John J. Rinehart

Effects of Corticosteroids on Human Monocyte Function

This report examined the effect of corticosteroids in vitro on human peripheral blood monocytes, essential cells in both immune and nonimmune cellular defense mechanisms. Monocyte chemotaxis in response to sera, Escherichia coli filtrate, and lymphokine chemotactic factor was markedly reduced (P < 0.01) by hydrocortisone succinate (HCS) at 16 mug/ml. Methylprednisolone succinate and unesterified hydrocortisone produced similar impairment of monocyte chemotaxis while two drugs which unmodified do not enter cells, hydrocortisone phosphate (HCP) and cortisone acetate, had no effect on chemotaxis. HCS also significantly impaired monocyte random migration at 16 mug/ml. Monocyte bactericidal activity was reduced by HCS at 16 mug/ml (P < 0.01)) but was not affected by HCP even at 120 mug/ml. In comparison, HCS did not alter granulocyte chemotaxis even at 500 mug/ml, and bactericidal activity was reduced at 16 mug/ml (P < 0.01). Monocyte phagocytosis of cryptococci was reduced only 20% (P < 0.05) at 120 mug/ml. HCS at 120 mug/ml did not alter monocyte base-line or postphagocytic hexosemonophosphate shunt activity, viability by trypan blue exclusion, adherence to tissue culture flasks, or surface binding of IgG globulin. These corticosteroid-induced defects in monocyte function may contribute to reduced cellular defense during corticosteroid therapy.

The effect of gamma interferon on IL-1 secretion of in vitro differentiated human macrophages.

After being cultured overnight, human monocytes lose their ability to secrete interleukin‐1 (IL‐1) when stimulated by lipopolysaccharide (LPS). However, when these monocytes were cultured for up to 9 days with various concentrations of interferon‐gamma (IFN‐gamma), these cells were found to retain their ability to secrete appreciable amounts of IL‐1 on LPS stimulation. However, the effect was observed only if the monocytes were exposed to the IFN before LPS stimulation and simultaneous addition of IFN and LPS to macrophages was ineffective. This effect of IFN‐gamma was related to the concentration of IFN added to the cultures and was completely neutralized by a monoclonal antibody to IFN‐gamma. In addition to inducing IL‐1 secretion, IFN‐gamma also appeared to increase the overall production of IL‐1, since reinduction of IL‐1 secretion was not associated with a decrease in intracellular IL‐1 content. When these macrophages were initially cultured with IFN‐gamma, washed, and further cultured with IFN free medium, these macrophages were found to progressively lose their capacity to secrete IL‐1 in response to LPS. Conversely, when monocytes were initially cultured in medium free of IFN, washed, and then further cultured in new medium, but now containing IFN‐gamma, these macrophages were found to regain their capacity to secrete IL‐1. However, the amount of reinduced IL‐1 secretion decreased as the length of the initial culture period without IFN increased, with less than optimal IL‐1 secretion occurring if monocytes were allowed to mature for 6 days before IFN‐gamma pretreatment. In summary, these studies suggest that IFN‐gamma may be important in enhancing IL‐1 production and secretion by maturing macrophages and tissue macrophages and consequently may play a role in regulating the accessory cell activity of these cells for a variety of immune responses in vivo.

Interleukin 1 secretion by human monocytes and macrophages.

Interleukin 1 (IL‐1) is generally regarded as a major regulator of T lymphocyte proliferation. Macrophages from animals and cloned tumor cell lines have been shown to produce this monokine in response to a variety of stimuli. The ability of human monocytes and macrophages to generate IL‐1 is much less well characterized. We previously demonstrated that human monocytes cultured for 1–6 days transformed to macrophages but retained their capacity to support concanavalin A‐driven T cell proliferation. However, cultured macrophage capacity to support antigen‐driven T cell proliferation began to decline after 3 days of culture and was markedly deficient by 6 days of culture. To determine if this loss of accessory cell function was due to the inability to secrete IL‐1, we measured the monokine produced by normal fresh human monocytes and macrophages cultured in vitro from monocytes. IL‐1 was assayed by the mouse thymocyte proliferation method. Fresh monocytes secreted IL‐1 readily in response to lipopolysaccaride and latex particles. Macrophages cultured from fresh monocytes, however, lost this ability after ≥2 days in culture. Mixing experiments failed to demonstrate an inhibitor present in the macrophage supernatants that would suppress thymocyte proliferation. Stimulated T cells incubated with monocytes and 3‐day cultured macrophages failed to prolong or promote IL‐1 secretion.

Central Nervous System Toxicity of Biological Response Modifiers

Exploiting the immune system has been an attractive and, for several decades now, an aggressively pursued approach for the treatment of cancer. The immune system is extremely complex, consisting of various cellular compartments such as Tcells, B-cells, macrophages, and natural killer (NK) cells under the regulation of a variety of cytokines. The term “biologic response modifier” has been applied to cancer treatments using cytokines, as well as other substances derived from both biological and non-biological sources, that alter the immune system and eventually the host-tumor interaction. The list of potential therapeutic agents is long and the potential for exploiting the immune system for the treatment of cancer is great. During the past decade, the focus in applying biological response modifiers to cancer therapy has been on evaluating cytokines. The development of recombinant technology has allowed for the production of large quantities of purified agent and has made, more than any factor, widespread testing in cancer patients possible. Clinical trials evaluating cytokines have demonstrated a different spectrum of toxicities for these agents from those of conventional cytotoxic chemotherapeutics. Whereas nausea, vomiting, and bone marrow suppression are often dose-limiting with conventional chemotherapeutics, biological response modifiers most often elicit a toxicity complex characterized by fever, chills, malaise, and anorexia. In light of the interaction between the central nervous and immune systems, it is not surprising that clinical trials evaluating biological response modifiers have also demonstrated that central nervous system (CNS) toxicity is very common. It has sometimes been difficult to determine the exact role of the biologic response modifier in producing CNS toxicity. Frequently patients are also receiving antiemetics, antihistamines, narcotics, or other agents with sedative effects. Preexisting brain metastases or other organic brain abnormalities may predispose patients to CNS toxicity from the use of a biologic response modifier or may be directly responsible for the symptomatology. In some instances, previously asymptomatic brain metastases have become apparent in conjunction with use of the biologic response modifier and all other adjuvant medication. Also, it is often difficult to determine whether CNS toxicity is primary or the consequence of other systemic effects produced by the biologic response modifier including often profound cardiovascular, renal, and hepatic dysfunction.

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation☆

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.

Modulation of adhesion molecules on human large granular lymphocytes by interleukin-2 in vivo and in vitro.

The modulation of adhesion molecules on human large granular lymphocytes (LGL) by interleukin (IL)-2 was investigated both in vivo and in vitro. Intercellular adhesion molecule-1 (ICAM-1; CD54) expression increased on LGL of cancer patients receiving IL-2 adoptive immunotherapy. ICAM-1 expression on LGL isolated by Percoll gradient centrifugation, LGL purified, and expanded by adherence to plastic surfaces and LGL identified by Leu 19 (CD56) monoclonal antibody were increased significantly in response to IL-2 in vitro. Exposure of LGL to IL-1, interferon (IFN)-gamma, and tumor necrosis factor (TNF) in vitro did not induce ICAM-1. The expression of LFA-1 (CD11a/CD18), a receptor for ICAM-1, and other leukocyte adhesion molecules, including Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), was only maintained by IL-2. IL-2 induction of ICAM-1 and the maintenance of CD18 complex expression on small lymphocytes separated by Percoll gradients were similar to that on LGL. We conclude that IL-2 enhances the expression of ICAM-1 on multiple human lymphocyte populations including LGL effectors. Expression of the CD18 complex on LGL does not appear to be highly regulated by IL-2. These findings may have implications relevant to the role of these adhesion molecules in the activities of LGL modulated by IL-2.

Human monocyte to macrophage differentiation in vitro: characterization and mechanisms of the increased antibody-dependent cytotoxicity associated with differentiation.

Human monocyte‐to‐macrophage differentiation in vitro is associated with marked enhancement in the capacity to bind and lyse antibody‐opsonized red blood cells (rbc). We previously demonstrated that this was due in part to an increased number of Fc receptors. The current study further characterized the mechanism of this enhanced macrophage as antibody‐dependent cellular cytotoxicity (ADCC). We observed that 1) macrophages but not monocytes lysed “innocent bystander” rbc as well as opsonized rbc; 2) macrophages exhibited an increased hexose monophosphate shunt activity compared to monocytes; 3) macrophage produced H2O2 but not OH·; and 4) macrophage lyses of opsonized rbc were 80% O2 dependent. We conclude that human monocyte‐to‐macrophage maturation in vitro is associated with an enhanced O2‐dependent cytotoxic mechanism normally present in monocytes. The enhanced H2O2 production noted in macrophages may reflect an increased generation of several other reactive oxygen species in these cells. One of these oxygen radicals may be the mediator of the enhanced macrophage cytotoxicity and of the “innocent bystander” phenomenon observed.

Biologic effects of the adoptive transfer of cells depleted of monocytes with L-phenylalanine methyl ester

Monocytes macrophages have negative regulatory effects on many immunologic responses. Depletion of monocytes from peripheral blood using the lysosomotropic agent, L-phenylalanine methyl ester (PME), has been shown to improve lymphokine activated killer (LAK) cell expansion in vitro. A pilot study of the adoptive transfer of LAK cells expanded with PME was performed in patients with metastatic renal cell carcinoma. Patients received interleukin-2 (IL-2) by continuous infusion for 5 days. Leukopheresis was performed daily for 4 days during the second week. Cells obtained from 8 patients were depleted of monocytes using PME in an one-step procedure; < or = 3% of the remaining cells were monocytes. All cells were expanded for 10 days in air-porous plastic bags with IL-2. Cells expanded 2.7-fold when depleted with PME and 1.7-fold when not depleted (P = 0.02). Expanded cells were administered together with IL-2. Patients received up to 60 x 10(10) PME-depleted cells (mean = 26 x 10(10)) with LAK activity (% lysis) of 60 +/- 12%. Lymphocyte phenotype and cytolytic activity were not modulated by PME-depletion, and clinical toxicities and systemic immunologic effects observed in patients receiving PME-depleted cells were similar to that of 5 patients receiving cells not expanded with PME. Thus, the use of PME to deplete monocytes ex vivo can result in the yield of large number of effectors that retain immunologic activity for potential clinical use. The process is convenient, efficient, and does not add clinical toxicity.

Expansion of lymphokine-activated killer cells for clinical use utilizing a novel culture device

Two major problems encountered in the application of lymphokine-activated killer (LAK) cell therapy in man are the massive culture volumes required for LAK cell induction and the paucity of LAK cells available for administration (human doses are less than or equal to 10% of effective murine LAK cell doses). We have, therefore, developed and tested a plastic porous culture device, Sclair plastic bags (E.I. DuPont De Nemours Co.), that can be utilized at virtually any volume and does not require rotation for optimal use. Normal or patient lymphocytes were cultured in the device or in plastic 16 mm wells at 1-20 X 10(6)/ml RPMI 10% human sera with 1500 pM interleukin-2 for 4 days: LAK cell activity did not decline despite high cell densities. The device was equal to the 16 mm wells in induction of normal donor and patient LAK cell activity when either autologous fresh tumor or Raji targets were used. In a non-therapeutic clinical evaluation we isolated and stored in liquid nitrogen autologous tumor cells from 11 patients with cancer. 2-6 weeks post-operatively lymphocytes and mononuclear cells from these patients and paired normal donors were obtained and LAK cells were induced in Sclair bags or standard culture wells. Autologous patient LAK cell activity and normal donor LAK cell activity against patients tumor cells were equivalent in the Sclair culture device and culture well system. Lymphocyte recovery and [3H]thymidine incorporation were also similar. Subsequently, we developed an expansion scheme utilizing the device in which cell density was maintained at optimal levels by changing media and reducing cell concentration after 6, 10 and 14 days of culture. We were able to expand LAK cell number 5-10-fold with no loss of LAK cell activity in this time frame utilizing both normal and patient cells. In this system plasma and sera were equivalent in their capacity to support LAK cell expansion but less than 10% plasma or sera supported suboptimal activation. Thus, we have developed a practical system to augment the number of LAK cells available for human LAK cell therapy and simultaneously reduce the complexity and volume of the induction system.

ACI-reductones enhance interleukin-2-induced lymphocyte cytotoxicity

Arachidonic acid (AA) metabolites and reactive oxygen species (ROS) are implicated in the suppression of interleukin-2 (IL-2) activity. We investigated the effects of aci-reductones, compounds that function both as inhibitors of AA metabolism and as scavengers of ROS, on the generation of IL-2-induced, lymphokine activated killer (LAK) activity. Aci-reductones belonging to the 4-aryl-2-hydroxytetronic acid system improved the in vitro generation of LAK activity from IL-2-treated human peripheral blood mononuclear cells (PBMC) approximately 4-fold. Those aci-reductones belonging to the 3,4-dihydroxyhenzofuranone class were less effective. LAK activity improvement was comparable to that produced by indomethacin with superoxide dismutase plus catalase and comparable to the improvement produced by depleting PBMC of monocytes. Aci-reductones completely suppressed the production of prostaglandin E2 from PBMC in response to IL-2 and partially suppressed superoxide anion production. Daudi cell and lymphocyte subset proliferation and monocyte viability were not affected. Less improvement in LAK activation was observed when PBMC depleted of monocytes were exposed to IL-2 and aci-reductones. We conclude that aci-reductones improve LAK generation from PBMC in vitro. This property may be mediated via effects on monocyte AA and ROS metabolism.