Genetics of the immune response to infectious pathogens.

In this selective review of the recent literature in the field of genetically determined host resistance to infection, we highlight five areas in which research is directed towards the search for proteins encoded by genes that function to maintain a 'resistant' phenotype in the face of challenge by a variety of pathogenic organisms. In particular, we discuss newly described genes that may regulate host resistance, newly described functions of genes previously identified, the reverse genetics approach to cloning an elusive gene, a direct genetics approach to a similar problem, and the role of the major histocompatibility complex in regulating our ability to resist challenge by infectious organisms.

Stromal-cell and cytokine-dependent lymphocyte clones which span the pre-B- to B-cell transition.

Five stromal-cell-dependent lymphocyte clones are described that correspond to late pre-B or early B-cell stages of differentiation. They are useful for determining the molecular requirements for pre-B replication, for studying the stromal cells that supply those factors, and for delineating the final sequence of differentiation events as newly formed lymphocytes prepare to exit the bone marrow. The efficiency of lymphocyte growth at limiting dilution varied substantially on different stromal-cell clones and may reflect functional heterogeneity of stromal cells. Most lymphocyte clones were similar to uncloned lymphocytes from Whitlock-Witte cultures in that they responded only transiently to interleukin-7 (IL-7) and then died, unless maintained on a stromal-cell clone. One unusual lymphocyte clone (2E8) was propagated for more than 1 year in IL-7 alone and was selectively responsive to that cytokine. Most of the lymphocyte clones were not tumorigenic in immunodeficient mice. However, one pre-B clone (1A9) grew autonomously in culture when held at high density, responded to conditioned medium from a number of cell lines, and was tumorigenic. Tumors derived from this clone were infiltrated by stromal cells and lymphocytes taken from the tumors' retained characteristics of the original clone. Ly-6 antigens were inducible on 2E8 and 1A9 cells, but the lymphocytes were otherwise arrested in differentiation. The 2E8 cells had rearranged and expressed kappa light-chain genes but displayed them on the surface along with surrogate light chains and mu heavy chains. Thus, expression of authentic light chain need not coincide with termination of surrogate light-chain utilization in newly formed B cells. Several glycoproteins have recently been demonstrated to be associated with surface immunoglobulin (Ig) on mature B-lineage cells and plasma-cell tumors. We now show that one member of this family (approximately 33 kD) was associated with the mu+surrogate light-chain complex on the 1A9 pre-B-cell clone. When compared to mature B lymphomas, fewer bands coprecipitated with the surface-labeled Ig isolated from pre-B- and early B-cell lines, suggesting that components of the antigen receptor are sequentially acquired during development. The normal replication and differentiation of pre-B cells is probably regulated by complex interactions with multiple cytokines and matrix components of the marrow microenvironment. Cloned lymphocyte lines that are dependent on stromal cells should continue to be important tools for molecular definition of those interactions.

Adipogenesis in a murine bone marrow stromal cell line capable of supporting B lineage lymphocyte growth and proliferation: biochemical and molecular characterization.

Recent advances in long-term bone marrow (BM) culture techniques have allowed investigators to dissect cellular components responsible for lympho hematopoiesis. Consequently, a number of "stromal" cell clones have been developed which are capable of supporting B lineage lymphocyte growth and proliferation in vitro by direct cell-cell interactions and the release of cytokines. While much work has focused on the support function of these cells, questions remain regarding their own differentiation potential. We have examined adipogenesis in the cloned BM stromal cell, BMS2. The presence of hydrocortisone, methylisobutylxanthine, or 30% fetal calf serum each accelerated adipocyte differentiation. This process was accompanied by the accumulation of triglycerides and cholesterol esters along with the induction of adipocyte-specific enzymes. Likewise, the steady-state level of mRNA transcripts increased for genes related to lipid metabolism. However, the pattern of mRNA expression in BMS2 adipocytes differed from that of a well-established, pre-adipocyte cell line, 3T3-L1, with respect to the following genes: glycerol phosphate dehydrogenase, CAAT/enhancer binding protein and angiotensinogen. Adipocyte BMS2 cells retailed the ability to support stromal cell-dependent B lineage lymphocytes in methylcellulose assays. The adipocytes continued to express macrophage-colony-stimulating factor mRNA constitutively and interleukin 6 mRNA in an inducible manner, similar to the BMS2 pre-adipocytes. Together, these data document a close developmental relationship between a specialized fibroblasts and adipocytes in the BM and suggest that adipocyte stromal cells may play an active role in lympho-hematopoiesis.

Characterization of murine bone marrow and spleen-derived stromal cells: analysis of leukocyte marker and growth factor mRNA transcript levels.

Stromal cells are believed to regulate lympho-hematopoiesis through direct cell-cell interactions and the release of growth factors. Many questions remain, however, about their lineage derivation and functional heterogeneity. We previously prepared a panel of stromal cell lines from murine spleen and bone marrow and characterized them based on their ability to support lymphocyte growth in long-term cultures. These cells are now compared with respect to their expression of various immunoglobulin superfamily and cytokine genes by Northern blot analysis. These results indicate that although stromal cells appear to be mesodermal in origin, they are not closely related developmentally to the hematopoietic progenitor cells they support. The potential production of at least six cytokines was demonstrated. All clones constitutively expressed mRNA for macrophage colony stimulating factor, interleukin-6, transforming growth factor beta and neuroleukin. The most potent lymphocyte supporting clones also made interleukin 7 constitutively. Previous findings had suggested that these clones responded to exogenous stimuli and this has now been demonstrated in terms of induced expression of IL-6 and G/M-CSF mRNA. Interleukin 6 mRNA levels were markedly upregulated by exposure of cells to LPS, TNF, IL-1, IL-6, IL-7, and EGF. G/M-CSF mRNA levels were "superinduced" by the combination of LPS and cycloheximide, a protein synthesis inhibitor. These responses are similar to ones documented by investigators working with endothelial cells and fibroblasts. Together, these data suggest that stromal cells are a multifunctional component of the lymphopoietic microenvironment and may be active participants in a complex, cytokine-mediated regulatory network.

Stromal cell lines which support lymphocyte growth: characterization, sensitivity to radiation and responsiveness to growth factors.

Stromal cells which grow as an adherent layer of Whitlock-Witte cultures are thought to be an essential component of the lymphohemopoietic microenvironment. Stromal cell lines from bone marrow (BM) and spleen have been obtained by treatment of cultures with 5-fluorouracil and selected for their lymphocyte support capacity by measuring the clonal growth of stromal cell-dependent lymphocyte lines in methyl cellulose. Established stromal cell lines differed significantly from stromal cells in primary Whitlock-Witte cultures with respect to expression of certain hemopoietic cell surface markers. For example, the Thy-1 and Mac-3 antigens were expressed by stromal cell lines obtained from BM and spleen, but not by stromal cells in primary cultures. Features common to all stromal cells include synthesis of actins, the neural adhesion molecule N-CAM, and a variety of collagens. Two types of common leukocyte antigens were not significantly expressed. The proliferation and total protein synthetic capacity of lymphocyte-supportive stromal cell lines was sensitive to ionizing radiation. After exposure of the cells to 200 rads, the incorporation of either [3H]thymidine or [3H]Leucine was reduced to less than 50% of control values, but the growth of lymphocytes was augmented in the presence of an irradiated stromal cell layer. The proliferation of stromal cell lines was also affected by exposure to a variety of growth factors. Addition of epidermal growth factor or endothelial cell growth factor augmented BM or spleen-derived stromal cell proliferation, while interferon-gamma had the opposite effect. In general, but not exclusively, lymphocyte growth was inhibited by factors which augmented the proliferation of stromal cells. Novel methods are described for isolating stromal cells and determining their capacity to support lymphocyte growth in vitro. Evidence is presented that this ability is not restricted to BM-derived stromal cells. The function of stromal cells was not dependent on their ability to proliferate, and this may be modulated by immunoregulatory and other growth factors.

In vivo localization and growth of the 70Z/3 pre-B-cell line.

The 70Z/3 pre-B-cell line has long been used as a model for understanding the nature and mode of action of differentiation-inducing stimuli as well as mechanisms which control immunoglobulin light chain gene expression. This study is a first appraisal of the localization, growth, and differentiation of the cell line in vivo. At 24 hr after intravenous injection, radiolabeled 70Z/3 cells localized efficiently to the bone marrow and analysis by flow cytometry revealed that fluorescein isothiocyanate-labeled cells localized to bone marrow and spleen in a ratio of 2:1. Growth of the cell line paralleled the localization pattern. When the cells were given intravenously, bone marrow contained 100% of tumor cells at a time when a majority of spleen cells were still normal. Tumor cells were found in the blood only at end-stage disease in a minority of animals. Because 70Z/3 cells differentiate in vitro in response to a variety of factors, it is possible that exposure to the in vivo environment would have a similar effect. When blast cells from heavily infiltrated bone marrow and spleen were analyzed for the expression of a panel of B-lymphocyte lineage surface antigens, however, there was no evidence for surface kappa induction. Inductive stimuli may be present in limiting quantities in vivo or overridden by by negative feed-back control mechanisms. This information provides a basis for in vivo experimentation with the inducible 70Z/3 cell line and raises issues concerning normal mechanisms which control B-lineage cell differentiation.

Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells.

The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis. Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by [3H]thymidine labeling and radioautography. In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains. The turnover of small lymphocytes also increased. These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC. In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects. The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production. Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.

Regulation of bone marrow lymphocyte production: IV. Altered kinetic steady state of lymphocyte production after chronic changes in exogenous stimuli.

A transient increase in B lymphocyte production in mouse bone marrow has previously been shown to follow a single administration of various exogenous agents. The effect of sustained changes in exogenous stimuli on the level of bone marrow B lymphocyte production has now been studied. In mice given multiple injections of sheep red blood cells (SRBC) for four weeks the production of bone marrow lymphocytes was augmented, as indicated by increased numbers of cytoplasmic mu-chain-bearing pre-B cells and of surface mu-chain-bearing B lymphocytes, as well as increased rates of pre-B cell proliferation and small lymphocyte turnover. In an attempt to reduce potential external stimuli, mice were raised on an elemental diet. When compared to conventionally reared mice, however, they showed little difference in bone marrow small lymphocyte production and an identical pre-B cell proliferation rate. In addition, the small lymphocyte production rate in the thymus was not consistently altered either in SRBC-treated mice or elemental diet-fed mice, whereas small lymphocyte renewal in the spleen showed changes reflecting those in the bone marrow. The results demonstrate that a chronic increase in exposure to extrinsic agents can produce a long-term elevation of the population size and production rate of bone marrow B lineage cells. This suggests that the level of the kinetic steady state of primary B lymphocyte production normally observed in the bone marrow may reflect the level of exposure to potential stimulants in the external environment.

Regulation of B-lymphocyte production in the bone marrow: role of macrophages and the spleen in mediating responses to exogenous agents.

B-Lymphocyte production in mouse bone marrow can be stimulated by administering a variety of foreign materials in vivo. The nature and location of cells mediating this effect have now been studied, using assays of lymphocyte renewal and pre-B-cell proliferation. Pretreatment of mice with silica, to depress macrophage function, abolished the stimulation of small lymphocyte renewal produced by administering either sheep red blood cells (SRBC) or mineral oil and reduced the response to bovine serum albumin. The response was still abolished when silica was given 6 or 24 hr, but not 48 hr, after SRBC. Splenectomy prevented the stimulation of marrow lymphocyte renewal when performed either 4 weeks before or up to 72 hr after SRBC injection. The stimulation of pre-B-cell proliferation was similarly prevented by pretreatment with either silica or splenectomy. The results indicate that the wave of increased B-lymphocyte production after SRBC injection depends for the first 2-3 days upon silica-sensitive, spleen-dependent mechanisms, suggesting an early mediation by splenic macrophages. Primary B-lymphocyte production in vivo may thus normally be stimulated by exposure to external environmental agents acting indirectly on bone marrow B-cell progenitors via cellular reactions in peripheral lymphoid tissues.

In vivo regulation of B lymphocyte production in the bone marrow: effects and mechanism of action of exogenous stimuli on pre-B cell proliferation and lymphocyte turnover.

The present studies demonstrate that a single administration of an extrinsic agent (SRBC) can stimulate increased production of B lymphocytes in mouse bone marrow as revealed by 2 in vivo assays which quantitate pre-B cell proliferation and small lymphocyte renewal, respectively. The mechanisms mediating this stimulatory effect are sensitive to silica in vivo and require the presence of the spleen. Early events are both silica-sensitive and spleen-dependent, while a subsequent stage appears still to be spleen-dependent but not silica-sensitive. Sustained exogenous stimulation by multiple SRBC injections for 4 wk in young mice produces an expanded population size and increased production of pre-B cells and B lymphocytes in the bone marrow, apparently an elevated kinetic steady state of B lymphocyte production. (Formula: see text). As depicted schematically in Figure 1, the results suggest that the magnitude of bone marrow B lymphocyte production in vivo may reflect a basal level, putatively regulated by microenvironmental and other endogenous factors, which is amplified by exogenous environmental stimuli mediated by the action of macrophages located in the spleen. Further questions about such an environmental amplification (Fig. 1) concern the nature of later events in the spleen, the identity of putative stimulatory factors or cells circulating from the spleen to the bone marrow, the receptive target cell stages in the bone marrow and the consequences of this process with respect to the size and diversity of B lymphocyte clones and of primary humoral immune responses in vivo.

Characteristics of mononuclear phagocytes mediating antilisterial resistance in splenectomized mice.

The characteristics of mononuclear phagocytes mediating resistance to infection with Listeria monocytogenes during the early phase (up to 48 h) of the response were investigated in mice of the A strain that had undergone splenectomy. Although irradiation in the sham-operated host had no effect on its antilisterial response when administered immediately before infection, it markedly reduced the ability of the splenectomized host to resist listerial challenge. This effect of radiation was demonstrable in the high-dose range (600 r) and could not be reversed immediately by repopulation with 20 x 10(6) syngeneic nucleated bone marrow cells. Administration of silica 24 h before infection profoundly enhanced the growth of L. monocytogenes in the liver of splenectomized mice. Shielding of the liver, but not the bone marrow, protected the splenectomized host against the effects of radiation, indicating that the cell population responsible for mediating the enhanced antilisterial resistance resides in the liver. The enhanced antilisterial resistance of splenectomized mice was specifically because of the absence of the spleen and not merely because of the removal of a favorable replicating environment for listeria organisms.

Measurement of 5'-nucleotidase in mouse peritoneal macrophages in listeriosis.

5'-Nucleotidase activity in peritoneal macrophages of mice was measured both before and after infection with Listeria monocytogenes in hosts which possessed high or low anti-listerial resistance either due to a genetically determined trait or as a result of splenectomy. Reduction in enzyme activity was directly related to the degree of infection that developed in the hosts and hence was inversely related to the level of anti-listerial resistance observed. Basal 5'-nucleotidase activity was significantly lower in noninfected hosts that possessed high anti-listerial resistance, possibly reflecting a higher level of native macrophage activation in these hosts.