Cellular immunity in man: correlation of leukocyte migration inhibition factor formation and delayed hypersensitivity

The formation of leukocyte migration inhibition factor (MIF) by the lymphocytes of 13 normal persons immune to the protein antigen keyhole limpet hemocyanin (KLH) has been investigated. KLH-induced MIF formation expressed as percent migration was compared with delayed hypersensitivity, antibody, and in vitro lymphocyte blastogenic responses to this antigen. Individuals were studied 404–840 days (median 540 days) after their last exposure to KLH. Nine persons had delayed hypersensitivity to KLH and 10 had circulating KLH antibody. The lymphocytes of 11 showed an in vitro blastogenic response to KLH stimulation, while the lymphocytes of nine produced MIF after KLH stimulation. The mean percent migration for the subjects with KLH delayed hypersensitivity was 48.2 (range 20.4–70.4) compared with 133 (range 120–161) for the four persons who did not have KLH delayed hypersensitivity (P < 0.05). The correlation coefficient between the precent migration and delayed hypersensitivity was ?0.78 (P < 0.01). No correlation was demonstrated between migration inhibition and the other parameters of immunity.     

Macrophage migration stimulation factor, migration inhibition factor and the role of suppressor cells in their production.

Guinea pig lymph node lymphocytes and human peripheral blood lymphocytes when stimulated by specific antigen or mitogen will release factors that affect in vitro macrophage migration. Migration inhibition factor production appears to be under the control of suppressor cells which are T lymphocytes. When suppressor cells are generated by stimulation with Con A for 4 days, migration stimulation factor (M.St.F.) activity is found. In other situations where M.St.F. is found this is thought to be due to increased suppressor cell activity. For example, young adults produce this lymphokine when stimulated with Con A, whereas aged individuals produce MIF. Concanavalin A appears to be the mitogen of choice for M.St.F. production, and phytohemagglutinin for MIF production. The release of this putative factor M.St.F. from suppressor T cells helps to explain some of the difficulties that have existed in studies of macrophage migration inhibition.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.     

Production of migration inhibitory factor (MIF) by human leukocytes following exposure to Epstein-Barr virus.

The ability of Epstein-Barr virus (EBV) to induce a cell-mediated immune response (CMI) in cultures of human leukocytes was investigated. Partially purified EBV, obtained from culture fluids of AV-1 cells, was inactivated by uv-irradiation. Inactivated virus was mixed with peripheral leukocytes from Hodgkin's disease (HD), infectious mononucleosis (IM) and malignant lymphoma patients as well as from normal individuals in an in vitro culture system. Production of migration inhibitory factor (MIF), as measured by guinea pig macrophage migration inhibition (MMI), was utilized as an indicator of CMI response. Significant differences in MIF response were observed subsequent to exposure of the cells to EBV. Leukocytes from patients in each of the disease categories tested exhibited greater MIF production than did those from the normal controls. There were significant differences in MIF production by leukocytes from the malignant and non-malignant disease categories. Serum from each subject was examined for immunoglobulin specific for EBV capsid antigen (anti-VCA). Although the majority of individuals within the disease categories tested had elevated anti-EBV serum titers, no correlation could be made between elevated anti-VCA titer and levels of MIF production.     

Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

Production of macrophage migration inhibitory factor and lymphotoxin by leukocytes from normal and Wiskott-Aldrich syndrome patients

The production of macrophage migration inhibitory factor (MIF) and lymphotoxin (LT) by cultured leukocytes from patients with Wiskott-Aldrich syndrome (WAS) and normal controls was studied. The presence of these lymphokines in leukocyte culture supernatants usually correlated directly with the dose of stimulant used. Doses of nonspecific mitogens and specific antigens, which produced maximal in vitro lymphocyte transformation, stimulated maximal production of these mediators. When the incorporation of tritiated thymidine by stimulated leukocyte cultures from patients with Wiskott-Aldrich syndrome (WAS) was deficient, they usually produced less MIF and lymphotoxin than normal. However, when their in vitro lymphoproliferative responses were normal, the lymphotoxin activity in supernatants of WAS leukocyte cultures was normal.     

Spectrum of in vivo and in vitro cell-mediated immune responses in coccidioidomycosis.

The cell-mediated immune responses of 12 healthy, coccidioidin skin-test positive subjects (Group I) were compared with those of 15 healthy, coccidioidin skin-test positive persons who had primary asymptomatic coccidiodomycosis, (Group II), 12 patients with active, pulmonary coccidioidomycosis (Group III), four patients with disseminated disease (Group IV), and five patients who had been in clinical remission for 1 year or longer (Group V). Lymphocytes from healthy subjects in Groups I and II responded in vitro to Coccidioides immitis antigen by undergoing an increased DNA synthesis (lymphocyte transformation) and/or by producing macrophage migration inhibitory factor (MIF). In contrast, patients in Groups III and IV failed to respond to Coccidioides antigens in vivo (skin tests) or in vitro (lymphocyte transformation and production of MIF). The responses of subjects in Group V with inactive disease fell in between those of healthy donors in Groups I and II and patients in Groups III and IV. The cellular immune defect, in terms of antigen recognition, appeared to be specific for C. immitis in all but one patient.     

Mathematical analysis of a capillary migration assay for cellular immune sensitivity: simple theory for dose-response.

A simple model is used to investigate parametric dependences of in vitro leukocyte assays for cellular immune sensitivity. The model is first tested by computing areas of cell migration occurring when migration inhibition factors (MIF) are absent from the culture medium. The theory subsequently is modified to include MIF production, and analytic expressions for the migration inhibition index $\text{I}$ are derived. These then are evaluated in order to study consequences of varying such factors as the ratios of constituent leukocytes or kinetic parameters pertaining to the ability of the culture medium to sustain migration.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Changes in leucocyte migration after renal transplantation

The leucocyte migration test, an in-vitro measure of cellular immunity, has been used to follow the changes in cell-mediated hypersensitivity to kidney and histocompatibility antigens in three patients after renal transplantation.Inhibition of leucocyte migration, indicating strong sensitization to the antigens used, occurred in each patient, starting five to seven days after transplantation. Satisfactory renal function had not been established in any of the patients at this time. In one case inhibition of leucocyte migration persisted almost continuously until the 24th day and was associated with poor renal function proved histologically to be due to rejection. Treatment with increased dosage of prednisone was associated with a rapid reversion to normal of the migration index and improvement in renal function. Later, inhibition of migration occurred again, and shortly afterwards the graft ceased to function. In the other two cases the migration index became normal without alteration in immunosuppressive therapy and a satisfactory diuresis followed. It is suggested that this simple test should prove useful in the specific diagnosis of rejection and in control of immunosuppressive therapy.     

In vitro studies on transplantation immunity. I. MIF production by sensitive lymphocytes in mice

Sensitized lymphocytes from mice immunized with skin homografts produce migration inhibitory factor upon incubation with lymphocytes (antigen) from the sensitizing strain. The MIF is produced within 14 hr following incubation of sensitized lymphocytes and antigen. In this reaction, antigenic specificity is a prerequisite for MIF production; however, the action of MIF transcends the strain barrier. Also, MIF produced in homograft reactions in mice inhibited the migration of peritoneal cells from normal guinea pigs. Finally, lymphocytes from mice bearing skin homografts do not develop the capacity to produce MIF prior to the rejection of the sensitizing skin grafts.     

The mediator of cellular immunity: XI. Origin and development of MIF producing lymphocytes

Thoracic duct lymphocytes obtained from rats infected with Listeria monocytogenes were characterized with respect to size, turnover and their capacity to release macrophage migration inhibitory factor (MIF). Cells responsive to Listerial antigens (LMA) in the MIF assay were identified in lymph during the first week of an immunizing infection. These were immunoblasts or large lymphocytes, as evidenced by their sedimentation with S phase lymphocytes at unit gravity. When labeled cells from the lymph of Listeria-infected donors were infused into similarly infected recipients, donor S phase lymphocytes localized rapidly, and in substantial numbers, in peritoneal exudates induced by the unrelated organism, F. tularensis. Within this immigrant population were cells which conferred immunity against L. monocytogenes and released MIF in cultures containing LMA. Exudates harvested 36 hr or 61 hr after stimulation contained labeled lymphocytes that were smaller than the S phase cells recovered during the early post-induction period. The observed shift of radioactivity from large to smaller lymphocytes was parallelled by a shift MIF production to exudate fractions containing smaller cells. The MIF producing cells in exudates of advancing age also exhibited increasing resistance to inhibition by vinblastine. These findings suggest that MIF is released by a family of lymphocytes—large, medium and small. LMA-responsive lymphocytes are delivered to the thoracic duct soon after their formation, at a stage in development when they can be stimulated to release only low levels of MIF. These mediator producing cells circulate briefly in the blood and differentiate fully only after they extravasate into inflammatory foci.     

Macrophage Migration Inhibition by Serum from Desensitized Animals Previously Sensitized with Tubercle Bacilli

MIGRATION of peritoneal exudate cells removed from guinea-pigs or mice exhibiting delayed hypersensitivity is inhibited by specific antigen^1–3. This in vitro macrophage migration inhibition has been regarded as a useful immunological test for delayed skin hypersensitivity^4,5. Studies of the mechanism of this phenomenon revealed that, in contact with specific antigen, lymphocytes from sensitized animals released into the medium a specific substance (migration inhibitory factor; MIF) capable of inhibiting the migration of normal macrophages^6,7. When injected intradermally into normal guinea-pigs, MIF elicits inflammatory reactions characterized by induration, erythema and mononuclear cell infiltration⁸.     

Genome-Wide Analysis in Swine Associates Corneal Graft Rejection with Donor-Recipient Mismatches in Three Novel Histocompatibility Regions and One Locus Homologous to the Mouse H-3 Locus

In rodents, immune responses to minor histocompatibility antigens are the most important drivers of corneal graft rejection. However, this has not been confirmed in humans or in a large animal model and the genetic loci are poorly characterised, even in mice. The gene sequence data now available for a range of relevant species permits the use of genome-wide association (GWA) techniques to identify minor antigens associated with transplant rejection. We have used this technique in a pre-clinical model of corneal transplantation in semi-inbred NIH minipigs and Babraham swine to search for novel minor histocompatibility loci and to determine whether rodent findings have wider applicability. DNA from a cohort of MHC-matched and MHC-mismatched donors and recipients was analysed for single nucleotide polymorphisms (SNPs). The level of SNP homozygosity for each line was assessed. Genome-wide analysis of the association of SNP disparities with rejection was performed using log-likelihood ratios. Four genomic blocks containing four or more SNPs significantly linked to rejection were identified (on chromosomes 1, 4, 6 and 9), none at the location of the MHC. One block of 36 SNPs spanned a region that exhibits conservation of synteny with the mouse H-3 histocompatibility locus and contains the pig homologue of the mouse Zfp106 gene, which encodes peptide epitopes known to mediate corneal graft rejection. The other three regions are novel minor histocompatibility loci. The results suggest that rejection can be predicted from SNP analysis prior to transplant in this model and that a similar GWA analysis is merited in humans.     

Antigen-dependent heterogeneity of human migration inhibitory factor

Human migration inhibitory factor (MIF) produced by peripheral blood mononuclear cells stimulated with purified protein derivative, tetanus toxoid, streptokinase-streptodornase, or Candida albicans antigen was analyzed by gel filtration and isoelectrofocusing. In all cases, supernatants harvested after a 24-hr exposure of the mononuclear cells to the antigen yielded only one MIF species with an isoelectric point of 5. In contrast, isoelectrofocusing of supernatants obtained from cells exposed to the antigen for an additional 24 hr demonstrated that different antigens induce the elaboration of different MIF species. Streptokinase-streptodornase and tetanus toxoid induced the production of one MIF species with an isoelectric point of 5 (pH 5-MIF). Stimulation of cells with Candida antigen elaborated a MIF species with an isoelectric point of 3 (pH 3-MIF). In contrast, stimulation of cells with purified protein derivative induced the production of both pH 3-MIF and pH 5-MIF.     

Desensitization V: Suppression of MIF production by lymphokine-activated macrophages

The systemic injection of high doses of antigen into a previously immunized animal results in a state of transient anergy with respect to cell-mediated immune reactions. This phenomenon is known as desensitization. We have previously shown that desensitization is a multistage process. The initial 24-hr period is characterized by excessive lymphokine production with a failure to express delayed hypersensitivity reactions due to abolition of local chemotactic gradients. Subsequent stages of desensitization involve failure of lymphokine production in vivo. The results presented here demonstrate that lymphocytes obtained from immunized and desensitized animals later than 24 hr after desensitization are markedly suppressed in their ability to produce MIF. In addition, it was found that lymphokine-activated macrophages can suppress in vitro MIF production by lymphocytes from immune, nondesensitized animals. In vitro and in vivo activation of macrophages were equally effective. Thus, it is likely that at least one mechanism for the inhibition of lymphokine production in the post-24-hr period of desensitization, involves activation of a population of suppressor macrophages by lymphokines produced during the initial 24-hr period.     

“Weak” histocompatibility antigens generate functionally “strong” humoral immunity

The kinetics and complexity of the immune responses generated by weak histocompatibility antigens on transplants of normal tissues were investigated by analyzing (1) the survival times of test skin grafts on mice that had been initially immunized with skin, liver, or kidney implants, (2) the survival times of test skin grafts on mice that had received putatively immune sera or lymphocytes harvested from an allografted host, and (3) the cytotoxic potential of these cells and sera in vitro. The data indicate that despite chronic rejection, weak antigens rapidly immunize hosts, and that the immune response includes a major humoral component that can either accelerate graft rejection or prolong graft survival.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Lymphocytes in transplantation immunity--in the relation to HLA antigens

Allograft rejection, in kidney transplantation, is initiated by recipient's lymphocytes with the recognition of genetically determined antigens, histocompatibility antigens, which are expressed on the graft, followed by killer T cell induction and antibody production against the graft. Recent advances in our knowledge of the human major histcompatibility complex antigens-HLA antigens, which have an important role in transplantation immunity, are herein reviewed briefly and the relation to allograft rejection is discussed.