types with such potential are stimulated to express these chemokine genes in vivo during hapten‐mediated DTH responses, implying the presence of subtle cell type‐ and tissue‐specific control mechanisms. J. Leukoc. Biol. 55: 452–460; 1994.

Immunosuppression in murine renal cell carcinoma. I, Characterization of extent, severity and sources

SummaryFour cell-mediated immunological responses related to tumor elimination have been examined in mice injected with a transplantable renal cell carcinoma (Renca). Lymphokine-activated killer (LAK) cells generatedin vitro from spleen cells of normal mice were capable of attacking Renca, EL-4, P815 and YAC-1 targets, but those from mice bearing Renca for 3 weeks could not. Natural killer activity, stimulatedin vivo by administering poly(I) poly(C), was less than 50% of normal in Rencabearing hosts. In addition, development of cytotoxic T lymphocytes to allogeneic targets was markedly inhibited in mice possessing the renal tumor. Finally, the delayed hypersensitivity response to a dermally applied hapten was approximately 70% less than normal in tumor-bearing mice, no matter whether the tumor existed subcutaneously or intrarenally. A kinetic study of the development of nonresponsiveness using the LAK assay showed onset of poor response at 1 week, which became maximal within 3 weeks following receipt of tumor subcutaneously. The immunological depression was seen to be attributable in part to suppressor cells present among spleen cells but not bone marrow cells of tumor-bearing hosts. The suppressor cells preventedin vitro LAK generation by normal spleen cells and, when adoptively transferred to normal mice, they inhibited natural killer stimulation and delayed hypersensitivity generation. Another source of immunological downregulation was provided by Renca cells themselves. Incorporation of Renca cells that had been X-irradiated with 30000 rad into cultures of normal and Renca-derived splenic cells suppressed replication of both almost completely. Furthermore, the presence of X-irradiated Renca cells in cultures of normal spleen cells prevented development of LAK cells. Thus, the suppression seen in Renca-bearing mice derives from multiple sources and whether each is in any way related to the other has been discussed. Identification of the phenotypes of cells responsible for the lymphoid cell-mediated suppression and examination of its elimination are communicated in the companion paper.

Immunosuppression in murine renal cell carcinoma. II, Identification of responsible lymphoid cell phenotypes and examination of elimination of suppression

SummaryIn our companion paper we have reported that cell-mediated immunity of mice bearing renal cell carcinoma is profoundly suppressed. The non-responsiveness of such animals was found to be attributable to Renca cells themselves and to splenic lymphoid cells that down-regulate other fully capable lymphoid cells. In this communication the lymphoid cell source of suppression within Rencabearing mice has been explored with the aim of identifying phenotypes of the responsible cells, the manner by which suppression is mediated, and initial ways by which suppression may be eliminated. A plastic-adherent cell bearing the Thy 1.2 surface marker as well as the Lyt1 and Lyt2 antigens has been found to operate, perhaps in conjunction with macrophages, to down-regulate lymphokine-activated killer (LAK) cell development for natural killer (NK) and non-NK targets that include Renca cells themselves. The splenic suppressor cells lost the capacity to suppress the NK response of normal recipient mice upon shallow irradiation (250 rad) prior to adoptive transfer. Spleen cells, presumably macrophages, from Renca-bearing mice were found to suppress the generation of LAK and NK cellsin vitro by synthesizing prostaglandins. Indomethacin, a prostaglandin synthetase inhibitor, blocked the induction of suppression bothin vitro andin vivo, suggesting the presence of endogenous prostaglandins in Renca-bearing mice. The suppression seen in Renca-bearing mice that derives from multiple sources and has been prevented by two separate methods has been discussed from the viewpoint of the inter-relatedness of the sources.

Suppression of Adjuvant Arthritis by Antibodies Specific for Collagen Type II

Immunization of Lewis rats with an alum flocculate of collagen type II, prior to the induction of arthritis by an intradermal injection of Mycobacterium butyricum in oil, caused reduced arthritic responses when compared with non-pretreated control animals. The majority of collagen-immunized animals that expressed diminished disease possessed low levels of serum antibody to collagen type II whereas most rats with undiminished disease did not. Passive immunization of Lewis rats with antibody to collagen type II also effectively reduced the severity of adjuvant-induced arthritis. Two possible mechanisms that might explain suppression of this disease by antibodies to collagen type II are discussed.

A guinea pig model of hypersensitivity to allergenic fractions of natural rubber latex

Allergy to natural rubber latex is a growing medical problem with life-threatening aspects. The aim of this study was to learn if guinea pigs could serve as a suitable model for immediate-type hypersensitivity to latex. Guinea pigs were immunized either with whole non-ammoniated latex extract, or with one of nine SDS-PAGE-separated components. Other animals were injected with electroeluted latex components localized on gel at 14, 24 and a cluster around 45 kD. Before and after immunization, sera from the animals were examined by ELISA, immunoblots, passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis. Latex-specific antibodies were detected by ELISA and immunoblots in sera from all immunized animals. PCA assays showed that the guinea pigs had homocytotropic antibodies dilutable to 1:250. PCA was abolished when sera from animals immunized with allergens in alum were heated at 56 degrees C for 30 min indicating the antibodies were of the E isotype. Passive systemic anaphylaxis was induced in 4 of 10 guinea pigs. The results show that guinea pigs are capable of making antibodies to latex protein components that mediate dermal and systemic anaphylaxis, paralleling the spectrum of clinical and laboratory findings of humans with immediate-type clinical latex hypersensitivity.

Use of Hapten‐Altered Self Moieties to Probe the Cell‐Mediated Lympholytic Response and Immunotolerance Interface

DTH, delayed-type hypersensitivity; MCH, membrane coupled hapten; CML, cell-mediated lytnpholysis; CTL, cytolytic T lytnphocyte; SSF, soluble suppressor factor; PSA, picryl sulfonic acid; TNP, trinitrophenyl; PFC. plaque forming cells; SRBC, sheep red blood cells; Mis, minor histocompatibility locus; KAF, killer assisting factor; TNBS. trinitrobenzene sulfonic acid; DNBS, dinltrobenzene sulfonic acid; TNCB, trinitrochlorobenzene; MLR, mixed lymphocyte reaction; S. I., stimulation index.

Suppression of contact sensitivity and cell-mediated lympholysis by oral administration of hapten is caused by different mechanisms

Oral administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), a water-soluble analog of trinitrochlorobenzene (TNCB), causes suppression of contact sensitivity (CS) to picryl chloride. We wished to determine what effect oral administration of TNBS (10mg), 10-12 days prior to sensitization, caused significant suppression of CS to picryl chloride. However, mice had to be fed at least three times at weekly intervals to cause suppression of the CTL response. CS was inhibited both at the afferent (induction) as well as at the efferent (effector) phase of the response. The unresponsiveness for CS was readily transferrable with cells from spleen, Peyers patches, and mesenteric lymph node cells from fed mice. In contrast, although the hapten specific CTL response was significantly suppressed in mice fed three times. suppression could not be transferred with any of the aforementioned lymphoid populations. Furthermore, spleen cells from fed mice showed a significantly enhanced in vitro CTL response after stimulation with hapten-modified syngeneic stimulator cells. The results indicate that suppression of CS and of the CTL response in TNBS fed mice is probably caused by different mechanisms.

Shared IgE-Binding Sites among Separated Components of Natural Rubber Latex

BACKGROUND IgE-mediated allergy to proteins present in natural rubber latex is a well-recognized problem. Latex contains a complex mixture of proteins ranging in molecular weight from 6 to > 200 kD. OBJECTIVE The aim of this study was to determine whether shared allergenic sites exist on separate latex components. METHODS Following electrophoresis, latex components at 14, 24, and 46 kD were electroeluted from SDS-polyacrylamide gels and used in ELISA inhibition and immunoblot inhibition studies of human latex-specific IgE antibodies. RESULTS A minimum of 4 major allergenic sites (for convenience labeled A through D) were found to exist in 3 components of nonammoniated latex. Minimally, 2 are present in the 14-kD component (A, B) and 3 in the 24-kD component (A-C). The 46-kD fraction has 3 or more antigenic sites (A, C, D) but lacks one (B) that is present in both the 14- and 24-kD components. CONCLUSIONS Four different IgE-binding moieties were detected among three latex protein components (14, 24 and 46 kD). Some of these allergenic sites were shared by two or more components. Recovery of these and others from fragmented latex components will allow identification of their amino acid composition and their availability will ultimately lead to better diagnostic and therapeutic options for patients with latex allergy.

Down-regulation of cytotoxic T lymphocyte generation by two distinct suppressor-cell systems.

Two distinct suppressor systems have been described that are capable of down-regulating in vivo generation of cytotoxic T cells directed toward haptenaltered self-antigens. One system, induced by hapten, involves three T cells that others have shown to function sequentially to suppress DTH. The initiator of this cascade is a T cell that is readily induced in spleens of mice injected intravenously with syngenic membrane-coupled hapten. This Ts, when triggered by the same syngeneic membrane-coupled hapten that induced it, elaborates a factor. The other two Ts arise in lymph nodes and spleens of mice painted epidermally with hapten. One of the two Ts in this set is readily armed by the factor of the first Ts. The factor confers its specificity and genetic restriction upon the accepting Ts. The latter, when properly triggered, makes a factor that is taken up by its companion Ts, which actually suppresses by way of a nonspecific factor. Whereas this Ts cascade is operative at the efferent limb of DTH, it mediates suppression only at the afferent phase of the CTL response. A distinctly different suppressor system is induced by minor locus (Mls) antigen. When Mlsd lymphoid cells are injected intravenously into Mlsc-possessing mice, an Lyt-1+ T-suppressor cell is generated that can be found in the spleen as well as among peritoneal exudate cells. This Ts interacts with macrophages to accomplish nonspecific suppression of the CTL response that is detectable both in vivo as well as in vitro. A Ts soluble product has been found to be effective to suppress CTL generation in vitro only when macrophages are present in culture. The macrophage that accomplishes suppression is I-A-. Although the afferent limb of the CTL response is down-regulated by this suppressor system, our in vitro culturing system is so structured as to make the helper T cell inactive. Thus, the mechanism of suppression must be oriented to the other early participants in the response, namely, precursor CTL, helper and differentiation factors, and/or the antigen-presenting cell.

Isogeneic and allogeneic lymphocyte interactions may be controlled by cell surface immunoglobulin tropism.

Abstract A study of allogeneic MLC (mixed lymphocyte culture) and two types of isogeneic MLC has been conducted in which subsets of B-cells were serologically removed from pools prior to using the remainder as stimulator cells. Cellular division in the two types of ILC (isogeneic lymphocyte culture) was found to be triggered by lymphocytes with IgG 1 on their surfaces. In contrast, the stimulator cells in ALC (allogeneic lymphocyte culture) possessed membrane-bound IgG 2A and/or possibly IgG 2B . Splenic T-cells were incapable of stimulating replication of splenic or lymph nodal T-cells in the absence of B-cells. Splenic T-cell preparations served as weak stimulators of other allogeneic T-cells but only when B-cells, either isogeneic or allogeneic to the stimulator T-cells, were present. We propose that stimulation in the MLC occurs in two distinct steps. First, immunoglobulins on cell surfaces may function to bring appropriate subsets of cells together. Next, antigenic recognition occurs that results in blastogenesis. Furthermore, the tropism or attraction that certain immunoglobulins have for some cell types may determine which subsets of cells participate in allogeneic and which take part in isogeneic MLC.