AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype.

CD163 is a member of the scavenger receptor cysteine-rich superfamily restricted to the monocyte/macrophage lineage and is thought to be a useful marker for anti-inflammatory or alternatively activated macrophages. In this study we used mass spectrometric analysis to determine that the antigen recognized by the antibody AM-3K, which we previously generated as a tissue macrophage-specific monoclonal antibody, was CD163. An anti-inflammatory subtype of macrophages stimulated by dexamethasone or interleukin-10 showed strong reactivity for AM-3K and increased expression of CD163 mRNA. Immunohistochemical staining of routinely processed pathological specimens revealed that AM-3K recognized a specialized subpopulation of macrophages. In granulomatous diseases such as tuberculosis, sarcoidosis, or foreign body reactions, tissue macrophages around granulomas, but not component cells of the granulomas such as epithelioid cells and multinucleated giant cells, showed positive staining for AM-3K. In atherosclerotic lesions, scattered macrophages in diffuse intimal lesions were strongly positive for AM-3K, whereas foamy macrophages in atheromatous plaques demonstrated only weak staining. We therefore suggest that, in routine pathological specimens, AM-3K is a useful marker for anti-inflammatory macrophages because these cells can be distinguished from inflammatory or classically activated macrophages. Because AM-3K cross-reacts with macrophage subpopulations in different animal species including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, bovine species, horses, monkeys, and cetaceans, it will have wide application for detection of CD163 in various animals.     

Strain variation in BCG-induced chronic pulmonary inflammation in mice. I. Basic model and possible genetic control by non-H-2 genes.

C57BL/6 mice (haplotype H-2b) responded in a dose-dependent fashion to killed BCG by marked enlargement of the spleen and lung. Neither CBA nor C3H mice (haplotype H-2k) responded to such treatment. Pulmonary inflammation in responder B6 animals was characterized by a marked chronic interstitial and alveolar granulomatous process, and was accompanied by occasional granulomata, hyperemia, and loss of architecture in the spleen. Inflammation in non-responder CBA and C3H animals was minimal in both the lung and spleen. The response does not appear to be controlled by genes within the major histocompatibility complex, but is associated with a C57 background. B10.BR mice (responder background, H-2k) were responder animals and C3H.SW mice (nonresponder background, H-2b) were nonresponders. In addition, all animals tested with a C57 background were responders even though two of these strains were not H-2b (C57BL/Ks, H-2d and C57Br/cd, H-2k). The resolution of the mechanism of genetic control of this response in mice may provide information relevant to possible genetic control of chronic pulmonary inflammation in man.     

Histological and ultrastructural study of the inflammation evoked by Paracoccidioides brasiliensis antigen in previously immunized mice

Bentonite particles uncoated and coated with soluble antigen of Paracoccidioides brasiliensis (Pb) were intravenously injected into mice with and without previous immunization with Pb antigen. The inflammatory reaction around the bentonite emboli in small lung vessels was quantitated and morphologically studied by light and electron (EM) microscopy, 2 to 8 days after challenge. In control nonimmunized animals, coated and uncoated bentonite particles caused mild and nonspecific inflammation made up by macrophages. By EM, they formed loosely aggregated clusters with cytoplasm containing few organelles and borders without interdigitation. In immunized mice injected with coated bentonite particles, the inflammatory area was significantly greater than that in nonimmunized animals in all periods of study with maximum difference at day 2. The inflammatory process at days 2 and 4 was characterized as mature granulomata, composed of macrophages with great number of organelles in the cytoplasm, large euchromatic nuclei and prominent nucleoli. Altogether these findings indicated a lesion with high metabolic activity, compatible with a granulomatous hypersensitivity reaction. At days 6 and 8, there was a change from mature to epithelioid granulomata, well demonstrated by EM which showed macrophages with characteristically interdigitated cytoplasmic borders. The results strengthen the importance of cellular immunity in the genesis of epithelioid granuloma in paracoccidioidomycosis and reinforce the usefullness of the present model in studies of the inflammatory cellular sequency and events in this mycosis.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

Phenotypic differentiation patterns of the human monocyte/macrophage system

Summary In the present study phenotypic properties of non-stimulated and stimulated blood monocytes and of their normal macrophage derivatives were studied applying enzyme cytochemistry, isoenzyme analysis of acid esterase (EC 3.1.1.6), and immunohistochemical staining using a panel of newly established monoclonal antibodies specific for the monocyte/macrophage lineage. Certain marker profiles could be established for the various normal subpopulations within the monocyte/macrophage system, which were also observable in epithelioid cells and U-937 cell line considered as reactive and neoplastic differentiation variants of monocytes, respectively. Alveolar macrophages, in contrast to the other analysed monocyte/macrophage populations, showed a highly activated phenotype comparable to lymphokine stimulated blood monocytes and epithelioid cells. The results underline the concept that the adaptation of monocytes/macrophages to their particular microenvironment is of decisive importance for their definitive differentiation.     

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.     

Modulation of F1 cytotoxic potentials by graft-vs-host reaction. Cooperative non-H-2- and H-2D region-gene control of F1 natural resistance to graft-vs-host reaction-associated immunosuppression

Our previous study revealed that in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congenic B10-series strains, parental H-2k spleen cells (SC) could not induce the graft-vs-host reaction (GvHR)-associated immunosuppression (GAIS). We also elucidated that a limited number of non-H-2 genes of parental C3H/He or AKR/J mice that had been incorporated into the F1 hybrids determined the F1 resistance to the GAIS, and the present study was done to explore the mechanism implicated in this type of F1 resistance to GAIS. SC from B10.AL mice carrying an rH-2 (K:k I:k S:k D:d) haplotype but not SC from H-2K B10.BR (k k k k) mice induced GAIS of in vitro CTL responses to third-party alloantigens in H-2k/d (C3H/He x B10.D2)F1 recipients mice. Further, SC from H-2k/a (C3H/He x B10.A)F1 mice carrying heterozygous C3H/B10 non-H-2 background but not SC from the same H-2k/a (B10.BR x B10.A)F1 mice but carrying homozygous B10/B10 background induced GAIS in H-2k/d (C3H/He x B10.D2)F1 recipients. Although C3H/He-, B10.BR-, and C3H.OH (d d d k)-SC were incapable of inducing GAIS in (C3H/He x B10.D2)F1 (k/d k/d k/d k/d) recipients, they were all good inducers of GAIS in (C3H.OH x B10.BR)F1 (d/k d/k d/k k/k) recipients. Exactly the same pattern of co-operative non-H-2 AKR and H-2D region-gene control of GAIS was observed on GvHR induced in H-2k/d (AKR/J x B10.D2)F1 recipients. These results suggest that the non-H-2 genes of C3H/He or AKR/J strain inhibit the functional expression of certain antigenic determinant(s) when it is encoded by heterozygous but not homozygous gene(s) linked tightly to H-2D region of k haplotype. Thus, the F1 resistance to GAIS is mediated by immune response of F1 recipients who miss the antigenic determinant(s) against that expressed on cell surface of GvHR-inducing T lymphocytes.     

An in vitro model of the leukocyte interactions associated with granuloma formation in Mycobacterium tuberculosis infection

The principal defense of the human host against a Mycobacterium tuberculosis infection is the formation of granulomas, organized collections of activated macrophages, including epithelioid and multinucleated giant cells, surrounded by lymphocytes. This granuloma can sequester and contain the bacteria preventing active disease, and if the granuloma is maintained, these bacteria may remain latent for a person's lifetime. Secretion of a variety of chemoattractant cytokines following phagocytosis of the bacilli by the macrophage is critical not only to the formation of the granuloma but also to its maintenance. To investigate this process of early granuloma formation, we developed an in vitro model composed entirely of human cells. Combining blood lymphocytes and autologous macrophages from healthy purified protein derivative skin test-negative individuals and mycobacteria resulted in the formation of small, rounded aggregate structures. Microscopic examination found macrophage-specific CD68(+) epithelioid macrophages and small round CD3(+) lymphocytes that in complex resembled small granulomas seen in clinical pathology specimens. Acid-fast staining bacteria were observed between and possibly within the cells composing the granulomas. Supernatants from the infected cells collected at 24 and 48 h and 5 and 9 days after infection were analyzed by a multiplexed cytokine bead-based assay using the Luminex 100 and were found to contain interleukin (IL)-6, IL-8, interferon-gamma and tumor necrosis factor-alpha, cytokines known to be involved in human granuloma formation, in quantities from two-fold to 7000-fold higher than supernatants from uninfected control cells. In addition, chemotaxis assays demonstrated that the same supernatants attracted significantly more human peripheral blood mononuclear cells than those of uninfected cells (P<0.001). This model may provide insight into the earliest stages of granuloma formation in those newly infected.     

Genetically determined resistance to lethal murine cytomegalovirus infection is mediated by interferon-dependent and -independent restriction of virus replication.

Susceptibility of 4-week-old mice of different strains to lethal murine cytomegalovirus (MCMV) infection was studied. Strains homozygous for H-2k and C57BL strains were resistant to greater than or equal to 10(5.5) PFU. B10.BR mice congenic for C57BL background genes and H-2k were about 10-fold more resistant than either C3H/HeN or C57BL strains. BALB/c mice (H-2d) were susceptible (50% lethal dose, 10(5.05) PFU). This susceptibility was dominant over resistance associated with H-2k but not that associated with C57BL background genes. The dominant susceptibility trait segregated in backcross mice as if carried by a single gene. Virus replication in spleen cells in vivo correlated with susceptibility to lethal infection. A similar trend was found in tests of salivary glands. Replication of MCMV in vitro in cultures of adherent spleen cells and primary mouse embryo cells correlated with replication in vivo. Neutralization of interferon (IFN) in cultures of adherent spleen cells reversed H-2k-linked restriction of viral replication but had minor effects on cells of other strains. Natural killer cell responses to infection were often higher in more resistant strains, but B10.BR mice developed minimal natural killer cell responses. Specific antibody and cytotoxic T cell responses in B10.BR mice were similar or lower than in other strains. Thus, resistance to lethal MCMV infection was not immunologically mediated, was dependent on and reflected by the capacity of cells from a given mouse strain to support replication in vivo and in vitro, and was IFN dependent and recessive if linked to H-2k but IFN independent when associated with C57BL background genes.     

CD4 is required for the development of a protective granulomatous response to pulmonary tuberculosis

To confirm the primary role of CD4 T cells in pulmonary tuberculosis, mice with a disruption of their CD4 gene (CD4 KO) were exposed to an aerosol of Mycobacterium tuberculosis and survival, cellular responses in the lung and granuloma development followed. CD8 and NK cells from the lungs of infected CD4 KO mice expressed IFN-gamma and were recruited in numbers similar to those seen in the C57BL/6 mice; recruitment correlated with initial control of bacteria. The major defect in mice lacking CD4 was the significant reduction in total cellular recruitment into the lungs. CD4 KO mice did not generate the typical mononuclear granulomatous lesions, instead the cellular influx was macrophage in character and was localized as perivascular cuffing. Early control of M. tuberculosis growth is therefore independent of CD4+ cells but such cells are required to ensure recruitment of mononuclear cells to the lung and thus ensure long-term survival.     

CD3- bone marrow cells augment the generation of cytotoxic T lymphocytes showing a preference for the X-chromosome linked gene product of stimulator cells

Responder cells, composed of both a limited number of nylon wool-passed lymph node (NW-LN) cells and an excess number of CD3+ cell-depleted bone marrow (CD3- BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3- BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW-LN cells. C3H/He (H-2k, C3H background) responder cells were stimulated with either B10.D2 (H-2d, B10 background) or BALB/c (H-2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 x BALB/c) F1 male target cells than (BALB/c x B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c) x B10.D2) F1 male than (B10.D2 x BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H-2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He x B10.BR) F1 male target cells than B10.BR or (B10.BR x C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X-chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.     

Primary in vitro cytotoxic response of F1 T lymphocytes against parental antigens.

Spleen cells from adult female (AKR/J x BALB/(c)F1 mice can respond to mitomycin C-treated spleen from AKR/J mice and can generate effector CTL in a 5-day primary in vitro culture. The response is comparable in magnitude to the response to allogeneic H-2K or H-2D antigens. The response is T cell mediated and is directed to antigen(s) present only on the parental cells. The target cell must be homozygous at H-2Kk to be lysed and H-2Dk antigens do not serve as a target in this response. Spleen cells from (B10.BR x B10.D2) hybrids that have been stimulated with AKR/J lyse B10.Br as well as AKR/J target cells. Similar H-2k/d hybrid F1 anti-H-2k parent responses are seen in certain other strain combinations. A number of possible interpretations of these responses are discussed.     

In vivo-induced suppression of T cell proliferation: the relationship between the specificity of induction and control

We have previously shown that sc immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and to peptide 8. In contrast, immunization of B10.BR (H-2k) mice either with TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous Ag. In the present article , we report that in the B10.BR (H-2k) strain, ip prepriming with (TMVP) 7 days prior to sc immunization with peptide 8 causes a drastic reduction in the in vitro proliferative response of peptide 8-specific T cells while no such effect is seen in the congenic C57BL/10 (H-2b) strain. This suppression of T cell responsiveness can be transferred with TMVP-primed spleen cells. Moreover, deleting T cells from the transferred spleen cells abrogates the suppressive effect. In both H-2 haplotypes, ip prepriming with peptide 8 causes suppression of the proliferative T cell response induced by subsequent immunization with peptide 8. This prepriming has no effect on the response to TMVP immunization of B10.BR mice but does effect the response of C57BL/10 mice. Using various synthetic peptides to analyze the specificity of the suppression, we have determined that (1) T cells involved in the suppression of the proliferative T cell response to a single peptide determinant do not suppress the proliferative T cell response to other determinants on the protein antigen and (2) these T cells with suppressor function, and proliferating T cells which are ultimately regulated, can exhibit specificity for the same epitope. These studies suggest that there may exist fundamental differences as to how T cells which participate in suppression an proliferating T cells (which include mainly T helper cells) recognize protein antigens.     

Functional differentiation in the genetic control of murine T lymphocyte responses to human fibrinopeptide B

The ability to generate proliferative and helper T lymphocyte responses in mice was compared by using the 14 amino acid peptide, human fibrinopeptide B (hFPB). Lymph node or peritoneal exudate T cells from mice immunized with hFPB were assessed for in vitro proliferation to soluble hFPB as determined by the uptake of 3H-thymidine. The T cell proliferative response to hFPB was found to be under MHC-linked Ir gene control; mice possessing the H-2a,k haplotypes were responders, whereas H-2b,d,q,s mice were nonresponders. The influence of non-H-2 genes on these responses was not investigated, so exclusive regulation by H-2 is provisional. The absence of a detectable lymph node and peritoneal exudate T cell proliferative response persisted in H-2b,d,q,s mice after immunization and boosting with several doses of hFPB. In addition, the capacity to produce a T cell proliferative response was inherited in an autosomal dominant manner and gene(s) controlling responsiveness to hFPB mapped to the I-A subregion of the H-2 complex. To measure peptide-specific helper T cell activity, an in vitro microculture assay in which hFPB-primed lymph node T cells and normal spleen B cells and macrophages were used was developed measuring anti-fluorescein isothiocyanate (FITC) IgM and IgG plaque-forming cell (PFC) responses after culture with FITC-conjugated peptide. Immunization of B10.BR, C57BL/10, B10.D2, and B6AF mice with hFPB primed for significant helper T cell activity as assessed by the ability to augment a primary in vitro IgM response to FITC. The normal B cell IgM responses were completely dependent on hFPB-primed T cells and required that hapten (FITC) and carrier (peptide) be linked. In addition, immunization with FITC-conjugated peptide elicited positive in vivo PFC responses to FITC in B10.BR and C57BL/10 mice, indicating similar genetic control of helper activity in both the intact animals and the in vitro microcultures. Thus, B10.BR mice show both T help and T proliferative responses to hFPB, whereas C57BL/10 mice show only T help and no T proliferative responses. In contrast to B10.BR mice, C3H and CBA mice immunized with hFPB were completely unresponsive when assayed for helper T cell activity in vitro despite their ability to generate positive lymph node T cell proliferative responses. These results indicate responsiveness to hFPB by T helper and proliferating cells is different and is under separate genetic control.     

Myelin proteolipid protein-induced experimental allergic encephalomyelitis. Variations of disease expression in different strains of mice

Strains of mice with diverse genetic backgrounds were tested for susceptibility to experimental allergic encephalomyelitis (EAE) induced by myelin proteolipid protein. EAE was elicited in all strains of mice tested, but the clinical and histologic features varied. SJL (H-2s) mice had a high incidence of both clinical and histologic disease characterized by early onset of clinical signs. Inguinal lymph node T cells from diseased animals responded specifically [( 3H]thymidine incorporation) to proteolipid protein and not to myelin basic protein. In contrast, BALB/c (H-2d), DBA/1 (H-2q), C57BL/6 (H-2b), AKR (H-2k), CBA (H-2k), C3H (H-2k), B10.BR (H-2k), and C57BR (H-2k) mice showed a later onset of clinical signs and typically a lower disease incidence. However, the most marked variations in disease incidence occurred among BALB/c (H-2d) substrains in which the incidence of EAE ranged from eight of nine (BALB/cPt) to complete resistance (BALB/cWt and BALB/cORNL). Because these BALB/c substrains were initially derived from the same inbred genetic source and are serologically identical at H-2, these results suggest that expression of proteolipid protein-induced EAE in the mouse involves additional loci outside the MHC.     

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.     

Immunity to leprosy. II. Genetic control of murine T cell proliferative responses to Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured after immunization of mice at the base of the tail with antigen and challenging lymphocytes from draining lymph nodes in culture with M. leprae. This T cell response to M. leprae has been compared in 18 inbred strains of mice. C57BL/10J mice were identified as low responder mice. The congenic strains B10.M and B10.Q were found to be high responders, whereas B10.BR and B10.P were low responders. F1 (B10.M X C57BL/10J) and F1 (B10.Q X C57BL/10J) hybrid mice were found to be low responders, similar to the C57BL/10J parent, indicating that the low responsive trait is dominant. Whereas B10.BR mice were shown to be low responders to M. leprae, B10.AKM and B10.A(2R) were clearly high responders, indicating that the H-2D region influences the magnitude of the T cell proliferative response. Gene complementation within the H-2 region was evident. Genes outside the H-2 region were also shown to influence the response to M. leprae. C3H/HeN were shown to be high responder mice, whereas other H-2k strains, BALB.K, CBA/N, and B10.BR, were low responders. Gene loci that influence the T cell proliferation assay have been discussed and were compared to known background genes which may be important for the growth of intracellular parasites. Because mycobacteria are intracellular parasites for antigen-presenting cells, genes that affect bacterial growth in these cells will also influence subsequent immune responses of the host.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to Mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.     

Requirement of I-E molecule for thymocyte apoptosis induced by staphylococcal enterotoxin B in vivo

In vivo administration of bacterial superantigen staphylococcal enterotoxin B (SEB) to BALB/c mice led to thymus atrophy resulting from thymocyte apoptosis. In this study, we demonstrated that SEB induced a substantial reduction in thymocyte numbers in BALB/c, B10. D2 (H-2(d) haplotype), B10.BR, C3H/HeJ, C3H/HeN (H-2(k)), and (BALB/c x B6)F1 (H-2(dxb)), but caused little or no effect in I-E- strains such as B6, B10, A.BY (H-2(b)), and A.SW (H-2(s)) mice. Elimination of CD4(+)CD8(+) cells predominantly accounted for the thymocyte loss, although the numbers of other subpopulations may also be reduced. Thymocyte apoptosis was shown by an increase in the level of DNA fragmentation in BALB/c but not in B6 mice after SEB administration. Treatment with anti-I-Ed monoclonal antibody to BALB/c mice blocked SEB-induced thymocyte apoptosis when anti-I-Ad exerted less effect. In contrast to SEB, staphylococcal enterotoxin A led to comparable levels of thymus atrophy in BALB/c and B6 mice. Studies on the surface marker expression indicated that CD25 expression was upregulated on BALB/c mouse thymocytes but with only a moderate increase in B6 mice. The CD4(+)CD8(+) cells were the major (>90%) population that expressed elevated levels of CD25 in BALB/c mice. An increase in the expression of TCRalphabeta, CD3, and CD69 surface markers was also observed on thymocytes from BALB/c mice, but not from I-E- strains. The differential response of I-E+ and I-E- mice to SEB may be exploited as a model for the study of apoptosis in the thymus.     

Macrophage chemotactic factor partially purified from granulomatous inflammation

Pathophysiological roles of macrophage chemotactic factor (MCF) in granulomatous inflammation were investigated. MCF was extracted in 10 mM phosphate-buffered saline, pH 7.4, from experimentally produced epithelioid cell granulomas in the liver and skin of mice. MCF activity reached a peak in the lesions prior to the time when granulomatous inflammation became maximal. MCF was then purified from 10-week-old hepatic granulomas and 2-week-old skin lesions by gel filtration, ion exchange column chromatography, and HPLC gel filtration. MCF from either liver or skin had a molecular weight about 650 kDa. MCF from hepatic granulomas was coupled to Affi-Gel beads and transplanted subcutaneously into naive mice. In vivo macrophage chemotaxis was observed around the beads and the cells formed a sheet, but organization of macrophages into granulomas did not occur with the MCF-active fractions. Macrophage chemotaxis alone is insufficient to elicit granulomatous inflammation.