Patrick M. Flood

Parathyroid hormone and lipopolysaccharide induce murine osteoblast-like cells to secrete a cytokine indistinguishable from granulocyte-macrophage colony-stimulating factor.

Osteoblasts are the cells responsible for the secretion of collagen and ultimately the formation of new bone. These cells have also been shown to regulate osteoclast activity by the secretion of cytokines, which remain to be defined. In an attempt to identify these unknown cytokines, we have induced primary murine osteoblasts with two bone active agents, parathyroid hormone (PTH) and lipopolysaccharide (LPS) and analyzed the conditioned media (CM) for the presence of specific cytokines. Analysis of the CM was accomplished by functional, biochemical, and serological techniques. The data indicate that both PTH and LPS are capable of inducing the osteoblasts to secrete a cytokine, which by all of the techniques used, is indistinguishable from granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF is not constitutive and requires active induction. Production of the cytokine is dependent on the dose of PTH or LPS added. It has been demonstrated that the addition of GM-CSF to bone marrow cultures results in the formation of increased numbers of osteoclasts. Therefore, these data suggest that osteoblasts not only participate in bone remodeling by formation of new matrix but may regulate osteoclast activity indirectly by their ability to regulate hematopoiesis.

An approach to the unification of suppressor T cell circuits: A simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM)☆

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiFs) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiFs which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

Regulatory responses in contact sensitivity: afferent suppressor T cells inhibit the activation of efferent suppressor T cells.

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.

Genetic, Serological and Functional Analysis of I-J Molecules

I-J determinants, defined by antibodies produced in intra-I region recombinant strains of mice, have been important markers in the analysis of immunoregulatory pathways. Study of these determinants has not only advanced our understanding of T cell subsets involved in the generation of suppressor, contrasuppressor, and helper amplifier activity, but has facilitated analysis of T cell-derived soluble mediators (factors) involved in cellular interaction and regulation. The observation that I-J molecules associated with suppressor factors determine the restricted functional activity of the factors provides evidence that I-J molecules are involved in information trafiBcking amongst immunoregulatory T cell subsets. In addition, the observation that some cellular interactions are I-J-restricted provides evidence that I-J molecules are important self structures recognized by interacting T lymphocytes. At present, the genetic basis for control of I-J determinants is unknown. Although it is clear that polymorphism in I-J molecules is controlled by genes mapping in the I region of the H-2 gene complex, molecular genetic studies provide evidence that I-J molecules are not encoded by I region genes. Formal proof of the latter must await isolation and characterization of I-J structural genes. However, these observations are compatible with the concept that I-J molecules are encoded by non-H-2 genes, but the expression of these non-H-2 genes is influenced by I region genes, eg, I-J molecules may be T cell receptors which recognize self Class II molecules and/or T cell receptors which recognize (or are recognized by) these self receptors. In the text which follows, our study of I-J determinants is summarized. Studies by other investigators are reviewed elsewhere in this volume. In the first part of

Immunological signals which control T cell responses

A number of identifiable immunological parameters can influence the elicitation and regulation of antigen-specific inflammatory responses to immunogenic epitopes. Injection of antigen in vivo can lead to the activation of type IV hypersensitivity responses, or to the induction of immunological tolerance to that antigen. We have used the hapten trinitrophenol as a model system for studying the factors which influence the generation and regulation of hypersensitivity responses to immunogenic epitopes in vivo. The generation of hypersensitivity or tolerance to trinitrophenyl depends on a number of immunological factors, including the form of the antigen, the route of immunization, and the presence of immune complexes of antibody and antigen on the surface of the antigen-presenting cell. Immunization with trinitrophenyl resulting in unresponsiveness can be the result of either the inability to prime inflammatory cells in vivo or the induction of suppressor T cells.

Modes of Communication Within the Immune System: Action or Reaction?

The immune system is a complex intertwining of individual components who collectively have one major purpose in mind: to recognize and eliminate antigens which are perceived by the immune system to be “different,” i.e. nonself. To do this, and to prevent the immune system from launching an attack on itself which may prove fatal, the immune, system has developed a complex mechanism of regulation which functions remarkably well (most of the time). This system of regulation is based on a system of cellular co-recognition and communication that theories of immunosemiotics are based.

The role of contrasuppressor T cells in the adoptive transfer of contact sensitivity responses to picryl chloride.

ConclusionIt is clear that the regulation of the immune response to contact-sensitizing agents is very complex. At least three functionally distinct cell types are involved in the initial response to antigen. These cells can be regulated by the activity of suppressor T cells directed at either the PCl-F-producing cell [17, 18], the Tinf cell [17, 18], or the natural contrasuppressor cell [27]. Within at least one of these populations are two subpopulations of suppressor cells, the Ts-afferent cells and the Ts-efferent cells [4, 13]. It is likely that not all of these regulatory cells are unique, but rather mediate different immunologic activities based on the circumstances of their surroundings. As an example, it has been found that unique functional activities can be ascribed to certain domains of regulatory molecules secreted by Ts cells [30–32], and it is possible that each Ts cell subset mediates multiple functions depending on its partner or target cell [33–35]. The similarities in functional activity between Ts and Tcs suggest that Tcs cells may also have a heterogeneity of functional activity.What is clear is that wherever there is suppression there is likely to be contrasuppression, and we have described at least two levels of contrasuppression active in the regulation of contact sensitivity. The challenge to all of us is to discover how the immune system activates and utilizes these diametrically opposed activities to function normally.

A Cell-Free Product Secreted by Ly−2+ Cells can Induce a Molecule Required for Ly2 Suppressor Cell Activity

The generation of suppression to SRBC in vitro involves the interaction of different T cell subsets in a well defined regulatory “feedback” circuit. The cells of this circuit have been identified by the correlation of function with a unique profile of cell surface glycoproteins (differentiation antigens). The induction signal is delivered by an I-J+ Ly1+2− cell to an Ly1+2+ I-J+ acceptor cell (1,2). The interaction between the Ly1 inducer cell and the Ly1,2 transducer cell is antigen-specific and restricted by genes linked to the V-region of the immunoglobulin heavy chain locus. The transducer cell then activates the effector cell of the suppressor circuit. One mechanism by which this takes place is the differentiation of transducer cells into effector cells (3). Other mechanisms may exist. The effector cell is so called because of its ability to suppress Ly1 cells directly, without the need for an Ly1,2 transducer cell (4). These effector cells are capable of suppressing not only helper cells, but also the feedback inducer cells. This results not only in a shutdown of antibody synthesis but abrogates additional T suppressor induction and thereby completing the feedback circuit. The phenotype of the effector cell is an Ly1−2+ and, unlike the other cells of the feedback circuit, it does not bear the I-J subregion gene products (4). This is especially important in light of the fact that the interaction between the suppressor effector cell and its target is partially restricted by genes mapping into this same I-J subregion of the MHC.

Cellular and Molecular Analysis of Immune Response to Tumor Cells: Demand for IL-2 and IL-4 in the Generation of Specific Anti-Tumor Cytotoxicity

Specific anti-tumor cytotoxic lymphocytes (CTL) are one of the effectors of cell-mediated responses, which are considered to be important for the control of tumor rejection (1 – 3). However, the factors which regulate tumor-specific CTL proliferation and differentiation have not yet been completely defined.

Infectious and noninfectious tolerance are blocked by a monoclonal antibody to T-suppressor factor

Two forms of hapten-specific unresponsiveness have been demonstrated following intravenous (iv) injection of hapten-conjugated syngeneic spleen cell based on the nature of the antigen-presenting cell (APC): I-J+, I-A- APC have been shown to induce T-suppressor cells (Ts cells) which are demonstrated upon adoptive transfer, while I-J-, I-A+ APC induce a nontransferable tolerance. In this paper we report that a monoclonal antibody specific for T-suppressor effector cells and factors (14-12) can block the Ts cells induced by I-J+, I-A- APCs and the tolerance induced by I-J-, I-A+ APCs. In addition, it sufficiently overcomes suppression such that injection of TNP-spl iv induces immunity rather than suppression. We show that the I-A+, I-J- TNP-spl, which induce nontransferable tolerance upon iv injection, are the cells which induce immunity in 14-12-treated recipients. These results demonstrate that injection of I-J-, I-A+ APC does not lead to clonal deletion and the tolerance induced by the iv injection of both I-J+, I-A- and I-J-, I-A+ APC operate via Ts cells.