Macrophage-T cell interactions involving Listeria monocytogenes--role of the H-2 gene complex.

This study reports on the in vitro interactions between T cells from Listeria-immunized mice, macrophages from normal mice, and heat-killed Listeria organisms. This interaction was assayed either by determing the amount of thymocyte mitogen in culture fluids after 24 hr, or by estimating the degree of T cell proliferation after 96 hr. Each assay depended on critical concentrations of macrophages, T cells, and heat-killed Listeria, points that were evaluated in a number of experiments. Both assays required specific Listeria-immune T cells. For an effective interaction, the T cells and the macrophages had to share the I-A region of the H-2 gene complex. Macrophages bearing Ia, which represented a minor population of macrophages, were essential for the proliferative response to macrophage-associated Listeria. Also, Ia-bearing macrophages were an important component in the interactions leading to increased secretion of mitogen. The immunogenic moiety associated with Listeria was short-lived, disappearing 24 hr after uptake of Listeria by macrophages. The interactions were not blocked by anti-Listeria antibodies but were partially sensitive to trypsinization.     

Control of macrophage Ia expression in neonatal mice--role of a splenic suppressor cell

The control of macrophage expression of I region-associated antigens (Ia) in neonatal mice was studied by comparing responses of neonatal and adult mice to immune vs nonimmune stimuli. Adults generated peritoneal exudates rich in Ia-bearing macrophages in response to i.p. injection of live Listeria monocytogenes, Listeria-immune T cells, and heat-killed Listeria, or a soluble mediator termed macrophage Ia-recruiting factor (MIRF). Neonates failed to respond to these stimuli. In contrast, both neonates and adults generated Ia-negative peritoneal exudates when stimulated with thioglycollate. A neonatal spleen cell that blocked the response of adults both to immune T cells and heat-killed Listeria and to MIRF was identified and characterized. Some of the suppressor cells appeared to be early precursors of the phagocytic lineage that develop into mature monocyte-macrophages. Suppression was apparently mediated by metabolites of arachidonic acid since indomethacin and aspirin in vivo blocked the effect. Similar suppressor activity was found in adult bone marrow and in adult resident peritoneal exudate cells. Thus, the phagocytic line autoregulates its surface expression of Ia in both neonatal and adult mice. This mechanism becomes particularly pointed during early development and could contribute to the lack of immunity during ontogeny.     

Regulation of macrophage Ia expression in mice with severe combined immunodeficiency: induction of Ia expression by a T cell-independent mechanism

We have studied the expression of Ia molecules by macrophages from mice with severe combined immunodeficiency (CB-17 scid) that lack demonstrable T cell and B cell functions. CB-17 scid mice had approximately normal numbers of Ia-bearing macrophages in the peritoneal cavity, spleen, and liver. Peritoneal macrophages responded in culture to T cell-derived lymphokines with enhanced expression of Ia molecules. However, unlike immunocompetent controls, SCID mice could not enhance Ia expression in an antigen-specific T cell-dependent manner after secondary challenge in vivo with a conventional protein antigen such as hemocyanin. Further demonstration of their T cell deficiency was the failure of CB-17 scid spleen cells to proliferate and produce IL 2 in response to the T cell mitogen, concanavalin A. Upon infection with Listeria monocytogenes, CB-17 scid mice developed chronically high loads of bacteria, whereas CB-17 control mice eliminated all viable bacteria and became resistant to secondary infection. However, Listeria-infected CB-17 scid mice did show, in parallel with the CB-17 controls, an unexpected and striking increase of Ia-positive macrophages. These data indicate that induction of Ia expression in macrophages can occur via a mechanism that is independent of mature T cells.     

The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. II. Differential Induction of Tumor Necrosis Factor-Alpha Production by Rickettsia tsutsugamushi and Rickettsia conorii

The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage-like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat-killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat-killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH-1 subset of T cells is predominant in immunity to R. tsutsugamushi.     

The role of interleukin 1 and 2 in generation of acquired resistance against mouse typhoid infection afforded by dialyzable factor from Salmonella typhimurium

Dialyzable factor (DF) prepared from a ribosomal fraction of Salmonella typhimurium was tested for its ability to induce interleukin 1 (IL 1) and 2 (IL 2) production, in relation to acquired resistance, after an intraperitoneal injection of DF. IL 1 production in vitro by peritoneal macrophages of DF-treated mice reached the maximum 4 days after injection, at the time when the nonspecific local resistance via macrophages directly activated with DF became apparent (Kita et al, Microbiol. Immunol. 28:807, 1984). Concanavalin A-induced IL 2 production by splenocytes of DF-treated mice reached the maximal level between days 6 and 8, and it could be enhanced even on day 14. Antigen-induced blastogenic responses of splenocytes from DF-treated mice reached the maximal level 14 days after treatment. Although DF did not show the mitogenic activity to normal splenocytes, T cells of DF-treated mice could respond to S. typhimurium. On the contrary, T cells of normal mice could respond to heat-killed cells of S. typhimurium when they were cultured with macrophages which had been directly stimulated in vitro with DF. Furthermore, T cells from DF-treated mice could respond to antigens of different species of bacteria, and especially to Listeria monocytogenes. These results suggest that T cells of DF-treated mice, being at the intermediate stage of activation via monokines including IL 1 which is produced by macrophages stimulated with DF, are able to proliferate immediately after the administration of challenging organisms as a second signal, and also that the specificity of the response may be defined by the challenging organisms.     

Cellular Aspects of the Resistance of Chickens to Eimeria tenella Infections*

SYNOPSIS. Sporozoites of Eimeria tenella were injected into the peritoneal cavity of normal chickens and chickens immunized against E. tenella. In some experiments normal scrum and serum from resistant chickens were injected prior to the injection of sporozoites. After 15 or 30 minute periods of intraperitoneal incubation, exudates were harvested and the occurrence of intracellular sporozoites was determined. Only macrophages and degranulated granulocytes were observed to contain sporozoites. There was no significant difference between the number of macrophages obtained from normal chickens (normal macrophages) which contained sporozoites and the number of macrophages obtained from immune chickens (immune macrophages) which contained sporozoites. Significantly fewer immune macrophages treated with immune serum contained sporozoites than untreated normal or immune cells, normal macrophages treated with either serum, or immune macrophages treated with normal scrum. Sporozoites in untreated normal macrophages did not appear to be harmed by the intracellular environment, based on structural observations. The majority of sporozoites in macrophages from all other groups were difficult to distinguish within the cytoplasm and were visibly distorted. It is hypothesized that the presence of fewer infected macrophages in exudates of immune chickens and serum-treated normal chickens was caused by an enhanced ability of these cells to destroy the parasite. Similar observations were noted in the case of sporozoites within degranulated granulocytes of experimental groups. The lack of understanding of the degranulation phenomenon makes it difficult to interpret these findings.     

BCG-induced suppressor cells. I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro.

Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.     

Specificity of the Anamnestic Response Produced by Listeria monocytogenes or Mycobacterium tuberculosis to Challenge With Listeria monocytogenes

When mice immunized with Listeria monocytogenes were given a second injection of listeria, they showed an anamnestic immune response to intravenous challenge with listeria, as measured by enumeration of the viable infecting organisms in the spleens of the infected animals. This response was independent of the effects of the challenge dose. When mice immunized with living or heat-killed attenuated mycobacterial cells were boosted with living H37Ra, there was also an accelerated response to listeria challenge. The response was greater in the mice given the primary immunization with living cells than in those immunized with heat-killed cells. The response to listeria challenge in mice immunized and boosted with mycobacteria was of less magnitude than that in the mice immunized and boosted with listeria. Growth of listeria in the mice immunized and boosted with mycobacteria was retarded only during the first 2 days of the infection, whereas the infecting listeria in mice immunized and boosted with listeria were permanently inactivated. Mice immunized with mycobacterial ribosomal fraction and restimulated with living mycobacterial cells showed no accelerated response to listeria challenge. It is evident from these results that resistance to these organisms is specifically evoked, but that once evoked it is not completely nonspecific in action. Also, the resistance produced by the mycobacterial ribosomal fraction to challenge with mycobacteria is completely specific in action. Therefore, it has been shown that there are two mechanisms involved in acquired immunity to facultative, intracellular parasites. One is nonspecific and mediated by activated macrophages. The other is specific and mediated by a mechanism as yet unknown.     

Inhibition of macrophage-mediated antigen presentation by hemolysin-producing Listeria monocytogenes

T lymphocytes and macrophages from Listeria-infected mice were used to evaluate the processing and presentation of live Listeria monocytogenes in vitro. Antigen presentation to T cells was quantitated by interleukin-2 production. In contrast to inert antigens such as heat-killed Listeria, live bacteria were processed and presented poorly. To evaluate the role of hemolysin (Hly), we used isogenic pairs of Hly+ and Hly- Listeria as antigens. In contrast to live Hly- bacteria, which were presented as well as heat-killed Listeria, live Hly+ bacteria were presented poorly. Hly+ bacteria also inhibited the presentation of heat-killed Listeria. This effect was apparent with as few as 10 bacteria/macrophage and was not due to loss of macrophage viability or decreased Ia expression after exposure to the live bacteria. With respect to murine listeriosis, the LD50 values for the Hly- strains were at least 1000 times higher than those for the Hly+ strains. These results suggest that the ability of Hly+ bacteria to inhibit antigen processing and presentation may be an important determining factor in Listeria infection and immunity.     

Attempts to characterize the T-cell population and lymphokine involved in the activation of macrophage oxygen metabolism in murine listeriosis

Cultures of T cells from Listeria monocytogenes-immune mice, macrophages, and heat-killed Listeria organisms produced a factor(s) capable of activating macrophage oxygen metabolism. The activity depended on the presence of Lyt 1+2,3- T cells in the primary culture. Macrophage oxygen metabolism could also be induced by a L. monocytogenes-specific T-cell clone which was recently shown to mediate anti-listerial protection in vivo and to secrete interferon-gamma (IFN-gamma) in vitro. Furthermore, macrophage activation was achieved by recombinant IFN-gamma. It is concluded that acquired resistance to facultative intracellular pathogens--at least in part--depends on the activation of macrophage oxygen metabolism by IFN-gamma derived from specific Lyt 1+2,3- T cells.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Allogeneic restriction of the delayed inflammatory reaction in the rat.

Delayed-type hypersensitivity (DTH) to Listeria monocytogenes was measured in rats that were recipients of syngeneic, semisyngeneic, and allogeneic immune thoracic duct lymphocytes (TDL). DTH could be transferred only to recipients that shared at least one haplotype with the TDL donors. The restriction was expressed in an inability of sensitized lymphoblasts to localize efficiently at antigen injection sites in the pinna of the ear and peritoneal cavity. Failure of allogeneic lymphoblasts to extravasate in more than trace numbers into Listeria-antigen-induced exudates was reflected in an absence of other lymphocyte-mediated expressions of DTH. Thus, lymphocyte-dependent MCA was not detected in Listeria-antigen-induced peritoneal exudates borne by recipients of allogeneic immune TDL and blood monocytes were not recruited in increased numbers into such exudates as they were in exudates borne by syngeneic rats. But allogeneic restriction of the delayed inflammatory response to Listeria antigen was overcome, at least in part, when antigen-presenting macrophages of the same MHC type as the immune TDL donors were implanted in the peritoneal cavity. The results encourage the belief that the observed failure of immune TDL to transfer DTH to allogeneic recipients is related to the inability of sensitized donor T cells to recognize antigen displayed by allogeneic macrophages.     

The role of B cells in the establishment of T cell response in mice infected with an intracellular bacteria, Listeria monocytogenes.

To clarify the role of B cells in the establishment of T cell response against intracellular bacteria, B-cell-deficient (muMT-/-) mice were infected with an intracellular bacteria, Listeria monocytogenes, and T cell response against the bacteria was analyzed. On day 6 of primary Listeria infection, spleen T cells of the muMT-/- mice showed significantly lower levels of proliferative response and IFN-gamma production than those of normal infected mice after in vitro stimulation with listerial antigen. Even in the secondary Listeria infection after immunization with viable bacteria, spleen T cells of the muMT-/- mice proliferated and produced IFN-gamma against listerial antigen at significantly lower levels than those of normal immunized mice. These results demonstrate participation of B cells in priming of Listeria-specific T cells in vivo. However, B cells failed to present Listeria antigen to Listeria-specific T cells in vitro unless Listeria antigen was solubilized. Furthermore, transfer of immune serum from Listeria-infected normal mice failed to enhance the Listeria-specific T cell response of muMT-/- mice. The results indicate that B cells support the T cell response against intracellular bacteria through a mechanism other than their Ig production or antigen presentation function.     

The interaction of gamma delta T cells with activated macrophages is a property of the V gamma 1 subset

Immunoregulation is an emerging paradigm of gammadelta T cell function. The mechanisms by which gammadelta T cells mediate this function, however, are not clear. Studies have identified a direct role for gammadelta T cells in resolving the host immune response to infection, by eliminating populations of activated macrophages. The aim of this study was to identify macrophage-reactive gammadelta T cells and establish the requirements/outcomes of macrophage-gammadelta T cell interactions during the immune response to the intracellular bacterium, Listeria monocytogenes (Lm). Using a macrophage-T cell coculture system in which peritoneal macrophages from naive or Lm-infected TCRdelta(-/-) mice were incubated with splenocytes from naive and Lm-infected alphabeta/gammadelta T cell-deficient and wild-type mice, the ability to bind macrophages was shown to be restricted to gammadelta T cells and the GV5S1 (Vgamma1) subset of gammadelta T cells. Macrophage adherence resulted in a 4- to 10-fold enrichment of Vgamma1(+) T cells. Enrichment of Vgamma1 T cells was dependent upon the activation status of macrophages, but independent of the activation status of gammadelta T cells. Vgamma1 T cells were cytotoxic for activated macrophages with both the binding to and killing of macrophages being TCR dependent because anti-TCRgammadelta Abs inhibited both Vgamma1 binding and killing activities. These studies establish the identity of macrophage cytotoxic gammadelta T cells, the conditions under which this interaction occurs, and the outcome of this interaction. These findings are concordant with the involvement of Vgamma1 T cells in macrophage homeostasis during the resolution of pathogen-mediated immune responses.     

Immunosuppression induced by attenuated Salmonella. Evidence for mediation by macrophage precursors

An attenuated aro A- strain of Salmonella typhimurium, SL3235, was previously shown to afford excellent protection in C3HeB/FeJ mice against challenge by virulent Salmonella and cross-protection against Listeria monocytogenes. In the present study, the immunologic status of immunized mice was evaluated by studying their ability to mount in vivo and in vitro plaque-forming cell responses to SRBC. SL3235-immunized mice exhibited marked suppression in their ability to generate anti-SRBC plaque-forming cell responses. Suppression was active and mediated by soluble factors as demonstrated by the capacity of immune cells to suppress the responses of normal cells in co-culture and across a membrane in transwell plates. Depletion of T cells from immune spleens did not alleviate the suppressive activity of the remaining cells. Depletion of splenic adherent cells resulted in partial alleviation of suppressive activity, demonstrating that mature macrophages are partly responsible for mediating the observed suppression. A sequential multi-step depletion procedure resulted in marked enrichment of a second suppressor cell population that was Mac1+, Thy-1.2-, surface Ig-, J11d-, non-adherent, non-phagocytic, and esterase negative. When cultured in vitro in the presence of L cell-conditioned medium, but not in the presence of Con A supernatant, these cells matured into typical macrophages within 72 h of culture. The cell population enriched for macrophage precursors (75%) retained the suppressive capacity of unfractionated splenocytes. Our data indicate that, in addition to the well-described involvement of mature macrophages in suppressing immune responses, bacterial infection may induce appearance of macrophage precursors that may also play an important regulatory role in the immune system.     

The mediator of cellular immunity. X. Interaction of macrophages and specifically sensitized lymphocytes.

Specifically sensitized lymphocytes have a focusing influence on mononuclear phagocytes that is expressed in the local accumulation and division of macrophages in bacteria-induced exudates. This was demonstrated by injecting Listeria monocytogenes into the peritoneal cavity of normal rats immediately before the animals were transfused with thoracic duct lymphocytes from either Listeria-immune donors or donors that had been infected with the unrelated parasite, Francisella tularensis. Sensitized lymphocytes originally present in the intravenous inocula were found later in the exudates. The arrival in the inflamed peritoneal cavity of specifically sensitized lymphocytes was associated with an exuberant influx of newly formed host cells and a local proliferative response that involved both immunoblasts and macrophages. These cytokinetic observations provide a plausible explanation of the delayed inflammatory response induced by the parasite, and imply that sensitized lymphocytes contribute to the host's defence by encouraging the prompt and purposeful deployment of monocyte-derived macrophages in centers of infection. In addition to their focusing influence on mononuclear phagocytes, specifically sensitized lymphocytes or their products can enhance the metabolic and microbicidal activity of macrophages. This activation process was revealed in the ability of rats infected with L. monocytogenes or BCG to control the growth of F. tularensis at a challenge site in the testis. But resistance was expressed only when the challenge organisms were injected with killed bacteria against which the recipients had been specifically immunized. The results accord with the view that macrophages are functionally activated by an immunological mechanism, and imply that the process is triggered locally by sensitized lymphocytes which are recruited from the blood.     

Dysfunction of Ia-positive antigen-presenting cells in autoimmune mice

Antigen-presenting activity of spleen macrophages for T-cell activation was studied in vitro using autoimmune prone MRL/Mp-lpr/lpr (MRL/l) mice, and their normal counterparts, MRL/Mp-+/+ (MRL/n) mice. In vitro induction of trinitrophenyl (TNP)-specific proliferative T-cell response in lymph node lymphocytes from picryl chloride-painted mice was markedly impaired in MRL/l mice. The presenting activity of TNP-hapten by spleen macrophages for proliferative T cells was also impaired in MRL/l mice. The impairment of both T-cell and antigen-presenting macrophage activities was marked in older MRL/l mice, but not in younger ones. The impaired antigen-presenting activity of macrophages was not due to the development of suppressor macrophages. In accordance with the impaired antigen-presenting activity of macrophages the population of Ia-positive cells in spleen macrophages and the production of interleukin 1 by spleen macrophages were also reduced in older MRL/l mice. These results suggest that Ia-positive macrophages are impaired in autoimmune prone mice and that the dysfunction of Ia-positive macrophages plays an important role in the pathogenesis of autoimmune diseases.     

Enhancement of macrophage bactericidal capacity by antigenically stimulated immune lymphocytes

The ability of antigenically stimulated immune lymphocytes to influence the bactericidal capacity of normal macrophages was studied in vitro. Purified lymphocytes were obtained from the lymph nodes and peritoneal exudates of guinea pigs immunized with bovine gamma globulin (BGG) and from control animals. Immune and control lymphocytes were added to normal macrophages and incubated overnight in the presence or absence of BGG. After washing, the macrophage monolayers were infected with Listeria monocytogenes; 4 hr later, the cells were lysed and the surviving intracellular bacteria quantitated. The macrophages which had been incubated with BGG-immune lymphocytes in the presence of BGG displayed a markedly enhanced listericidal capacity. In parallel experiments, these same antigen-stimulated lymphocytes were shown to inhibit the migration of normal macrophages. Lymphocytes derived from peritoneal exudates were more active than lymph node lymphocytes in both assays.     

Effect of Bacillus Calmette-Guérin infection on delayed footpad reaction to Listeria monocytogenes

The role of peritoneal macrophages induced by Bacillus Calmette-Guérin (BCG) in the induction of immune responses to Listeria monocytogenes was studied in mice. The peritoneal macrophages from mice treated with BCG 14 days previously contained a high proportion of Ia-bearing macrophages (approximately 56%) and the cells showed not only a high level of listericidal activity but also a strong ability for presentation of listerial antigen to Listeria-immune T cells. An intraperitoneal inoculation with a low dose of Listeria, which can induce the maximal level of delayed footpad reaction (DFR) and positive migration inhibitory activity of macrophages in untreated mice, did not induce a detectable level of such responses in BCG-treated mice. The bacterial growth at an early stage of infection was suppressed by scavenger macrophages in these mice. On the other hand, BCG-treated mice showed the early development of DFR and macrophage migration inhibitory activity after an inoculation with a high dose of Listeria. It is revealed in transfer experiments that Listeria-pulsed peritoneal exudate cells induced by BCG elicited the highest level of DFR and positive migration inhibition of macrophages in normal mice at the earlier period of injection compared with Listeria-pulsed resident peritoneal cells. These results suggested that the increased activities of macrophages acting as scavenger cells and as antigen-presenting cells play important roles in the modification of immune responses to Listeria in BCG-treated mice.     

Dissociation of macrophage accumulation and local chemotactic activity in delayed inflammatory sites.

A dissociation between the in situ generation of lymphocyte-dependent macrophage chemotactic activity (MCA) and the accumulation of macrophages in peritoneal inflammatory exudates was demonstrated in rats stimulated intraperitoneally with a saline suspension of killed Listeria monocytogenes (LM). Treatment of specifically immunized animals with cobra venom factor (CoVF) erased the hemolytic activity of serum complement (C) and the generation of peritoneal MCA. Such C-deficient rats nonetheless marshaled a substantial number of monocyte-derived macrophages into LM-induced exudates. The results suggest that MCA does not have an obligatory role in the attraction of macrophages into lesions in which there is a delayed inflammatory component. CoVF not only abrogated lymphocyte-dependent MCA in antigen-induced exudates but also decreased MCA of fresh and of heated normal rat serum. The serum of venom-treated animals could not be rendered chemotactic by C activation. It remains to be determined whether lymphocyte-dependent MCA is a product of antigen-stimulated T cells or is generated extracellularly by the interaction of T-cell factors with a humoral precursor. In any event, lymphocyte-dependent MCA differs from C-dependent MCA insofar as it is inactivated by heating (30 min at 56 °C).